EXAMINING HOW STAPHYLOCOCCUS AUREUS ENGAGES THE SULFUR REGULON TO MEET NUTRITIONAL DEMANDS By Paige Jennifer Kies A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics – Doctor of Philosophy 2023 ABSTRACT The age of antimicrobial resistance has driven a sharp demand for new routes that inhibit opportunistic pathogens such as Staphylococcus aureus. In response to this has been a steady rise in research characterizing pathways essential to pathogen proliferation, such as those relating to essential nutrient acquisition and metabolism. S. aureus employs a flexible metabolism that promotes colonization throughout most mammalian tissues. This characteristic, combined with an ability to develop antibiotic resistance, incites nosocomial infections that range from skin and soft tissue infection to osteomyelitis. Regarding essential nutrients, the foundation for S. aureus metabolism for the critical element sulfur is still being molded. The current thesis provides a series of investigations that expand our understanding of how S. aureus engages sulfur metabolism to meet nutritional demands. Expression of the machinery involved with sulfur acquisition and catabolism is under control of the transcriptional repressor, CymR. We investigate the transcriptional response of S. aureus to varying states of sulfur supplementation in vitro with a special focus on the sulfur regulon. We note that during sulfur starvation there is both a CymR- dependent and -independent response that occurs; notably, the majority of differentially expressed genes during sulfur depletion are influenced by the presence of CymR. Found within the sulfur starvation response is the upregulation of iron metabolism and oxidative stress response genes. Glutathione was used to exemplify this observed connection between sulfur, iron, and oxidative stress—the S. aureus sulfur source also protects against toxic levels of heme (iron source) and the oxidative stressor, H2O2. We further show that S. aureus expresses unique responses when exposed to organic (cysteine, reduced and oxidized glutathione) and inorganic (thiosulfate) sulfur sources. With this 2 series of transcriptomics, the thiosulfate uptake protein A (TsuA) is shown as the sole importer associated with growth on thiosulfate as a sulfur source. Interestingly, each sulfur source induced upregulation of sulfur-associated transporters, suggesting that regulatory actions within the sulfur regulon are more complex than previously appreciated. Upon examination of the sulfur regulon, we next focused on the redundancy surrounding transportation mechanisms of S. aureus sulfur sources. Glutathione is one example, having at least two associated transporters in this pathogen. We identify that the di-tripeptide transporter, DtpT, supports proliferation of S. aureus on nutritional glutathione. This line of inquiry also describes cysteinyl-glycine, the glutathione breakdown product, as a DtpT-mediated sulfur source for S. aureus. DtpT contributions to S. aureus physiology is likewise demonstrated through its impact on colonization of the murine liver. We also initiated a new field of study that characterizes the distribution of host sulfur sources using matrix-assisted laser desorption/ionization imaging mass spectrometry. With this technique we confirm the presence of several known sulfur sources during S. aureus infection of the murine kidney, including reduced and oxidize glutathione as well as cysteinyl-glycine. An additional metabolite, cysteine-glutathione mixed disulfide, was found to be highly abundant in the S. aureus-infected kidney. We establish that this compound is a sulfur source for this organism and that growth on cysteine-glutathione disulfide is associated with three transporters: the glutathione import system (GisABCD) as well as the cysteine/cystine transporters, TcyABC and TcyP. This work is concluded with thoughts surrounding future research which, alongside the data presented here, will further enhance our understanding of the nutritional sulfur interface between the host and pathogen during S. aureus infection. 3 This thesis is dedicated to those who have supported me. Thank you for helping me make dreams a reality. iv ACKNOWLEDGMENTS I would like to begin by expressing my heartfelt gratitude to my mentor, Dr. Neal Hammer. His exceptional guidance and the nurturing environment he provided have been instrumental in fostering my confidence and honing my skills as a scientist. I am immensely thankful for his unwavering support and encouragement to challenge myself! My committee members, Drs. Sean Crosson, Robert Quinn, Gemma Reguera, and Chris Waters, have also made fundamental contributions to my development as a scientist. I am thankful for all of their invaluable insights over the years, which have nurtured my critical thinking skills and overall scientific abilities. I also want to extend my appreciation to all the members of the Hammer laboratory, both current and past. Nick, Jessica, and Gabby have not only been fantastic colleagues but have also contributed to creating a relaxed and enjoyable lab atmosphere. I'd like to offer special recognition to the undergraduates I've had the privilege of working with during my time at Michigan State University: Abigail Kuplicki, Emily Cleary, Jessica Liu, and Rosemary Northcote. Learning how to be an effective mentor is an ongoing process that demands time, patience, understanding, and open communication from all parties involved. I'm truly thankful to each undergraduate who has assisted me in my journey toward becoming a better mentor. A special mention goes to Rosemary for her dedicated benchwork assistance during the crucial period leading up to my defense. She's an exceptional individual with a bright future ahead in her own graduate career, and I wish her nothing but the best! My fellow graduate students and post-doc in the lab provided a support and camaraderie which was indispensable throughout my graduate school journey. Joelis Lama Díaz, with boundless energy and a gift for bringing out the best in everyone, played v a crucial role in helping our lab break out of its shell. I am especially grateful to Joelis for generously allowing me to include her S-lactoylglutathione data in Figure 4.3 of this dissertation. Troy Burtchett, a true embodiment of the term “cool as a cucumber”, remained unflappable under all circumstances and kept the lab entertained with his reservoir of dad jokes. Cristina Kraemer Zimpel, an amazing post-doc in the lab, has been a constant source of guidance as I wrap up my tenure at MSU. I can't express enough how much I value her assistance, from training me on RNAseq analysis for Chapter 2 to creating a phylogenetic tree for Chapter 3. Rajab Curtis, a former PhD student in the lab who joined the program alongside me, has been an incredible peer on this journey. We've faced numerous challenges together, and I couldn't have asked for a better bench buddy during the pandemic. I've always enjoyed our discussions, whether they revolved around scientific theories or the playlists on Rajab's Spotify. Lastly, Beth Ottosen...she's simply the best, or should I say the 'be(th)st'? While Beth only rotated in the lab, she became my closest friend here in Michigan and has stood by me through every obstacle, often with a pop-tart in hand. The Department of Microbiology and Molecular Genetics at MSU is home to a remarkable group of individuals who consistently provide unwavering support for one another. I must extend my gratitude to Roseann Bills from the MMG office, who has been instrumental in guiding me through the intricacies of graduate school's technical aspects. I would have undoubtedly found myself lost on numerous occasions if it weren't for her irreplaceable help. The fifth floor of BPS holds a special place in my heart. I cannot begin to express how many times someone on this floor came to my aid, whether it was by providing a much-needed reagent, lending a friendly ear for scientific discussions, or vi simply stopping for a quick chat. I consider myself incredibly fortunate to have pursued my graduate studies within this department. I additionally want to thank my family and friends. They always say it takes a village and I firmly believe in that sentiment. My extended family members, who have checked in on me throughout the years, always brought a warm smile to my face and provided the encouragement I needed during tough times. I also want to acknowledge the incredible support I've received from two dear friends, Emily Baldwin and Akshaya Warrier, who have remained with me over many years, reaching out and forever encouraging me during our many video chats. My sister, Lily, played a crucial role in reminding me that life exists beyond graduate school, often through phone calls and conversations about topics unrelated to my work! She too is one of my oldest supporters and I cannot thank her enough for that. I would also like to recognize my brother Ben and his wife, Kami, for all their efforts in helping me set up the next chapter of my life in Minnesota. Above all, I want to express my deepest gratitude to my parents. No words can adequately capture the depth of my love for them. They both worked tirelessly to ensure that I had the opportunity to attend college, setting me on a life path that has consistently exceeded my expectations. My parents offered unwavering support throughout my journey. I can't fathom where I would be without them. vii TABLE OF CONTENTS CHAPTER 1: A resourceful race: bacterial scavenging of host sulfur metabolism during colonization..................................................................................................................... 1 CHAPTER 2: Defining the transcriptional adaptation of Staphylococcus aureus to a range of nutritional sulfur supplementation……….................................................................... 28 CHAPTER 3: Staphylococcus aureus DtpT supports nutrient sulfur acquisition of γ- glutamyl cycle intermediates........................................................................................... 79 CHAPTER 4: Employing MALDI-IMS to visualize the host-pathogen nutritional sulfur interface during Staphylococcus aureus systemic infection......................................... 118 CHAPTER 5: Summary and Future Directions............................................................. 139 REFERENCES..............................................................................................................150 APPENDIX A: Chapter 2 sulfur starvation Tables........................................................ 172 APPENDIX B: Chapter 2 sulfur source Tables............................................................. 283 viii CHAPTER 1: A resourceful race: bacterial scavenging of host sulfur metabolism during colonization Work presented within this chapter has been published as: Kies PJ, Hammer ND. 2022. A Resourceful Race: Bacterial Scavenging of Host Sulfur Metabolism during Colonization. Infect Immun 90:e00579-21. 1 ABSTRACT Sulfur is a requirement for life. Therefore, both the host and colonizing bacteria must regulate sulfur metabolism in a coordinated fashion to meet cellular demands. The host environment is a rich source of organic and inorganic sulfur metabolites that are utilized in critical physiological processes such as redox homeostasis and cellular signaling. As such, modulating enzymes dedicated to sulfur metabolite biosynthesis plays a vital role in host fitness. This is exemplified from a molecular standpoint through layered regulation of this machinery at the transcriptional, translational, and posttranslational levels. With such a diverse metabolite pool available, pathogens and symbionts have evolved multiple mechanisms to exploit sulfur reservoirs to ensure propagation within the host. Indeed, characterization of sulfur transporters has revealed that bacteria employ multiple tactics to acquire ideal sulfur sources, such as cysteine and its derivatives. However, bacteria that employ acquisition strategies targeting multiple sulfur sources complicate in vivo studies that investigate how specific sulfur metabolites support proliferation. Furthermore, regulatory systems controlling the bacterial sulfur regulon are also multifaceted. This too creates an interesting challenge for in vivo work focused on bacterial regulation of sulfur metabolism in response to the host. This Chapter examines the importance of sulfur at the host-bacterium interface and the elegant studies conducted to define this interaction. 2 INTRODUCTION Bacteria impact the health of complex, multicellular organisms such as humans. This concept is well recognized and remains an area of intense scientific study. One focal point within this domain is understanding the nutritional relationship between the host and colonizing bacteria (i.e., pathogens or symbionts). Over the past few decades, there has been exceptional progress in describing the dynamic host-bacterium interplay surrounding essential nutrients such as iron, magnesium, manganese, zinc, and carbon (1–3). Sulfur is another such essential nutrient that has started to receive more attention regarding its impact on bacteria during colonization. Within biological systems, sulfur is largely stored as a thiol group (R-SH) within organosulfur compounds. Due to the reactive property of sulfur, thiols partake in numerous cellular reactions, a characteristic that demands cells maintain an optimal supply of this nutrient (4). At the center of sulfur metabolism and distribution within a cell is the amino acid cysteine (Cys). In fact, intracellular Cys concentrations indicate whether a cell has sufficient quantities of sulfur. When Cys levels are below the threshold, cells import exogenous sulfur-containing metabolites and assimilate them to Cys. From there, Cys contributes to cellular processes such as maintenance of protein structure or ion coordination, synthesis of critical metabolic cofactors (e.g., coenzyme A, biotin), and redox balance (5–9). Here, we will discuss the diverse nutritional sulfur reservoir generated through host sulfur metabolism and the strategies bacteria utilize to exploit this diversity to meet the sulfur requirement during colonization. 3 HOST SULFUR METABOLISM Given the essential nature of sulfur, it can be hypothesized that the host modulates sulfur metabolism in response to bacterial colonization. However, to our knowledge there are no investigations that directly support this hypothesis. Thus, to address whether and how the host alters sulfur metabolism during infection, it becomes pertinent to first understand metabolism of this element from the perspective of a healthy host. As more information is presented regarding the host-pathogen nutritional sulfur interface, models then can be developed that reflect how these pathways operate during various types of infections. Furthermore, understanding how the host controls flux of sulfur during infection will require investigating alterations of enzymatic expression or activity within sulfur metabolism pathways, generating further interest for discussion of the proteins involved in sulfur distribution. Consequently, we will (i) briefly describe enzymatic pathways involved in generating sulfur-containing metabolites that are known sources of nutrient sulfur for bacteria and illustrate their diverse regulatory systems, (ii) address the contribution of pathways or enzymes to host physiology, and (iii) consider the consequences that arise from dysfunction of the major enzymes within these pathways. Transmethylation and reverse transsulfuration pathways. As the fulcrum of sulfur metabolism, free Cys can be synthesized from methionine via the transmethylation pathway (Fig. 1.1, orange) (10). The product of transmethylation, homocysteine, is subsequently used as a substrate in the reverse transsulfuration pathway, which requires the critical enzymes cystathionine β-synthase (CBS) and cystathionine γ-synthase (CSE; Fig. 1.1, green) (11–13). In animals this pathway is irreversible, meaning neither methionine nor homocysteine is produced from Cys. As the final product, Cys can 4 autoxidize to cystine (CSSC) or be acetylated, forming N-acetylcysteine (NAC) (Fig. 1.1, asterisk). Furthermore, reverse transsulfuration may flux such that Cys undergoes CBS- or CSE-mediated desulfhydration, resulting in production of hydrogen sulfide (H2S) and, in the case of CBS, a net loss of homocysteine (11). A common thread is that the enzymes participating in these two pathways are widely distributed, if not ubiquitous, throughout mammalian tissues (Table 1.1). In general, though, the pathways are predominantly active in the liver or kidney. In keeping with their importance to host physiology, products and substrates of the trans-methylation and reverse transsulfuration pathways are established sources of nutrient sulfur for symbiotic and pathogenic bacteria (14) (Figure 1.1, boldface, underlined). As a consequence of their expansive tissue localization, CBS and CSE regulation is complex. These two proteins are constitutively expressed in most cell types with a layered regulation system, from gene expression to posttranslational modifications, that determines enzyme concentrations throughout the host (15–18). As a result, CBS is abundant in the liver, brain, kidney, and pancreas compared to moderate expression in the muscles, endocrine, heart, or lungs. CSE also displays higher expression in the liver and kidney. In contrast to CBS, CSE is more prevalent in the cardiovascular system as well as the lungs and in the brain (15–18). Transmethylation and reverse transsulfuration enzymes as a whole localize to the cytosol of cells under normal conditions. However, some of the enzymes may move to other organelles under specific circumstances. CBS and CSE, for instance, can both be relocated to the mitochondria under conditions such as hypoxia (CBS) or an increase of intracellular Ca2+ (CSE) (19). 5 Dysfunction within either pathway leads to serious host impairments, another over- arching theme of host sulfur metabolism. For example, S-adenosylmethionine (SAM) accumulation from disrupted methionine adenosyltransferase (MAT) in the transmethylation pathway contributes to an array of ailments such as methionine accumulation in the blood (i.e., hypermethioninemia), neurodegenerative diseases (e.g., Parkinson), and cancer (20). Dysfunction of CBS and CSE are also associated with disease (15–17). CBS is known to be upregulated in Down syndrome and in breast, colon, or liver cancers. Additionally, some naturally occurring mutations in CBS render the host incapable of metabolizing methionine, a disease known as homocystinuria. This leads to a buildup of homocysteine, leading to a condition known as hyperhomocysteinemia. Continuing this trend, decreased CSE activity contributes to a variety of host maladies, including those with diabetes, asthma, or chronic kidney disease. On the other hand, increased CSE activity also causes hyperhomocysteinemia. The g-glutamyl cycle. Cys can be stored within the stable tripeptide, glutathione (GSH). This versatile low-molecular-weight thiol consists of g-glutamate-cysteine-glycine and can reach millimolar concentrations within a cell (12, 21). The g-peptide bond between glutamate and Cys can only be cleaved by select enzymes such as g-glutamyl transpeptidase (GGT) to ensure a controlled Cys repository within the host (12, 22– 24).One benefit from this strategy is increased control over the generation of reactive oxygen species (ROS) that result when Cys autoxidizes to CSSC. However, if Cys must be replenished, GSH can be catabolized via the g-glutamyl cycle (Figure 1.1, blue) (25– 27). Interestingly, the dipeptide formed in this pathway, cysteinyl-glycine (Cys-Gly), is not solely dedicated to the g-glutamyl cycle, as it is thought to impact the redox status of the 6 plasma (28–30). This supports the notion that thiol-containing compounds participate in multiple aspects of host physiology. Given the impact of this cycle (discussed below), the presence of GSH catabolism enzymes in most or all host tissues is unsurprising (Table 1.1). As described above with the reverse transsulfuration enzymes CBS and CSE, the host exerts a complex regulation system for expression of proteins in this aspect of the g-glutamyl cycle. GGT, a well-studied example, is constitutively expressed with transcriptional control exerted through the use of multiple promoters for the corresponding gene; this encourages selective expression throughout different host-tissue environments (31, 32). Furthermore, translational regulation at the mRNA 5’ end acts as a second layer of management for GGT production across different tissues (31). Within mammalian hosts, this results in GGT being highly expressed in regions such as the kidney (particularly the proximal tubule), pancreas, intestine, brain capillaries, or various types of white blood cells (33, 34). As expected, GGT expression is comparably low in the liver, given that this organ is the main site for GSH production (33, 34). As is typical of any enzyme with major contributions to host physiology, dysregulation of GGT imposes health disorders. In fact, alterations to GGT concentrations have long been associated with increased cardiovascular disease (35–39). GGT levels in the liver are also used as a marker for one of the most common ailments for the organ, nonalcoholic fatty liver disease (NAFD) (40, 41). Synthesis of GSH from Cys and/or NAC precursors completes the g-glutamyl cycle (Figure 1.1, blue) (42, 43). Continuing the concept of multifactorial expression, production of glutamate-cysteine ligase (GCL) and GSH synthase is influenced at the posttranscriptional and translational levels. As this is a well-reviewed area, information regarding GSH synthesis enzyme 7 expression and dysfunction is provided from the following review (42). As a select example, we consider the similar features of GCL and GSH synthase expression. Gene activity for both increases in response to a variety of compounds, such as tumor necrosis factor (TNF-alpha) or hepatocyte growth factor (HGF), in cultured rat hepatocytes. Furthermore, expression of both GCL and GSH synthase partially involves activator protein (AP-1), whose activity is induced by TNF-alpha. However, bear in mind that there are a number of other transcription factor binding sites located at the promoters of these genes, such as an ARE consensus sequence (GCL) or nuclear factor erythroid 2 motif (GSH synthase). Additionally, it is noteworthy that hydrogen peroxide (H2O2)-induced oxidative stress increases GCL transcription as well. Although GSH synthesis occurs in most host tissues (e.g., kidney, pancreas, lungs, plasma, or brain), its production is highest in the liver, indicating robust activity of GSH synthase, GCL, or even GSH reductase (GR), which reduces oxidized GSH (GSSG) (Figure 1.1, blue) (44–46). Dysfunction of GSH biosynthesis enzymes contributes to a number of host diseases (42). Polymorphisms in GCL can contribute to decreased cardiovascular health or asthma; decreased expression of this protein complex is known to contribute to disorders such as cystic fibrosis or alcoholic liver injury. Additionally, buildup of g-glutamylcysteine in those with GSH synthase deficiency can have impacts on the central nervous system or result in conditions such as hemolytic anemia. Given that GSH contributes immensely to host physiology beyond the role of Cys storage, it is important to discuss some of its additional roles. Importantly, GSH is a major detoxification metabolite. GSH combats ROS stress, particularly H2O2, through its own oxidation via GSH peroxidase (Figure 1.1, blue, GP) (47). This is an important reaction given that H2O2 endogenously results from 8 mitochondrial respiration and can lead to hydroxy radical formation (48). Furthermore, GSH is the substrate for GSH S-transferases, which detoxify electrophilic xenobiotics by conjugating GSH to the harmful metabolite (12, 22, 48). These enzymes are also greatly expressed in the liver compared to other tissues, making the organ a critical site of GSH metabolism. Changes to protein activity can also result from the process of glutathionylation (49). In this instance, the GSH reduced-to-oxidized ratio influences S- glutathionylation of protein Cys residues. Considering this, it is easy to see how malfunction of GSH synthesis and catabolism enzymes result in a variety of host disorders, epitomizing the vital impact of the g-glutamyl cycle on host physiology. Taurine and sulfate biosynthesis. Aside from GSH, Cys is also a precursor for synthesis of the nonproteinogenic amino acid taurine and inorganic sulfate (Figure 1.1, pink) (18, 50–54). Note that in this pathway, glutamate oxaloacetate transaminase (GOT) is also known as cysteine aminotransferase (CAT) or aspartate aminotransferase (AST) due to its ability to bind cysteinsulfinate, cysteine, and aspartate. Typically GOT nomenclature is associated with taurine or sulfate biosynthesis, whereas CAT is linked to H2S synthesis (see below). As such, this protein will be denoted GOT/CAT. Taurine, though it does not directly contribute to protein synthesis, is one of the most abundant amino acids throughout host organs and has a multitude of functions. For example, it is known for its participation in bile acid formation and controls intracellular Ca2+ levels as well as alleviating glutamate toxicity in the brain (50). Sulfate also contributes to important cellular reactions within the host. Similar to GSH, sulfate conjugation is an important step in the activation/detoxification of exogenous and endogenous compounds, such as steroids or xenobiotics (55). Maintenance of cysteine dioxygenase (CDO) is imperative, 9 as it contributes to the balance of Cys levels and ensures production of taurine and sulfate as the first synthetic step of these two metabolites. Unlike other enzymes discussed, CDO is unique in that the prevalence of this enzyme is more heavily influenced by diet than gene expression: when Cys from ingested protein is abundant, CDO is as well (56). This compares to increased CDO ubiquitination and degradation when Cys is scarce (i.e., in a lower protein diet). The CDO-deficient murine line underscores the impact of this protein on host physiology, as these mice have a high postnatal mortality rate, growth impairment, and connective tissue pathology (57). With this in mind, it is interesting to speculate how the scavenging of Cys by a colonizing bacterium might adversely affect CDO abundance and the resulting effects on taurine and sulfate biosynthesis. H2S production and oxidation. Cys also contributes to formation of H2S (Figure 1.1, black) (58–61). It is noteworthy that CBS and CSE activity within cells are also major sources of H2S production during Cys desulfhydration (11, 62). H2S is an increasingly studied gaseous signaling molecule. It is a neurotransmitter with implicated roles in vasorelaxation, angiogenesis, inflammation, oxygen sensing, and cytoprotection (63–70). However, H2S can also be toxic to mammals, perhaps being most infamously known for its inhibitory effects on mitochondrial cytochrome c oxidase (71). With this assorted influence on host physiology, regulation of H2S is critical. Thus, H2S concentrations are tightly controlled and as a result become a hub for thiosulfate and sulfate production, which are also known inorganic sulfur sources for colonizing bacteria. Both of these sulfur compounds may be generated as a result of H2S oxidation in the mitochondria (Figure 1.1, purple) (72–74). Interestingly, the oxidation product thiosulfate can replenish this H2S pool (Figure 1.1, red) (75–77). Therefore, thiosulfate plays an integral role in production and 10 break-down of H2S within the host. Thiosulfate also detoxifies cyanide, underscoring its importance to management of volatile compounds (78). Due to this activity, thiosulfate is commonly administered to those with acute cyanide poisoning (75). H2S toxicity necessitates that its production is strictly controlled; this is demonstrated best by the previously described layered regulation of CBS, CSE, and other enzymes involved in H2S production. Naturally, H2S catabolism is a notable step of regulation for sulfur metabolism. Sulfide-quinone oxidoreductase (SQR) represents the first enzymatic step in H2S oxidation in the mitochondria, and activity of this enzyme contributes to normal function of this organelle in the model eukaryotic organism Schizosaccharomyces pombe (79). In vertebrates, SQR has been identified in the kidney, liver, heart, colon, podocytes, and even leukocytes (Table 1.1) (80). Although less is known about the regulation of SQR, it has been demonstrated that H2S increases expression of this enzyme (80, 81). On the other hand, Cys reduces transcriptional levels of SQR, which emphasizes its involvement in host sulfur metabolism (79). As with CDO, it is interesting to consider whether scavenging of Cys by bacterial colonizers influences SQR expression in the host. It has recently been reported that deleting SQR results in mitochondrial dysfunction due to cysteine deficiency in S. pombe (79). Given the importance of SQR in yeast, it is somewhat unsurprising that studies are beginning to link defects in this enzyme to mammalian disease as well. It has been proposed that altered SQR activity contributes to reduced H2S concentrations observed in people afflicted with Alzheimer’s disease or ethylmalonic encephalopathy (80, 82, 83). Furthermore, an association between SQR overexpression and postmenopausal osteoporosis has been implicated (84, 85). Collectively, these facts illustrate the vital nature of sulfur metabolism enzymes and the diverse ways in which they 11 impact the host. Additionally, determining whether and how sulfur scavenging by a colonizing bacterium encourages or exacerbates host diseases associated with dysfunctional sulfur metabolism will be of immense value to the health care system. COLONIZING BACTERIA EMPLOY MULTIPLE STRATEGIES TO ACCESS THE DIVERSEHOST SULFUR RESERVOIR. A colonizing bacterium must acquire essential nutrients using metabolites available in a dynamic environment. This point is emphasized in a previous review that discusses the wide range of sulfur acquisition strategies employed by bacterial pathogens (14). However, different host tissues present various concentrations or types of sulfur metabolites that bacteria are capable of catabolizing (Figure 1.1, boldfaced and underlined). This poses a unique challenge that a colonizer can surmount by employing multiple acquisition strategies for various sulfur metabolites. Here, we provide an update on implicated in vivo mechanisms employed by colonizing bacteria to exploit physiological sulfur reservoirs. Advances in pathogen sulfur acquisition: Staphylococcus aureus and Francisella tularensis. S. aureus is a Gram-positive bacterium and leading cause of hospital-acquired infections in the United States (86, 87). S. aureus persistently colonizes 20% of humans as a commensal on areas such as the skin or nasopharynx. However, the pathogenic potential of this organism greatly stems from an ability to propagate in most host tissues, indicating that S. aureus encounters most host sulfur metabolism compounds. Remarkably, S. aureus does not encode various enzymes required in sulfate, sulfonate, or taurine assimilation and is not capable of de novo methionine or Cys synthesis. As such, from a nutritional sulfur standpoint, import of compounds such as Cys 12 and its derivatives, g-glutamyl cycle intermediates, thiosulfate, and H2S, represent the host metabolites that this organism may capitalize on in vivo. In support of this, one critical study describes Cys, CSSC, thiosulfate, sulfide, and GSH as viable nutritional sulfur sources for S. aureus in vitro (88). A more recent study has also demonstrated GSSG as a source of nutrient sulfur for this opportunistic pathogen (89). Interestingly, a novel ABC- transporter (glutathione import system ABCD) was implicated as the major GSH and GSSG importer in S. aureus. As is typical for ABC transporters, this system hydrolyzes ATP as a source of energy for substrate import. GisABCD seems to be conserved within close relatives of the pathogen, excluding Staphylococcus epidermidis. In consideration of the individuals who mare persistently colonized with S. aureus, it has been proposed that GisABCD-dependent GSH acquisition is an evolutionary development to compete for sulfur resources in the presence of S. epidermidis. Employing this transporter during infection could then greatly affect the host GSH-to-GSSG thiol balance. Such a disruption could have considerable impacts both on the production of downstream host sulfur metabolites as well as other important physiological aspects, like ROS management. However, a gisBCD-deficient mutant displays no overt virulence defect during murine systemic infection. It is postulated that S. aureus expresses an alternative GSH transporter and that the activity of this theoretical protein masks overt phenotypes during infection. Should this be the case, encoding multiple GSH transporters would underscore the importance of GSH acquisition to S. aureus propagation within host tissues. It is well established that a number of colonizing bacteria encode multiple Cys/CSSC transporters, again emphasizing the importance organisms have placed on these organic compounds throughout evolution. Transportation of these metabolites in S. aureus has recently been 13 elucidated and investigated in a biological context (90). In this study, it was also determined that both NAC and homocystine, the oxidized form of homocysteine, are viable sulfur sources for S. aureus. The transporters implicated in their collective acquisition, TcyABC and TcyP, were further demonstrated to have biological impacts on S. aureus propagation in vivo. TcyABC is an ABC transporter, while TcyP belongs to the sodium- dicarboxylate symporter family (91). The latter family of proteins is widely distributed throughout eukaryotes and prokaryotes; members include those that import dicarboxylic acids and some amino acids, including those that are small, nonpolar, and neutral, such as Cys (92). Notably, S. aureus TcyABC and TcyP function similarly to their Bacillus subtilis homologs, which have been previously characterized (93). In S. aureus, TcyABC and TcyP are necessary for maximal fitness in colonization of the murine host for both the methicillin-sensitive strain Newman and resistant strain LAC. Specifically, the Newman tcyABC tcyP double mutant displayed significantly reduced colony forming units (CFU) numbers in the heart when in competition against the wild type (WT). However, in the liver in activation of only tcyP results in impaired Newman colonization. Additionally, TcyP of LAC was found to be required for maximal competitive fitness in the murine heart and liver. In addition to increasing foundational knowledge, this study accentuates the fact that intraspecies variance exists for how some bacteria meet the sulfur nutritional requirement in the host. Identification of NAC and homocystine as sources of nutrient sulfur for S. aureus warrants a brief discussion regarding their distribution and impact within a healthy host. Both metabolites have been detected in human plasma, a location where thiols are commonly found in their oxidized form (21, 43, 94, 95). It is noteworthy that the balance between reduced and oxidized homocysteine has long been associated with 14 cardiovascular disease (e.g., forms of atherothrombotic disease) (96, 97). Thus, future in vivo data implicating the use of homocysteine or homocystine by a pathogen could have interesting implications regarding how the bacterium affects the cardiovascular system of mammalian hosts. In addition to being part of the plasma redox pool, NAC is known to be a GSH precursor within mammals, either through its own deacetylation or by its role in reducing CSSC to Cys; due to this, NAC is a clinically relevant supplement used to increase GSH levels within the host (43, 98). It is clear from these studies that the flexible nature of S. aureus sulfur metabolism and the burden this plasticity enforces on the host is just beginning to emerge. Sulfur acquisition and metabolism of the Gram-negative, facultative intracellular pathogen F. tularensis has also seen advances in recent years. This organism is the etiological agent of tularemia, a disease with various clinical manifestations depending on the route of entry (99). As a Cys auxotroph, F. tularensis depends on GSH as a source of Cys for successful intramacrophage survival (100). However, F. tularensis Ggt periplasmic localization implies that GSH catabolism initiates in this subcellular environment, resulting in Cys-Gly being transported into the cytosol as the nutrient sulfur source. In support of this notion, a Tn-Seq screen for genes required for replication in J774A.1 murine macrophage-like cells revealed a proton-dependent oligopeptide transporter (POT) family gene, dptA, as a top hit (101). This observation was validated through monoinfection of J774 macrophages with a dptA mutant. Furthermore, in a murine model of pulmonary infection, DptA significantly impacts the ability of F. tularensis to colonize the lungs and spleen. Reduced accumulation of radiolabeled GSH was also observed with a dptA mutant, supporting its function as a Cys-Gly transporter. Carbonyl cyanidem-chlorophenyl hydrazone (CCCP) abolition of WT F. tularensis cell 15 membrane potential also reduced intracellular radiolabeled GSH abundance to that of a dptA mutant, exemplifying this protein as a POT family member. Overall, these findings are the first to demonstrate Cys-Gly as a viable nutrient sulfur source to bacteria within multiple host environments. Progress regarding symbiotic sulfur acquisition: Staphylococcus hominis and Vibrio fischeri. Like S. aureus, S. hominis is a commensal of the human underarm skin (axilla) microbiome and has pathogenic potential, albeit to a lesser extent than S. aureus (102). Though little is known regarding sulfur metabolism in this species of staphylococci, one can predict capabilities similar to those of S. aureus. A current study clearly demonstrates the ability of S. hominis to import a sulfur-containing metabolite as well as accentuating another physiological role for Cys-Gly in the host (103). Here, the g- glutamyl cycle is highlighted for its participation in the production of an axilla malodor precursor, S-[1-(2-hydroxyethyl)-1methylbutyl]-L-cysteinylglycine (S-Cys-Gly-3M3SH). This pathway begins when glutathione S-transferase preforms a conjugation reaction, forming glutathione 3-methyl-3-sulfanylhexanol (SG-3M3SH) (104). From there, host GGT generates S-Cys-Gly-3M3SH. At this point it is postulated that S. hominis imports S-Cys- Gly-3M3SH through the POT family protein, PepT. Once in the cytoplasm, 3-methyl-3- sulfanylhexan-1-ol (3M3SH) is liberated and released back into extracellular milieu, which produces malodor. Although it has not been demonstrated, it is tempting to speculate that release of Cys during this process aids in the maintenance of nutrient sulfur demands in S. hominis. In light of the fact that host-bacterium interactions are not limited to mammals, the Gram-negative marine bacterium V. fischeri serves as a growing model of sulfur metabolism for nonmammalian symbionts. V. fischeri is a bioluminescent symbiont located 16 in the light organ of the bobtail squid, Euprymna scolopes (105). Both organic (Cys, CSSC, and GSH) and inorganic (sulfate and thiosulfate) sulfur sources are capable of supporting V. fischeri proliferation in vitro, and pioneering efforts have defined the nutritional sulfur interface between E. scolopes and this bacterium (105).In the first study, various mutants of the sulfur regulon regulator, CysB, were utilized (105). The authors determined that symbiosis of a cysB mutant is not dependent upon the ability of V. fischeri to assimilate sulfate in the squid light organ. Furthermore, a cysB variant grows similarly to the WT when CSSC was present in vitro and in vivo. However, due to redundancy of encoded CSSC transporters, the authors could not completely determine how V. fischeri acquires this nutrient from the host. As mentioned above, this could be a strong indication of the importance CSSC has to V. fischeri colonization. To that end, when the CSSC transporter TcyP is ectopically expressed in a cysB mutant, the strain establishes WT-like symbiosis (106). Additionally, expression of tcyJ, which encodes a CSSC-specific periplasmic binding protein, is highly upregulated in a mutant defective for sulfate assimilation (106). Collectively these data indicate an association between CSSC utilization as a sulfur source and V. fischeri symbiosis. In this environment, CSSC is predicted to arise as a result of Cys oxidation upon degradation of host proteins in the light organ crypt space (105). The importance of sulfate acquisition to establishment of symbiosis was further elucidated (106). Sulfate is a dominant sulfur source in seawater. Given the prevalence of seawater in the squid light organ, it is a substantial supplier of sulfate in this environment. Therefore, bacteria that colonize this host organ likely utilize sulfate as a nutrient sulfur source. Wasilko et al. identified that a sulfate assimilation mutant displays lower numbers of CFU per milliliter than the WT, suggesting that sulfate is an impactful sulfur source during 17 symbiosis. To examine this concept from another angle, the authors identified that V. fischeri encodes a novel sulfate transporter, YfbS (106). YfbS belongs to the solute carrier 13 (SLC13) protein family, which consists of sodium-coupled anion symporters with substrates such as sulfate, thiosulfate, or dicarboxylates (107). A yfbS mutant exhibits lower bioluminescence and number of CFU in the light organ compared to the WT, emphasizing the importance of sulfate acquisition to V. fischeri symbiosis. Overall, it is predicted that in this location the abundant sulfate pool provides ample sulfur to an expanding population. Bacterial regulation of sulfur acquisition and assimilation machinery. To meet its nutritional requirement, a colonizing bacterium must employ sulfur acquisition and assimilation systems in dynamic host environments. However, importing too much sulfur can be toxic due to the involvement of intracellular Cys in Fenton chemistry (i.e., production of hydroxyl radical formation) (108, 109). Therefore, it is critical for bacteria to regulate sulfur metabolism genes such that ideal propagation conditions are maintained within the host. For simplicity, this review will discuss two models of regulation that have been tested in vivo, recognizing that other influencing factors have also been described (110–112). Collectively, genes dedicated to achieving the nutritional sulfur requirement are largely collected within the sulfur regulon. Depending on the Gram status of a bacterium, expression of this regulon will depend on either a positive or negative transcriptional regulator (Figure 1.2). However, in both models the Cys status of the cell is indirectly communicated to a transcriptional regulator through O-acetylserine (OAS). Regulation of sulfur metabolism in Gram-negatives has been thoroughly characterized (Figure 1.2, A and B). In this system, there are two key enzymes: the positive LysR-family 18 transcriptional regulator CysB and serine transacetylase (CysE). CysE is the product of a gene that is constitutively expressed (113, 114). The catalytic activity of CysE is responsible for providing the critical metabolite, OAS. To do so, CysE binds to L-serine and acetyl-coenzyme A substrates, and the resulting acetyl group transfer produces OAS and CoA (115). OAS may then act either as an intermediate in sulfate or thiosulfate assimilation to Cys or as a signaling molecule. It is noteworthy that OAS spontaneously converts to N-acetylserine (NAS) (115). As such, this pool will be denoted O/NAS. Furthermore, CysE activity is directly controlled by concentrations of intracellular Cys (115, 116). Abundant Cys inhibits CysE activity, and the diminished O/NAS pool renders CysB inactive, repressing sulfur regulon genes (Figure 1.2, A) (115). Alternatively, during Cys limitation O/NAS concentration grows as a result of increased CysE activity (Figure 1.2, B). O/NAS association with CysB fosters binding to promoters, allowing expression of nutrient sulfur acquisition strategies, and Cys production ensues (114). While work regarding effects of CysB on bacterial colonization is relatively sparse, the aforementioned V. fischeri study underscores the significance of this protein during colonization of the bobtail squid light organ (105). Monitoring green fluorescent protein (GFP) production from the promoter of a CysB-responsive gene revealed that CysB activity varies throughout the colonizing population within select locations of the E. scolopes light organ. It is suggested that this reflects the preference of V. fischeri to utilize sulfate where CysB activity is greater than CSSC acquisition in regions with lower regulator action. In other words, this work exemplifies the ability of a colonizing organism to modulate its sulfur regulon in response to available host sulfur sources. Gram-positive bacteria employ negative transcriptional regulation through the cysteine metabolism repressor, CymR (Figure 1.2, C and D) (117). 19 Although less studied than CysB, there is a good understanding of how this regulator functions in S. aureus (118). OAS-thiol-lyase B (CysM; also known as O-acetylserine sulfhydrylase B) also plays an impactful role in addition to CysE and CymR. When CysE activity is inhibited due to replete intracellular Cys concentrations, CysM associates with CymR. In this complex, CymR is then proposed to bind DNA, repressing expression of the sulfur regulon (Figure 1.2, C). As Cys becomes limiting, O/NAS binds to CysM, destabilizing the CysM-CymR complex or reducing the complex’s affinity for DNA (118, 119). The former is a more plausible outcome given that CysM also functions in thiosulfate assimilation to Cys. At this point CymR is inactive and releases from the DNA, promoting transcription of sulfur acquisition and assimilation genes (Figure 1.2, D). It should be noted that in B. subtilis, OAS-thiol-lyase A (CysK) has been shown to interact with CymR rather than CysM (119, 120). However, it is unclear why this occurs or what subgroups of Gram- positive species utilize CysM or CysK for this regulatory process. CymR is also rendered inactive upon oxidation of its sole Cys residue (Cys25), suggesting that this regulator also senses the oxidation state of the cell (e.g., Cys levels) to regulate Cys metabolism (121). It would be of interest for future work to characterize how oxidation of CymRC25 affects binding of CysM or CysK. Interestingly, a S. aureus cymR deletion mutant is more resistant to macrophage oxidative stress and yet is less virulent in a murine intraperitoneal or bacteremia infection model (122). Further investigation deter-mined that inactivation of cymR resulted in reduced d-hemolysin activity, which was pro-posed to be the factor contributing to the virulence defect. These investigations clearly illustrate the complex nature of CymR, from the factors that regulate its activity within the sulfur regulon to its connections with virulence. Recently another LysR-family protein, GigC, was described in 20 Acinetobacter baumannii (123). Through transposon sequencing, GigC was first noted as being critical for colonization of Galleria mellonella (124). lacZ reporter assays further determined that this protein acts as both an activator and repressor for several sulfur assimilation genes. Specifically, promoters for cysI and the cysDN operon showed increased expression in a gigC deletion mutant, whereas activity of cysH and cysQ promoters was decreased. The authors demonstrated that GigC binds to the cysI promoter and proposed that it binds other sulfur regulon genes, such as cysDN, cysH, and cysQ. Considering this information, one could speculate that the bifunctional nature of this regulator serves to fortify A. baumannii sulfur preference throughout the host. Consistent with this, a gigC mutant shows decreased virulence in murine models of intraperitoneal and peritoneal infection. Future work on the model of GigC function and assessments of its sulfur regulation activity in other bacterial species will undoubtedly exemplify the diverse control that can be exerted over this critical regulon. In doing so, questions such as why these distinct regulatory factors have evolved can be more aptly addressed. OUTLOOK It is clear from this collective body of literature that colonizing bacteria interact with the host to procure a variety of sulfur sources. However, there remains a pivotal knowledge gap regarding whether the host actively redistributes or limits sulfur as a result of bacterial colonization and whether bacterial sulfur acquisition systems are an evolutionary response to this action. To that end, the nutrient sulfur status (i.e., excess or limited) of bacteria during various stages of colonization is currently at a nascent stage. Defining in vivo importance of transporters to bacterial propagation within the host, however, helps clarify the relative nutrient sulfur source hierarchy for colonization. Furthermore, expanding our 21 view of how import and assimilation mechanisms are regulated assist in explaining bacterial responses to the host environment during colonization. Specifically relating to pathogens, expanding our knowledge regarding these sulfur acquisition and assimilation pathways serves to broaden the list of possible therapeutic targets in the age of multidrug resistance. However, a holistic under-standing of these bacterial studies is incomplete without considering the host perspective. As can be seen above, there is currently little to no characterization of how host pathways respond to bacterial colonization. This represents unexplored territory within the nutrient sulfur relationship at the host-bacterium interface. ACKNOWLEDGMENTS We thank members of the Hammer laboratory for their insightful input and discussions. We sincerely apologize to the colleagues we were unable to cite due to space limitations. This work was supported by National Institutes of Health no. R01AI139074 and R21AI142517. 22 Table 1.1. Localization of sulfur metabolism enzymes in mammalian tissues. TABLES Pathway Enzyme Host 𝛾-glutamyl transpeptidase (GGT) human 𝛾-glutamyl cycle dipeptidase (DP) porcine, rat Transmethylation pathway Transsulfuration pathway Taurine & sulfate production glutamate-cysteine ligase (GCL) mouse GSH synthetase rat methionine- adenosyltransferase (MAT) methyltransferase (MT) rat n.d. SAH hydrolase human, mammals cystathionine b- synthase (CBS) cystathionine g- synthase (CSE) human human, mouse, rat cysteine dioxygenase (CDO) rat, mouse cysteinesulfinate decarboxylase (CSD) rat, mouse 23 Example tissue localization kidney, pancreas, intestine, liver wide tissue distribution, largely expressed as brush boarder enzyme (kidney, small intestine) and in cytosol of liver liver, kidney, pancreas, lungs, plasma, brain kidney, liver, heart, lungs liver, pancreas, kidney, lungs n.d., likely same tissues as MAT all tissues, plasma liver, brain, kidney, pancreas, heart, lungs, muscles, endocrine cytosol; liver, kidney, heart, lungs, brain liver, adipose, kidney, brain, lung, pancreas, colon liver, kidney, brain, adipose, lung, pancreas, colon References (12, 22–24, 31–34) (22–27) (44–46) (46) (11) (11) (11, 13) (15, 16, 18) (16–18) (18, 50–52) (18, 50, 51) Table 1.1 (cont’d) H2S production from cysteine H2S oxidation glutamate oxaloacetate transaminase (GOT); aka cysteine aminotransferase (CAT) rat sulfite oxidase (SO) human, mouse 3- mercaptopyruvate sulfurtransferase (3-MST) mouse thioredoxin mammals sulfide-quinone oxidoreductase (SQR) sulfide dioxygenase (SDO) thiosulfate sulfurtransferase (TST) mammals mammals mammals liver, arterioles, lymphocytes, kidney, arteriolar endothelium, anterior pituitary (cytosolic and mitochondrial); two isoforms intermembrane space of mitochondria, largely found in liver, kidney widespread (high expression in liver, intestine, kidney), mostly localized in mitochondria ubiquitous; depending on isoform will be found in cytosol (Trx-1) or mitochondria (Trx-2) ubiquitous, matrix of inner mitochondrial membrane ubiquitous, mitochondrial matrix mitochondrial matrix, mostly found in liver, stomach/intestin es (50, 51, 53, 54) (51, 74) (50, 58, 60) (61) (51, 73) (51) (51) n.d. = not discussed 24 FIGURES respectively) involves methionine adenosyltransferase Figure 1.1. The host environment is rich with sulfur-containing metabolites. Cys and H2S influence production of other sulfur-containing metabolites through their interconnected participation in several metabolic pathways (denoted by colored arrows) within a eukaryotic cell. Note that these pathways are simplified versions of enzymatic reactions (i.e., only the sulfur-containing enzymatic substrates and products are shown). Known sulfur sources for colonizing bacteria are boldfaced and underlined. Biosynthesis of Cys results from the transmethylation and reverse transsulfuration pathways (orange and green, (MAT), S- adenosylmethionine (SAM), methyltransferase (MT), S-adenosyhomocysteine (SAH), SAH hydrolase, cystathionine β-synthase (CBS), cystathionine γ-synthase (CSE), and homocysteine S-methyltransferase (HMT). An asterisk denotes the ability of (i) Cys to oxidize to CSSC or methylate to form NAC and (ii) for homocysteine to oxidize to homocystine. Free Cys can be released from GSH via the γ-glutamyl cycle (blue), consisting of glutathione reductase (GR), glutathione peroxidase (GP), γ-glutamyl transpeptidase (GGT), and dipeptidase (DP) enzymes. This cycle may also result in production of GSH from Cys with the use of glutamate-cysteine ligase (GCL) and glutathione synthase (GSH synthase). Factors involved in the formation of sulfate and taurine (pink) include cysteine dioxygenase (CDO), cysteinesulfinate decarboxylase (CSD), nonenzymatic oxidation (dashed arrow), glutamate oxaloacetate transaminase, also known as Cys aminotransferase (GOT/CAT), and sulfite oxidase (SO). Cys can also 25 Figure 1.1 (cont’d) be directly metabolized to form H2S (black) with the activities of GOT/CAT, 3- mercaptopyruvate sulfurtransferase (3-MST), and thioredoxin (TRX). H2S can be oxidized to sulfate or thiosulfate (purple) using sulfide-quinone oxidoreductase (SQR), sulfur dioxygenease, also known as ETHE1 (SDO), and thiosulfate sulfurtransferase (TST). Thiosulfate is also a source of H2S as a result of TST or 3-MST activity (red). Image created with BioRender.com. 26 Figure 1.2. Intracellular cysteine modulates the bacterial sulfur regulon. (A to D) Regulation of the sulfur regulon in Gram-negative (A and B) and Gram-positive bacteria (C and D) under high (A and C) or low (B and D) intracellular Cys concentrations. (A) When abundant in a Gram-negative bacterium, intracellular Cys inhibits activity of serine transacetylase (CysE). O-acetylserine (OAS) production is reduced and cannot facilitate CysB binding to DNA. Consequently, CysB fails to induce expression of the sulfur regulon. (B) Upon depletion of Cys, CysE produces OAS, which spontaneously converts to N- acetylserine (NAS). Either NAS or OAS binds to CysB (denoted O/NAS), allowing the positive regulator to induce expression of the sulfur regulon. (C) Sulfur-replete conditions in Gram-positive bacteria inhibit CysE, which leads to decreased OAS levels and promotes CysM or CysK (denoted CysM*) interaction with CymR. CymR functions as a negative regulator that binds DNA and inhibits expression of the sulfur regulon. (D) Limited intracellular sulfur leads to increased CysE-mediated OAS generation, destabilizing the CysM*-CymR complex and promoting transcription of sulfur acquisition systems. Oxidation of CymRC25 during oxidative stress conditions also contributes to an inactive state. In the inactive state, CymR no longer binds DNA, allowing expression of sulfur acquisition and Cys biosynthesis genes. Image created with BioRender.com. 27 CHAPTER 2: Defining the transcriptional adaptation of Staphylococcus aureus to a range of nutritional sulfur supplementation 28 ABSTRACT Bacterial pathogens must adapt to dynamic environments to proliferate within host tissues. Accordingly, elegant regulatory systems evolved to combat the immune response, coordinate virulence factor production, and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcriptional changes of a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur- starvation response in chemically defined growth conditions. Notably, these results expand the set of genes within the sulfur starvation regulon and support previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. Validation of results is highlighted by the ability of sulfur-containing glutathione (GSH) to mitigate heme and peroxide toxicity in S. aureus. Since a variety of compounds fulfill the S. aureus sulfur requirement, we monitored transcriptional profiles in response to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic (thiosulfate) metabolites. This analysis reveals sulfur source-specific mRNA alterations, with thiosulfate inducing the largest transcriptional shift. Consequently, the sole thiosulfate transporter (SAUSA300_RS10985) has been established as it is essential for S. aureus growth when thiosulfate is the nutritional sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth of this bacterium on thiosulfate. Collectively, a resourceful 29 transcriptomics framework is provided which underscores the versatile nature of S. aureus sulfur metabolism. 30 IMPORTANCE The opportunistic pathogen Staphylococcus aureus proliferates within numerous host environments and consequently is a leading cause of hospital-acquired infections. During colonization S. aureus must acquire essential nutrients like sulfur to survive. Regulation of machinery to procure host-derived metabolites occurs to satisfy nutritional requirements and maintain homeostasis to circumvent detrimental impacts on bacterial physiology. This report investigates the S. aureus regulatory response during various states of sulfur supplementation in vitro, enhancing our knowledge of staphylococcal sulfur metabolism and represents a repository of information that will guide future research surrounding S. aureus sulfur acquisition and metabolism. 31 INTRODUCTION Staphylococcus aureus harbors considerable pathogenic potential given it adapts to and proliferates within most vertebrate organs (86, 125). Such capabilities demand bacteria sense and respond to the extracellular milieu to procure essential nutrients from the host. For example, regulatory mechanisms that control S. aureus transition metal acquisition and homeostasis are well defined (126). The transcriptional repressors Fur, Zur, and MntR modulate genes involved in acquisition and utilization of iron, zinc, and manganese, respectively. Each regulator has dedicated binding pockets for their cognate metal ion (i.e., Fe2+, Zn2+, and Mn2+) which senses intracellular concentrations, allowing the regulator to switch to an active, repressive conformation in replete environments (126). Activity of these metalloregulators is tightly coordinated given that transition metals, though essential, are detrimental when in excess (126–128). Sulfur represents another essential nutrient whose coordinated acquisition by S. aureus involves similar regulation. Within cells sulfur is stored in the form of cysteine (Cys) which partakes in numerous cellular reactions such as protein and co-factor synthesis or redox balance (4, 5, 7, 8, 129–132). Given this amino acid is the crux of sulfur distribution within a cell, all metabolites used to meet this nutritional requirement must be catabolized (organosulfur) or assimilated (inorganic) to Cys. As with iron, in Escherichia coli it has been demonstrated that an overabundance of intracellular Cys contributes to Fenton chemistry (108). Thus, stringent management of this element in coordination with iron is needed to accommodate proliferation (133). The major regulatory factor influencing S. aureus sulfur homeostasis is the cysteine metabolism repressor, CymR (117, 118, 134). In contrast to the metalloregulators, CymR indirectly senses Cys levels to modulate DNA 32 binding activity via two methods (Fig. 2.1). Intracellular Cys levels are communicated through the sulfur assimilation intermediate O-acetylserine (OAS) whose abundance will drop under high Cys concentrations. OAS-thiol-lyase B (CysM, aka MccA) responds to this OAS depletion by complexing with CymR, activating repression (Fig. 2.1A). Alternatively, CymR is also known to respond to the oxidation state of S. aureus through its sole Cys residue at position 25 (Fig. 2.1B). To maintain intracellular sulfur, metabolites—including Cys, cystine (CSSC), N- acetylcysteine, homocysteine, reduced and oxidized glutathione (GSH and GSSG, respectively), thiosulfate (TS), and hydrogen sulfide— are acquired by S. aureus and converted to Cys (88–90). These compounds participate in host sulfur metabolism across numerous tissues (134). However, even with advances in our understanding of sulfur metabolism in this pathogen, how S. aureus synchronizes internal demands for this element throughout the host and the mechanisms by which S. aureus prioritizes sulfur metabolite utilization remain outstanding questions. This study defines how S. aureus responds to sulfur starvation by establishing sulfur replete and deplete transcriptional profiles that emerge in a CymR-dependent and - independent manner. Using a chemically defined medium (CDM) supplemented with various sulfur sources, our findings expand upon work performed by Soutourina et al. who describe a CymR-dependent transcriptional response of S. aureus cultured in rich medium supplemented with 2 mM CSSC (118, 122). Like the previous study, we observe a CymR-independent upregulation of iron transport and oxidative stress genes when S. aureus is sulfur starved (122). The importance of connected regulation is established by showing that S. aureus scavenging exogenous nutrient sulfur in the form of glutathione 33 (GSH) combats heme-induced oxidative stress as well as enhancing survival in response to hydrogen peroxide (H2O2). Additionally, growth on inorganic TS elicited the greatest number of differentially expressed genes compared to proliferation on organic metabolites (Cys, CSSC, GSH, and GSSG). The RNAseq was substantiated by addressing the role of upregulated genes in response to TS. This investigation confirms that SAUSA300_RS10985, a YeeE/YedE family transporter, is required for S. aureus growth on TS along with a protein of unknown function, SAUSA300_RS10980. Together with their high degree of homology to the E. coli thiosulfate uptake proteins A and B (TsuAB), these findings suggest the reannotation of SAUSA300_RS10985 and SAUSA300_RS10980 to tsuA and tsuB, respectively (135). Overall, this work validates the importance of transcriptional links between sulfur starvation, iron homeostasis, and oxidative stress responses as well as accentuating intricacies within the S. aureus sulfur regulon. RESULTS Staphylococcus aureus sulfur starvation response includes altered transcription within the CymR regulon. A direct assessment of the S. aureus sulfur starvation response has not been established. Previous work defined the CymR regulon using a cymR deletion mutant (ΔcymR), an isogenic SH1000 wild type (WT) strain, and microarray hybridization (118). Both strains were cultured in rich medium (tryptic soy broth; TSB) supplemented with 2 mM cystine (CSSC). We sought to compare the sulfur starved transcriptome of WT S. aureus with a sulfur deprived cymR transposon mutant (cymR::Tn) to define the CymR-dependent and -independent response. This assessment required growth conditions distinct from those employed previously (118). WT JE2, a 34 derivative of the current endemic USA300 LAC strain, and an isogenic cymR::Tn were cultured in CDM supplemented with 25 µM CSSC to mid-exponential phase. Cells were washed, resuspended into CDM supplemented with or without 25 µM CSSC, and cultured for an additional 2 h prior to isolating RNA and performing RNAseq (Fig. 2.2). The use of WT and cymR::Tn provides a robust methodology to discern between the respective S. aureus CymR-dependent and -independent responses to sulfur starvation. Comparisons between culture conditions and the total number of differentially regulated genes (>2-fold) are outlined in Table 2.1. Overall, the WT sulfur starvation response included 425 upregulated and 142 downregulated genes, resulting in a total of 567 differentially expressed transcripts (Table 2.1a and Table A-1). The number of genes responding to sulfur starvation (n=567) is far greater than the previously described CymR regulon (n=79), supporting the hypothesis that sulfur starvation induces CymR-dependent and -independent responses (118, 122). To define CymR-dependent transcripts, we compared the sulfur replete CymR regulon to the previously reported CymR regulon, which was also generated in sulfur-replete culture conditions (18,22). Specifically, genes differentially expressed in cymR::Tn when cultured in CSSC-supplemented CDM were identified and compared to WT grown in the same condition (Table A-2). This analysis revealed that cymR inactivation altered the expression of 67 genes, 45 upregulated and 22 downregulated (Table 2.1b). For comparison, 79 genes were upregulated when ΔcymR was cultured in CSSC- supplemented TSB (118, 122). With the WT sulfur starvation and CymR regulons in hand, we sought to compare upregulated sulfur metabolism genes across the three conditions (118). Notably, 15 out of the 16 published sulfur acquisition and metabolism genes are 35 shared between sulfur starved WT (Table A-1) and the previously reported CymR regulon (118, 122) with a total of 28 common genes between the two Tables overall (Table 2.2 and Fig. 2.3A). Adding upregulated genes from the sulfur-replete cymR::Tn condition (Table 2.3) transcriptome to this comparison reveals 10 total shared genes (Fig. 2.3A), eight of which are published sulfur metabolism genes (Table 2.2c) while the remaining two, SAUSA300_RS01875 and SAUSA300_RS15260, are described as cell wall associated by Soutourina et al. (118). To more strictly define sulfur metabolism associated genes within our conditions, cluster of orthologous groups (COGs) were assigned to the WT sulfur starvation and cymR::Tn sulfur replete Tables and then reassessed against Soutourina et al. (118). Sulfur-metabolism associated genes within the sulfur-deplete WT (n=26), sulfur-replete cymR::Tn (n=14), and the Soutourina et al. published (n=16) Tables were compared, revealing eight shared genes (Fig. 2.3B, Table 2.2) (118). Soutourina et al. also identified cell-wall associated genes in the CymR regulon and therefore COG assigned cell-wall associated genes were also isolated and analyzed; however, cell-wall associated genes were not shared across the three Tables (Fig. 2.3C). A direct comparison of the WT sulfur starvation and cymR::Tn sulfur replete conditions reveals 35 shared genes (Fig. 2.3D), 16 of which are sulfur metabolism associated (Fig. 2.3D, asterisk). This suggests that CymR influences expression of these 35 genes. The disparity between the total number of genes differentially regulated in response to sulfur starvation (n=567) and the sulfur replete CymR regulons (n=79 and n=67) underscores the importance of accounting for the sulfur status of cells. We surmise that CymR-dependent and -independent transcriptional alterations comprise the WT 36 sulfur starvation response. Therefore, identifying differentially expressed transcripts that are similar between sulfur starved WT and cymR::Tn will define transcripts regulated independently of CymR as those transcripts change in abundance regardless of whether CymR is present. Induction of sulfur starvation in cymR::Tn cells results in the differential expression of 344 genes, with 169 and 175 being up- and downregulated, respectively (Table 2.1c and Table A-3). Of those 344 genes, 138 are also differentially regulated in sulfur starved WT cells (Fig. 2.4A, Tables A-1 and A-3). These genes contribute to several cellular pathways with amino acid transport and metabolism, inorganic ion transport and metabolism, and genes of unknown function being most represented (Fig. 2.4B). Two transcriptional regulators are also differentially expressed in this CymR-independent sulfur starvation response (Tables A-1 and A-3). SbnI is a heme-responsive regulator for staphyloferrin B synthesis (136) while Fur is the master iron regulator. While these 138 genes represent the CymR-independent transcriptional response to sulfur starvation the remaining 429 genes are CymR-dependent, underscoring the importance of CymR in the sulfur starvation response. In keeping with this, a range of cellular processes were described with the COG analysis such as energy production and conversion, amino acid transport and metabolism, transcription, as well as carbohydrate transport and metabolism (Fig. 2.4C). Lastly, a subset of genes are differentially expressed solely in the cymR::Tn sulfur starvation response (n=117). Though these genes are considered CymR-independent, their differential expression is more likely a compensatory response to the loss of CymR. Within this subset of genes there are only three COG categories that have more than 10 genes represented: those with unknown function, amino acid transport and metabolism, and inorganic transport and metabolism 37 (Fig. 2.4D). Together, these comparisons reveal an intricate S. aureus starvation response that engages transcriptional alterations within and beyond the sulfur regulon. Sulfur starvation induces changes in numerous S. aureus transcriptional regulators and the upregulation of iron acquisition and oxidative stress response genes. In addition to their initial report of genes differentially expressed in a ΔcymR mutant cultured in TSB + 2mM CSSC (118), Soutourina et al. observed 18 upregulated genes involved with oxidative stress and metal ion homeostasis (122). Genes associated with oxidative stress and iron metabolism are also upregulated in response to sulfur deprivation in this study (Table A-1). Of the 567 differentially expressed genes in sulfur starved WT, 34 encode transcriptional regulators, of which 28 are upregulated while 6 are downregulated (Table 2.4). Notably, cymR, fur, and perR (major peroxide sensor) were upregulated (Table 2.4). Increased fur and perR expression were also observed in Soutourina et al. (122). Interestingly, fur upregulation occurred in both of our sulfur starvation Tables despite iron supplementation (Tables A-1 and A-3). Consistent with activation of the Fur and PerR regulons, both the WT and cymR::Tn sulfur starvation responses include an upregulation of seven oxidative stress genes as well as 29 iron acquisition and Fe-S cluster assembly genes (Table 2.5). Some iron acquisition genes are upregulated in both the WT and cymR::Tn sulfur starvation conditions, indicating that their expression is CymR-independent (Table 2.5, asterisk). Within these shared genes are those found to be upregulated by Soutourina et al. such as alkyl hydroperoxide reductase subunits C and F as well as ftnA (122). Some genes are uniquely upregulated only during WT sulfur starvation (e.g., fhuB, fhuG) which would suggest differential expression in response to the presence of CymR 38 (Table 2.5). Fourteen genes, largely involved in Fur-regulated siderophore biosynthesis and Fe-S cluster assembly, are unique to the cymR::Tn starvation condition as well as a regulator of redox-stress, HypR (137) (Table 2.5). Upregulation of hypR solely during cymR::Tn sulfur starvation led us to question whether other transcriptional regulators are altered in this condition. Two such regulatory systems were found: sarR (SAUSA300_RS12390), which modulates the virulence regulatory sar system (91, 138), is downregulated while SAUSA300_RS11905 (MerR family of metal sensing regulators) is upregulated (91, 139). Several of the CymR-independent upregulated genes belong to the Isd system (iron-regulated surface determinant) which promotes the release of iron from heme (140). The observed interconnection between sulfur, iron, and oxidative stress should benefit S. aureus because numerous enzymes require Fe-S clusters to function and homeostasis of these two nutrients is crucial given their contributions to Fenton chemistry (108, 141, 142). Additionally, GSH (a sulfur-containing metabolite with a free thiol) protects Streptococcus species against oxidative damage upon exposure to copper or paraquat (143, 144). These facts support the hypothesis that S. aureus employs a similar method of GSH scavenging to manage oxidative stress (144). Given the Isd system is important for heme-mediated iron acquisition and the fact that erythrocytes contain approximately 21 µM of free heme and 1-2 mM GSH (145, 146), we explored S. aureus sensitivity to hemin (oxidized form of heme) upon supplementation with reduced or oxidized GSH. WT and a hrtA::Tn mutant—which is hypersensitive to heme due to impaired efflux (147, 148)—display identical growth profiles when cultured in CDM with 50 µM GSH (Fig. 2.5A). However, toxicity is observed as an increased lag phase upon supplementation of the WT 39 culture with 10 µM hemin while the hrtA::Tn mutant fails to proliferate (Fig. 2.5A). To reflect GSH levels S. aureus likely encounters during infection, GSH was increased to a more physiologically relevant concentration (42); supplementation with 750 µM GSH abolishes any impact of hemin on WT propagation (Fig. 2.5B). The hrtA deficient strain experiences a longer lag phase, but eventually reaches a WT-like OD600 (Fig. 2.5B), indicating an alleviation of heme toxicity. To assess whether this protection is specific to the free thiol, reduced GSH was replaced with oxidized GSH (GSSG) using equimolar concentrations of sulfur. Exposing S. aureus supplemented with low (25 µM) or high (375 µM) concentrations of GSSG to hemin inhibited growth of both WT and the hrtA deficient strain (Fig. 2.5C, D respectively). Taken together, this indicates that S. aureus scavenging of GSH sufficiently combats hemin-mediate oxidative stress. To further demonstrate that GSH protects against ROS, we quantified the viability of S. aureus cells that were preincubated with GSH or GSSG and subsequently exposed to H2O2 (Fig. 2.5E). WT cultured in 750 µM GSH exhibited significantly increased viability upon H2O2 challenge compared to WT grown in 50 µM GSH or 375 µM GSSG. Collectively, these data indicate that sulfur starvation and iron homeostasis coordination protect S. aureus from oxidative stress (122). Adaptation of S. aureus to distinct nutrient sulfur sources induces unique transcriptional responses. Given that the absence of sulfur drives transcriptional changes, we hypothesized that discrete S. aureus transcriptome alterations occur when cultured in the presence of organic or inorganic sulfur sources. To test this, S. aureus was grown in CDM supplemented with 50 µM Cys, 25 µM GSSG, 50 µM GSH, or 50 µM sodium thiosulfate (sTS) and differentially expressed genes were identified (Table B-1, 40 B-2, B-3, and B-4, respectively). CSSC was used as the comparator because it is typically supplemented as the sulfur source in CDM (149, 150). In total, 776 genes alter expression when S. aureus proliferates in the presence of Cys, GSSG, GSH, or sTS when compared to CSSC (Tables B-1 to B-4). Only six genes were differentially expressed in WT supplemented with Cys (Table B-1). One of the two downregulated genes in this condition, SAUSA300_RS04580, encodes a putative pyridine nucleotide disulfide oxidoreductase (91), suggesting a role in reducing CSSC. However, inactivation of SAUSA300_RS04580 does not impact the ability of S. aureus to utilize CSSC as a sulfur source (Fig. 2.6). In stark contrast to Cys supplementation, sTS led to the greatest shift in differentially expressed genes. In this condition, a total of 231 genes were upregulated and 175 downregulated (Fig. 2.7A); compared to growth in GSH and GSSG, 135 upregulated and 83 downregulated genes are unique to growth on sTS (Fig. 2.7B, C). Supplementation with GSH or GSSG results in a total of 193 upregulated and 177 downregulated genes (Fig. 2.7A); 19 of these genes had unique differential expression when GSH was the provided sulfur source whereas 26 were specific to GSSG (Fig. 2.7B, C). An explanation for this disproportional transcript abundance across sulfur sources is variant transcriptional regulator expression. In total, 21 regulators experienced altered expression in the presence of sTS, GSH, and GSSG. Twelve of these regulators were solely altered in sTS while three were found to have differential expression in GSSG (Fig. 2.7D). Four regulators were differentially expressed in both GSH and sTS while one regulator was shared between GSSG and sTS (Fig. 2.7D). Only one regulator, vraR, shared differentially expression across all three sulfur sources (Fig. 2.7D). The 12 41 regulators uniquely expressed in sTS support the notion that altered gene expression in the presence of this sulfur source is related to transcriptional regulation. Consequently, we hypothesize that sTS supplementation leads to altered nutritional requirements which manifest through increased expression of transporters compared to GSH and GSSG. Consistent with this, 53 differentially transporters across all the sulfur conditions were found (Table B-5). Seventeen were specifically upregulated in sTS compared to the eight and four transporters uniquely upregulated in GSSG and GSH, respectively (Table B-5). Several transporters in Table B-5 are differentially expressed in just two sulfur sources— GSH and GSSG (n=1), GSH and sTS (n=5), or GSSG and sTS (n=1)—while 15 experience transcriptional alterations in all three conditions. Of these 15 genes, 12 are upregulated (Fig. 2.8 “+” symbols), six of which are were also upregulated in the previously described CymR regulon (Fig. 2.8, bold) (118). The remaining three shared transporters (i.e., SAUSA300_RS01625, SAUSA300_RS13340, and SAUSA300_RS13345) are downregulated (Table B-5). Collectively, these observations indicate that growth on inorganic sulfur sources alters S. aureus physiology to a greater extent than when this organism proliferates on organic sulfur sources. To substantiate these Table, we further assessed the sTS condition given that supplementation with this metabolite led to the largest transcriptional shift. SAUSA300_RS10985 and SAUSA300_RS10980 encode for a YedE/YeeE family transporter and oxidoreductase, respectively (91, 118, 151). Recently, E. coli homologs of SAUSA300_RS10985 and SAUSA300_RS10980 were found to import TS (TsuA, SAUSA300_RS10985 homolog, referred to as 985) and support its assimilation to Cys (TsuB, SAUSA300_RS10980 homolog, referred to as 980) (135, 152). This is in 42 accordance with previous predictions that 985 is the S. aureus TS transporter (118). To investigate whether 985 supports S. aureus utilization of TS as a source of nutrient sulfur, a 985::Tn mutant was employed. The Tn insertion likely has polar effects on the downstream 980 gene (Fig. 2.9A). As such, complementation was performed using the pKK22 vector (153). Both genes were cloned, under native promoter control, into pKK22 individually or as an operon. The resulting strains were then cultured in CDM supplemented with either 50 µM sTS or 25 µM CSSC (Fig. 2.9B, C respectively). In comparison to WT harboring an empty vector (EV), the 985::Tn EV mutant is incapable of proliferation when TS is the sulfur source (Fig. 2.9B). Expression of 985 in trans partially restores propagation of the mutant while complementation with the entire operon completely rescues the observed growth defect. Ectopic expression of 980 does not restore proliferation of the mutant in CDM supplemented with sTS. These results demonstrate that 985 is necessary, but not sufficient for maximal growth of S. aureus on sTS while 980 is sufficient, but not necessary. Each phenotype is sulfur specific given that strains demonstrate WT-like growth on the control sulfur source, CSSC (Fig. 2.9C). In accordance with the nomenclature proposed by Morigasaki et. al. (135) we propose to rename 985 and 980 to thiosulfate uptake proteins A and B (TsuA, TsuB), respectively. DISCUSSION As knowledge regarding S. aureus sulfur acquisition and assimilation mechanisms grow, it is vital to understand how these strategies are regulated during infection (89, 90). Initial observations defined the S. aureus CymR regulon using an isogenic mutant cultured in sulfur replete media (118, 122). Here, we examine how the CymR-dependent and - independent transcriptome fluctuates under sulfur replete and deplete conditions. To do 43 so, RNAseq was employed due to its increased sensitivity over the microarray technology applied by Soutourina et al. Additionally, WT and cymR::Tn cells were cultured in a defined medium in the presence or absence of CSSC allowing us determine the sulfur starvation transcriptome. Our study also assessed a methicillin resistant, USA300 LAC derivative (JE2) which is reflective of the current endemic strain, whereas previous work utilized the methicillin susceptible SH1000 strain. Comparing our WT sulfur starvation and cymR::Tn sulfur replete transcriptomes to the DcymR sulfur replete transcriptome generated by Soutourina et al. (118), we observe a greater number of differentially regulated genes related to sulfur metabolism and fewer cell-envelope associated genes (Fig. S2B and S2C). These differences are likely due to increased sensitivity, contrasting growth conditions, and variations in cell wall composition and structure given that different S. aureus strains were assessed (154, 155). Querying sulfur-starved WT (Table A-1), a CSSC-supplemented cymR::Tn mutant (Table A-2; Table 2.3), and the sulfur replete ΔcymR strain described in Soutourina et al. (118, 122) reveals overlap in upregulated sulfur-associated transporters. Notably, S. aureus encodes two Cys and CSSC transporters, TcyP and TcyABC (90). Upregulation of tcyP was consistent across all three Tables as was tcyA, the gene encoding the TcyABC substrate binding protein. However, the remaining tcyB-encoded permease and ATPase encoded by tcyC are upregulated in WT sulfur starvation and Soutourina et al., but not in the CSSC replete cymR::Tn mutant. Heightened expression of a recently described glutathione import system (gisABCD-ggt; SAUSA300_RS01055-RS01075) was also observed across the three analyses (89). The ABC transporter is encoded by divergently transcribed gisA and gisBCD. The terminal gene of the gisBCD operon is ggt, 44 which encodes a γ-glutamyl transpeptidase. All five genes exhibit increased expression in sulfur starved WT. Both gisA and gisB are expressed in CSSC supplemented cymR::Tn while Soutourina et al. observe upregulation of gisA (118, 122). The genes encoding for a predicted sulfate/sulfonate transport system, ssuABC, were also upregulated in both the CSSC supplemented cymR::Tn and WT sulfur starvation conditions; however only ssuB was upregulated in Soutourina et al. Despite these subtle inconsistencies across presumably operonic genes, expression of the gisABCD-ggt and ssuABC loci further solidifies the overlap within the compared Tables. Lastly, upregulation of tsuA, the verified TS transporter, occurred across all three Tables. A portion of the sulfur starvation response involves genes outside of the CymR regulon (Fig. 2.4) such as those involved in iron metabolism and oxidative stress (Table 2.5 and Table B-1). A potential link between iron and sulfur regulons seems intuitive considering the number of enzymes that require Fe-S clusters. In Pseudomonas aeruginosa the sulfur regulon transcriptional regulator, CysB, directly promotes expression of pvdS, an alternative sigma factor involved in the iron response (156), suggesting beneficial effects of balancing iron and sulfur levels. This coordination is further exemplified in E. coli where Fur binds an [2Fe-2S] cluster to sense intracellular free iron (133) though, whether Fur is also responsive to CysB in this organism is currently unknown. The fact that iron and sulfur potentiate Fenton chemistry also underscores a link between sulfur acquisition, iron acquisition, and oxidative stress. Scavenging host GSH, a low molecular weight thiol antioxidant, to satisfy the nutritional sulfur requirement may also offset toxicity associated with iron or heme-iron acquisition. We demonstrate that GSH protects S. aureus from hemin toxicity, supporting 45 propagation in an otherwise inhibitory environment (Fig. 2.5). Though not fully understood, heme can induce bactericidal effects at high enough concentrations. To combat heme accumulation, S. aureus employs the heme-regulated ABC transporter, HrtAB, which effluxes heme from the cell. Therefore, genetic inactivation of hrtA or hrtB results in heme hypersensitivity (147, 148). Here we observe that GSH scavenging promotes proliferation of both WT and a hrtA::Tn mutant in the presence of hemin. The free thiol of GSH is a crucial aspect of this protection given that GSSG is not sufficient to alleviate the toxicity. Additionally, we demonstrate that toxicity of another stressor, H2O2, is decreased upon culturing S. aureus in the presence of physiologically relevant GSH concentrations. This supports the notion that the restored growth observed in response to heme is likely due to management of cell-associated stress induced by heme rather than a direct, extracellular GSH-heme interaction (157, 158). The relevance of these results towards S. aureus management of heme-induced stress during bacteremia stem from the facts that the pathogen is hemolytic, and erythrocytes harbor abundant concentrations of GSH and heme (159, 160). Thus, coordination of the sulfur, iron, and oxidative stress regulons should benefit S. aureus. Due to the numerous metabolites that satisfy the S. aureus sulfur requirement, it is unknown whether this pathogen encounters sulfur-limited environments during infection. Nonetheless, S. aureus has evolved several elegant mechanisms to pillage host-derived sulfur reservoirs (134). Sulfur-containing metabolites vary in abundance or composition depending on tissue types and sub-cellular localization (134). Given this, it is accordingly plausible that S. aureus adapts to the local sulfur milieu by altering its sulfur acquisition and metabolism transcriptome. Growth of S. aureus on four different sulfur 46 sources (Cys, GSH, GSSG, and sTS) reflects this. In comparison to the CSSC condition, each sulfur source resulted in at least six (Cys) and a maximum of 248 (sTS) differentially expressed genes. The function of CymR when S. aureus meets threshold intracellular Cys levels provokes the thought that providing CSSC, Cys, GSH, GSSG, or TS to S. aureus would cause CymR to repress target genes. However, we observe upregulation of sulfur regulon genes in each condition, save Cys (Tables B-2 to B-4). This finding suggests that the provided sulfur concentration is sufficient for growth in vitro but does not maintain sufficient intracellular Cys concentrations that would result in CysE inhibition (Fig. 2.1). Quantifying kinetic expression of target genes while monitoring OAS levels throughout S. aureus growth will address this. Alternatively, CymR might respond to stimuli not currently appreciated. In fact, CymR has two defined inputs that affects its DNA binding capacity, OAS-thiol-lyase B (CysM) and oxidation of the Cys residue at the position 25 (Fig. 2.1) (118, 121). Another possibility is the influence of coordinated regulation on the sulfur regulon. Indeed, our Tables demonstrate upregulation of transcriptional regulators in both sulfur-limited and -replete environments, implicating connections between sulfur metabolism and other metabolic processes (e.g., iron, oxidative stress). Finally, the ΔcymR mutant used in the Soutourina et al. study exhibited decreased hemolytic activity and virulence though differential expression of virulence factors was not reported (118, 122). Similarly, our conditions did not reveal altered expression of virulence genes. We attribute this to harvesting RNA from mid-exponential cells when Agr, the major virulence regulator, activity is low. 47 Collectively, our investigation into the S. aureus sulfur response reveals intricacy surrounding CymR and the sulfur regulon. This manifests when examining the molecular mechanisms of TS assimilation. Extensive characterization of TS assimilation in E. coli and Salmonella typhimurium has generated a model where an ABC transporter, CysPUWA, imports TS into the cell (161–163). S. aureus does not encode a CysPUWA homolog (91). Here we demonstrate that S. aureus employs TsuA to support growth on TS (Fig. 2.9B). Additionally, TsuB is thought to be produced from the gene operonic with tsuA and contributes to the efficient assimilation of TS (Fig. 2.9B). Given that TsuB belongs to the TusA family of proteins, we predict that it is involved in guiding TS from TsuA to the first enzyme in assimilation, CysM. These observations mirror recent work in E. coli and, together, are reshaping our understanding of how bacteria use this inorganic metabolite to meet the nutritional sulfur requirement (135, 152). Most importantly, however, is that these data support the RNAseq by demonstrating that upregulated genes with predicted sulfur metabolism function indeed contribute to meeting this nutritional requirement. Overall, this work provides compelling Tables that highlight the S. aureus response to various states of sulfur supplementation and can be utilized to probe the nuances of sulfur metabolism to better understand this persistent threat to global health. ACKNOWLEDGEMENTS We thank Jeffery Bose for supplying the pKK22 vector. Transposon mutants were acquired from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH, and the Nebraska Transposon Mutant Library (NTML) Screening Array NR-48501. This work is funded by the National Institutes of Health R01 AI139074 and R21 AI142517. 48 MATERIALS AND METHODS Strains and Primers. A complete list of strains and plasmids as well as primers used in this study can be found in Tables 2.6, 2.7, and 2.8 respectively. JE2, a derivative of the community acquired USA300 LAC, is the WT S. aureus strain used in these studies (164). Bursa aurealis Tn inactivated strains were generated by transducing the Tn inactivated gene from the Nebraska Transposon Mutant Library into JE2 using a previously described protocol (164–166). Tn insertions and chromosomal deletions were verified using PCR. Plasmids were confirmed using Sanger sequencing (58, 59). Complementation studies were done with the pKK22 vector (153). All pKK22 vector derivatives were generated via Gibson assembly in the E. coli DH5α strain and then transformed into S. aureus RN4220, an intermediate strain, before JE2. Media and growth conditions. All strains were cultured overnight in 5 mL tryptic soy broth (TSB; Remel) at 37°C, shaking at 225 rpm. A base chemically defined medium (CDM) was prepared using a previously described recipe with slight modifications (149, 150). Base CDM included the following salts: K2HPO4 (7 mg mL-1), KH2PO4 (2 mg mL-1), (NH4)2SO4 (1 mg mL-1); amino acids: Phe (0.04 mg mL-1), Iso (0.03 mg mL-1), Tyr (0.05 mg mL-1), Glu (0.1 mg mL-1), Lys (0.01 mg mL-1), Met (0.07 mg mL-1), His (0.03 mg mL- 1), Trp (0.01 mg mL-1), Leu (0.09 mg mL-1), Asp (0.09 mg mL-1), Arg (0.07 mg mL-1), Ser (0.03 mg mL-1), Ala (0.06 mg mL-1), Thr (0.03 mg mL-1), Gly (0.05 mg mL-1), Val (0.08 mg mL-1), Pro (0.01 mg mL-1); nucleotides: adenosine (0.005 mg mL-1), cytosine, guanine (0.005 mg mL-1), thymine (0.02 mg mL-1), uracil (0.005 mg mL-1); vitamins: thiamine (0.001 mg mL-1), nicotinic acid (0.0012 mg mL-1), biotin (5e-6 mg mL-1), calcium pantothenate (2.5e-4 mg mL-1); MgSO4 (0.1024 mg mL-1); and FeCl3 (0.008 mg mL-1). This 49 medium lacks CSSC, asparagine, and glutamine. Freshly prepared D-glucose (5 mg mL- 1) was supplemented into the base medium and, depending on the condition, either no sulfur source, 25 µM cystine (CSSC), 50 µM cysteine (Cys), 25 µM reduced glutathione (GSH), 50 µM oxidized glutathione (GSSG), or 50 µM sodium thiosulfate (sTS) prior to inoculation. CSSC and Cys stocks were dissolved in 1 N HCl. Stocks of GSH, GSSG, and sTS were made fresh by dissolving in ddH2O prior to filter sterilization. Cultures for biological duplicates for each RNAseq experiment were conducted on independent days. Sulfur starvation was induced by washing WT or cymR::Tn S. aureus overnight cultures in 1X phosphate buffered saline (PBS) to a normalized optical density at 600 nm (OD600) of 1. Cells were then sub-cultured 1:100 (i.e., OD600 equal to 0.01) into two 250 mL Erlenmeyer flasks containing 50 mL CDM supplemented with 25 µM CSSC. Flasks were incubated at 37°C, shaking at 225 rpm, for 4 h (Fig. 2.2). The duplicate flasks were then combined, centrifuged at 4°C, 4700 rpm for 10 min, washed with PBS, centrifuged again, and resuspended in 100 mL CDM harboring no sulfur source. The resulting resuspension was separated by pipetting 50 mL into two 250 mL Erlenmeyer flasks. One flask of WT and cymR::Tn was supplemented with 25 µM CSSC while the other remained sulfur deplete. Flasks were incubated at 37°C, shaking at 225 rpm, for 2 h prior to RNA isolation. To assess transcriptional changes between organic (Cys, CSSC, GSH, and GSSG) or inorganic (sTS) sulfur sources, WT cells were prepared identically as described above up to OD600 normalization. Cells were then subcultured 1:100 in CDM supplemented with either 50 µM Cys, 25 µM CSSC, 25 µM GSSG, 50 µM GSH, or 50 µM sTS. Flasks were incubated at 37°C, 225 rpm, for 4 h prior to RNA isolation. 50 Cells were prepared for growth analyses in 96-well plates growth curves as follows: overnight cultures were washed in 5 mL PBS and normalized to an OD600 equal to 1. Cells were diluted 1:100 into 150 µL CDM supplemented with 25 µM CSSC, 50 µM sTS, 50 µM GSH, 750 µM GSH, 25 µM GSSG, 375 µM GSSG, or without a source of sulfur. Where described, a 30 mM hemin stock (dissolved in 1.4 M NH4OH) was diluted into the CDM at a final concentration of 10 µM. OD600 was monitored every hour for 24 h using a 96- well round bottom plate in an Epoch2 Biotek microplate spectrophotometer (Agilent, Santa Clara, CA) at 37°C, shaking continuously. For each strain, growth curves were conducted in technical triplicate with the average of the triplicates constituting a single biological replicate. Growth curves were conducted three independent times. RNA isolation and sequencing. 50 mL cultures from the respective growth conditions were centrifuged at 4°C for 10 min at 4700 rpm. RNA was isolated from the resulting pellet as previously described (90). The RNA was then treated with Turbo DNase following manufacturer’s instructions (ThermoFisher, Waltham, MA). Total RNA was sent to Genewiz Inc. (South Plainfield, NJ) who conducted rRNA depletion with Illumina Ribo- Zero Plus (Illumina, Inc., San Diego, CA) and then generated libraries using stranded total RNA kit and sequenced using Illumina HiSeq (Illumina, Inc.) with 2x 150 bp paired-end read technology. RNA-seq data processing and visualization. Raw reads for this project were submitted under bioproject number PRJNA989516. Gene expression was analyzed using Geneious Prime 2022.0.2 (Dotmanics, Boston, MA). Briefly, paired end fastq files were trimmed using Bbduk plugin, and mapped and annotated against the reference genome S. aureus USA300_FRP3757 (NC_007793.1) using Geneious mapper with default settings. The 51 expression levels were calculated within the software using default parameters, and DESeq2 was implemented to determine the differential expression with the comparisons listed in figure legends (167). Data was filtered using a log2 ratio greater than or equal to 1 and less than or equal to -1 representing a 2-fold change and an adjusted P-value of less than 0.05. Differential expression analysis was conducted using transcripts per million (TPM). Low TPM values (<10) were manually removed to eliminate bias. Variance was then controlled for by either discarding replicates with TPM differences >5 and/or if the TPM values between conditions were to close (≤5). Cluster of orthologous groups (COG) categories were determined using eggNOG-mapper v2 (168, 169). Briefly, protein sequences from USA300_FPR3757 (NC_007793.1) were used as input for eggNOG- mapper v2 to assign functional annotations. Protein sequences that were recognized by eggNOG-mapper but had no resulting COG assignment were denoted as “not classified”. The protein sequences that were not recognized by eggNOG-mapper and were double checked for any COG assignments as follows: the USA300_FPR3757 locus tag was used to identify the corresponding homolog in S. aureus NCTC8325 using Aureowiki (91). The NCTC8325 homolog locus tag was then queried against EggNOG v6.0 (170). When this did not return any COG assignment the gene was denoted as “no homolog found”. H2O2 peroxide killing assay. This assay was modified from a previously defined protocol (171). Overnight WT TSB cultures were washed in PBS and normalized to an OD600 of 1. Cells were then sub-cultured 1:100 into two 250 mL Erlenmeyer flasks containing 50 mL CDM supplemented with either 50 µM GSH, 750 µM GSH, or 375 µM GSSG. Cultures were incubated at 37°C, shaking at 225 rpm, for 4 h. Cells were then transferred to a 50 mL falcon tube and spun at 4000 rpm and then resuspended in 50 mL PBS. 1 mL of the 52 resuspension was utilized to obtain the OD600 while the remaining cells were spun at 4000 rpm. After decanting the supernatant cells were gently resuspended in residual PBS. One falcon tube for each growth condition was then resuspended in PBS to a final OD600 of 0.7. The other tube was resuspended to the same OD600 with PBS containing 1 M H2O2 (freshly made). Tubes were then placed at 37°C, static, for 20 min. Afterwards 3x 50 µL aliquots (i.e., 3 technical replicates) from each condition were resuspended in 950 µL PBS containing approximately 2,000-5,000 units mg-1 catalase (Sigma-Aldrich Cat No: C9322-5G; resuspended at 1 mg mL-1 and not filter sterilized). Samples were then incubated at 37°C, static, for 5 min. For each strain, three technical replicates were then serial diluted in PBS and 10 µL was spot plated onto TSA to enumerate for CFU. Note that the average of these technical triplicates is considered as one biological replicate. Plates were incubated overnight at 37°C. Each assay was performed in biological triplicate. Data visualization. Growth curves, heat maps, and bar graphs were generated using GraphPad Prism software version 10.0.0 (153). All finalized figures were generated using the open source Inkscape 1.2.2 (b0a8486, 2022-12-01). Available at: https://inkscape.org. 53 TABLES Table 2.1. Transcriptional changes resulting from sulfur starvation and CymR regulation. Culture condition Comparator Total DE genes Upregulated genes Downregulated genes 425 567 WT + CSSC WT no sulfur supplementationa cymR::Tn + CSSCb WT + CSSC cymR::Tn + cymR::Tn no sulfur supplementationc CSSC Genes were differentially expressed (DE) with a Log2 fold change at least equal to +/-1 with a P value <0.05 from DESeq2 output. aSulfur starvation response bCymR sulfur-replete regulon cStarvation-induced changes independent of CymR 255 170 142 22 85 45 67 54 Table 2.2. Previously identified sulfur metabolism genes identified within the upregulated sulfur starvation transcriptome and the CymR regulon. cymR +S Adjusted P-valueb 1.335E-06 WT -S Adjusted P-valueb 6.368E-33 5.563E-24 1.586E-27 4.827E-07 1.017E-03 1.637E-19 1.024E-06 6.023E-15 5.072E-08 9.671E-27 Locusa Gene WT -S Log2 FC cymR +S Log2 FC fdh 3.059 3.430 5.169E-07 2.266 2.699 3.735E-03 1.428E-04 5.396 4.636 5.543 2.108 1.617 4.815 2.679 3.664 2.907 4.815 SAUSA300_RS00910c SAUSA300_RS00915d ssuB SAUSA300_RS00930c SAUSA300_RS00935e SAUSA300_RS00940e SAUSA300_RS01055c gisA SAUSA300_RS02035c tcyP SAUSA300_RS02320d mccA SAUSA300_RS02325c mccB SAUSA300_RS02330c gmpA SAUSA300_RS02635f cysK SAUSA300_RS10985c SAUSA300_RS12345d ydbM SAUSA300_RS13015d tcyC SAUSA300_RS13020d tcyB SAUSA300_RS13025c tcyA aAll 16 sulfur metabolism genes from Table 1 of (118) represented as the respective JE2 homologs. that are shared between Soutourina et al. bAdjusted P-value from DESeq2 output from the WT sulfur starvation (denoted as WT - S, Table 1) and cymR::Tn sulfur replete ( represented as cymR +S, Table 2) conditions. cShared between the published CymR regulon (118), WT -S, and cymR +S dGenes that are downregulated in cymR +S and does not meet the adjusted P-value cutoff of <0.05 (data not shown). eDenotes genes that are upregulated in cymR +S but does not have a Log2 ratio ≥1 and/or does not meet the adjusted P-value cutoff of <0.05 (data not shown). fRepresents the gene that is upregulated in WT -S but does not have a Log2 ratio ≥1 and/or does not meet the adjusted P-value cutoff of <0.05 (data not shown). 1.132E-52 2.724E-03 7.411E-06 1.932E-03 5.380E-06 2.250E-02 1.135E-02 1.199E-02 5.131E-07 6.339 1.676 2.063 1.681 2.083 1.988 1.904 2.035 3.435 2.663E-02 1.693 55 Table 2.3. cymR mutant upregulated genes under sulfur replete conditions. Locus Gene Product SAUSA300 _RS10985 SAUSA300 _RS00910 SAUSA300 _RS10980 SAUSA300 _RS00925 SAUSA300 _RS00930 SAUSA300 _RS13915 SAUSA300 _RS02035 SAUSA300 _RS05050 SAUSA300 _RS02045 SAUSA300 _RS01055 SAUSA300 _RS02340 SAUSA300 _RS12610 SAUSA300 _RS00915 SAUSA300 _RS05345 SAUSA300 _RS02635 SAUSA300 _RS09430 SAUSA300 _RS02325 SAUSA300 _RS02335 SAUSA300 _RS09440 YeeE/YedE family protein DUF4242 domain-containing protein sulfurtransferase TusA family protein Log2 FCa 3.435 3.430 3.325 ssuC ABC transporter permease 3.109 acyl-CoA/acyl-ACP dehydrogenase 3.059 isaA lytic transglycosylase IsaA 2.807 tcyP L-cystine transporter gisA gmpC sdpC DoxX family protein hypothetical protein ABC transporter ATP-binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein CPBP family intramembrane glutamic endopeptidase SdpC ABC transporter ATP-binding protein YkyA family protein cysK cysteine synthase A hypothetical protein mccB gmpB bifunctional cystathionine γ- lyase/homocysteine desulfhydrase methionine ABC transporter permease crcB2 CrcB family protein 2.699 2.369 2.290 2.266 2.194 2.104 2.053 2.048 2.035 2.020 1.988 1.977 1.976 56 Adjusted P-valueb 5.131E- 07 1.335E- 06 9.084E- 07 2.476E- 06 5.169E- 07 1.255E- 03 1.428E- 04 6.815E- 04 2.041E- 04 3.735E- 03 6.966E- 03 2.280E- 03 1.266E- 02 1.382E- 02 1.199E- 02 1.388E- 02 2.250E- 02 2.036E- 02 9.513E- 03 COGc S S O P I M U S A P P S P L E not classifie d E P D Table 2.3 (cont’d) SAUSA300 _RS01875 SAUSA300 _RS13605 SAUSA300 _RS00920 SAUSA300 _RS02330 SAUSA300 _RS12685 SAUSA300 _RS01060 SAUSA300 _RS13940 SAUSA300 _RS04485 SAUSA300 _RS02855 SAUSA300 _RS07460 SAUSA300 _RS13025 SAUSA300 _RS13320 SAUSA300 _RS10230 SAUSA300 _RS05540 SAUSA300 _RS12705 SAUSA300 _RS11450 SAUSA300 _RS00605 SAUSA300 _RS12440 1.404E- 02 1.654E- 02 2.249E- 02 1.135E- 02 3.211E- 02 3.649E- 02 4.623E- 02 1.364E- 02 2.250E- 02 2.352E- 02 2.663E- 02 3.853E- 02 3.008E- 02 3.382E- 02 4.532E- 02 2.554E- 02 2.782E- 02 3.896E- 02 low temperature requirement protein A ATP-binding cassette domain- containing protein ABC transporter substrate- binding protein methionine ABC transporter ATP-binding protein alpha/beta hydrolase 1.933 1.906 1.904 1.904 1.878 ssuA gmpA gisB ABC transporter permease 1.834 DUF896 domain-containing protein DUF3055 domain-containing protein 1.825 1.822 M20 family metallopeptidase 1.785 tcyA lnsB isdA sirA 1.725 1.693 1.654 1.646 1.646 1.620 1.618 1.608 1.588 hypothetical protein transporter substrate-binding domain-containing protein CPBP family lipoprotein N- acylation protein LnsB hypothetical protein LPXTG-anchored heme- scavenging protein IsdA TetR/AcrR family transcriptional regulator DUF2529 domain-containing protein staphyloferrin B ABC transporter substrate-binding protein SirA CHAP domain-containing protein N- acetylglucosaminyldiphosphou ndecaprenol N-acetyl-β -D- mannosaminyltransferase TarA 57 SAUSA300 _RS03340 tagA 1.532 3.008E- 02 S V P P I EP S S E no homolog found ET S not classifie d M K S P S M Table 2.3 (cont’d) SAUSA300 _RS07465 SAUSA300 _RS04060 clpP SAUSA300 _RS00985 brnQ1 cmk (d)CMP kinase ATP-dependent Clp endopeptidase proteolytic subunit ClpP branched-chain amino acid transport system II carrier protein 1.530 1.525 1.503 SAUSA300 _RS04205 lnsA lipoprotein N-acylation protein LnsA 1.498 SAUSA300 _RS07075 brnQ3 branched-chain amino acid transport system II carrier protein 1.469 SAUSA300 _RS15260 hypothetical protein 1.454 2.784E- 02 4.532E- 02 4.983E- 02 3.766E- 02 4.532E- 02 4.532E- 02 F OU E not classifie d E no homolog found S 1.454 4.532E- 02 PTS transporter subunit IIC SAUSA300 _RS09900 aFold change (FC) expression of cymR::Tn grown in medium containing CSSC relative to WT grown in same medium. bAdjusted P-value from DESeq2 output. cCOG assignments using eggNOG-mapper. A: RNA processing and modification; D: cell cycle control and mitosis; E: amino acid metabolism and transport; F: nucleotide metabolism and transport; I: lipid metabolism; K: transcription; L: replication and repair; M: cell wall/membrane/ envelope biogenesis; O: post-translational modification, protein turnover, chaperone function; P: inorganic ion transport and metabolism; S: function unknown; T: signal transduction; U: intracellular trafficking and secretion; V: defense mechanisms . Not classified: genes with an eggNOG-mapper homolog, but no associated COG. No homologs found: neither eggNOG-mapper nor Eggnog v6 could find a homolog for these proteins. Genes highlighted in grey indicate overlap with Soutourina et al. (118). 58 Table 2.4. S. aureus sulfur starvation induces differential expression of genes encoding transcriptional regulators. Locus Gene Description Log2 FCa Adjusted P-valueb Upregulated SAUSA300_ RS00650 SAUSA300_ RS04840 SAUSA300_ RS07905 SAUSA300_ RS05125 SAUSA300_ RS08020 SAUSA300_ RS12240 SAUSA300_ RS03710 SAUSA300_ RS13930 SAUSA300_ RS03330 SAUSA300_ RS12705 SAUSA300_ RS10810 SAUSA300_ RS13640 SAUSA300_ RS00590 SAUSA300_ RS10675 SAUSA300_ RS13480 SAUSA300_ RS09835 SAUSA300_ RS12900 SAUSA300_ RS12510 SAUSA300_ RS01375 sbnI bifunctional transcriptional regulator/O- phospho-L-serine synthase SbnI 2.787 4.361E-11 spxA transcriptional regulator SpxA 2.710 2.553E-09 fur Fur family transcriptional regulator 2.692 9.202E-11 MarR family transcriptional regulator 2.488 6.488E-06 argR transcriptional regulator ArgR 2.348 2.264E-07 sarV saeR HTH-type transcriptional regulator SarV response regulator transcription factor SaeR TetR/AcrR family transcriptional regulator metal-dependent transcriptional regulator TetR/AcrR family transcriptional regulator 2.319 6.725E-06 2.241 2.563E-06 2.009 8.284E-06 1.998 4.804E-06 1.954 5.729E-05 XRE family transcriptional regulator 1.937 5.507E-06 MarR family transcriptional regulator 1.908 1.274E-04 sarS HTH-type transcriptional regulator SarS 1.798 1.302E-04 transcriptional activator RinB 1.720 1.793E-03 scrA SaeRS system activator ScrA 1.647 1.974E-03 helix-turn-helix transcriptional regulator 1.627 3.238E-03 MerR family transcriptional regulator 1.516 3.018E-03 MurR/RpiR family transcriptional regulator 1.506 2.981E-04 GntR family transcriptional regulator 1.481 5.905E-04 59 Table 2.4 (cont’d) lexA nrdR perR mgrA cymR 1.466 1.744E-03 1.432 1.425E-03 1.227 9.413E-03 1.224 1.972E-02 1.360 1.701E-03 transcriptional regulator transcriptional regulator NrdR transcriptional repressor LexA Rrf2 family transcriptional regulator Crp/Fnr family transcriptional regulator 1.131 1.960E-02 helix-turn-helix transcriptional regulator 1.179 2.229E-02 HTH-type transcriptional regulator MgrA peroxide-responsive transcriptional repressor PerR SAUSA300_ RS08625 SAUSA300_ RS08905 SAUSA300_ RS06710 SAUSA300_ RS03605 SAUSA300_ RS10805 SAUSA300_ RS10800 SAUSA300_ RS10060 SAUSA300_ RS14275 SAUSA300_ RS12730 Downregulated SAUSA300_ RS06210 SAUSA300_ RS03665 SAUSA300_ RS10935 SAUSA300_ RS03250 SAUSA300_ RS13560 SAUSA300_ RS02550 aFold change (FC) expression of WT grown in sulfur deplete conditions relative to WT grown in sulfur replete conditions. bAdjusted P-value from DESeq2 output. GTP-sensing pleiotropic transcriptional regulator CodY DeoR/GlpR family DNA-binding transcription regulator MarR family transcriptional regulator MerR family transcriptional regulator global transcriptional regulator SarA accessory gene regulator AgrB septation regulator SpoVG -2.386 4.644E-09 -1.696 2.131E-04 -1.867 4.348E-05 1.032 2.767E-02 -2.413 5.295E-08 -2.301 1.061E-06 -3.183 3.373E-12 1.134 2.713E-02 codY agrB sarA 60 Table 2.5. S. aureus sulfur starvation induces expression of iron acquisition and oxidative stress genes. Locus Gene Product H-type ferritin FtnA fer fur isdI ftnA snbI sstD isdG fhuA fhuD2 ferredoxin Iron acquisition and metabolism SAUSA300_ RS10250* SAUSA300_ RS07500* SAUSA300_ RS12340* SAUSA300_ RS05570* SAUSA300_ RS03395* SAUSA300_ RS00650* SAUSA300_ RS00885* SAUSA300_ RS07905* SAUSA300_ RS03880* SAUSA300_ RS03400 SAUSA300_ RS11755* SAUSA300_ RS11750* SAUSA300_ RS13050 SAUSA300_ RS11760 SAUSA300_ RS04430 SAUSA300_ RS03405 Oxidative stress SAUSA300_ RS02020* SAUSA300_ RS02025* ahpC ahpF fhuG fhuB htsD htsA htsB sufB ABC transporter substrate-binding protein staphylobilin-forming heme oxygenase IsdG ABC transporter ATP-binding protein bifunctional transcriptional regulator/O- phospho-L-serine synthase SbnI staphylobilin-forming heme oxygenase IsdI Fur family transcriptional regulator siderophore ABC transporter substrate- binding protein iron ABC transporter permease iron ABC transporter permease iron chelate uptake ABC transporter family permease subunit cation diffusion facilitator family transporter Fe(3+) dicitrate ABC transporter substrate-binding protein Fe-S cluster assembly protein SufB iron ABC transporter permease alkyl hydroperoxide reductase subunit F alkyl hydroperoxide reductase subunit C Log2 FCa Adjusted P-valueb 4.859 8.351E-36 3.830 1.039E-20 3.640 3.636E-14 3.569 2.570E-13 2.842 7.910E-10 2.787 4.361E-11 2.708 1.950E-09 2.692 9.202E-11 2.494 1.217E-08 2.401 6.004E-09 2.288 3.617E-08 1.805 1.631E-05 1.699 7.185E-05 1.683 3.142E-04 1.592 2.898E-04 1.079 1.426E-02 2.890 6.579E-11 2.847 2.870E-12 61 Table 2.5 (cont’d) frp isdF katA perR bsaA hypR sbnD 2.170 2.994 1.134 2.994 2.025 1.228 4.455 1.909 sfnaA sfnaC catalase 7.710E-08 2.713E-02 7.125E-03 1.969E-05 3.400E-09 4.050E-06 9.670E-19 2.779E-05 glutathione peroxidase gpxA2 glutathione peroxidase NAD(P)H-dependent oxidoreductase peroxide-responsive transcriptional repressor PerR redox-sensitive transcriptional regulator HypR staphyloferrin A biosynthesis protein SfaC staphyloferrin A export MFS transporter staphyloferrin B export MFS transporter hemin ABC transporter permease protein IsdF SAUSA300_ RS06680* SAUSA300_ RS14205 SAUSA300_ RS13655 SAUSA300_ RS06465 SAUSA300_ RS10060 Genes unique to sulfur depleted cymR::Tn SAUSA300_ RS03085 SAUSA300_ RS11770 SAUSA300_ RS11780 SAUSA300_ RS00625 SAUSA300_ RS05560 SAUSA300_ RS11775 SAUSA300_ RS14545 SAUSA300_ RS03875 SAUSA300_ RS05555 SAUSA300_ RS04425 SAUSA300_ RS03865 SAUSA300_ RS04410 SAUSA300_ RS04420 SAUSA300_ RS04415 aFold change (FC) expression of WT grown in sulfur deplete condition relative to WT grown in sulfur replete condition (Table A-1). The cymR::Tn unique genes are the expression ratio of cymR::Tn grown in sulfur deplete conditions relative to cymR::Tn grown in sulfur replete conditions (Table A-3). bAdjusted P-value from DESeq2 output. heme ABC transporter substrate- binding protein IsdE SUF system NifU family Fe-S cluster assembly protein sfnaB staphyloferrin A synthetase SfaB ABC transporter ATP-binding protein ABC transporter ATP-binding protein Fe-S cluster assembly ATPase SufC Fe-S cluster assembly protein SufD ABC transporter permease cysteine desulfurase 2.990E-06 1.325E-02 5.562E-04 2.651E-03 2.110E-07 1.951E-04 1.190E-05 6.940E-06 2.513E-03 1.014E-02 2.970E-07 2.922 2.109 1.581 2.679 2.816 1.534 2.090 2.249 2.710 1.661 2.728 sufD sufU sufC sufS sstC isdE sstA 62 Table 2.5 (cont’d) *Also upregulated in sulfur starved cymR::Tn. 63 Table 2.6. Strains used in this study. Strain wild type cymR::Tn hrtA::Tn tsuA::Tn SAUSA300_RS04580::Tn Description USA300 LAC derivative JE2 Erythromycin resistant (Ermr) Bursa Tn insertion at JE2 chromosome position 1733227 Ermr Bursa Tn insertion at JE2 chromosome position 2479408 Ermr Bursa Tn insertion at JE2 chromosome position 2156268 Ermr Bursa Tn insertion at JE2 chromosome position 927895 Reference (164, 166) (164, 166) (164, 166) (164, 166) (164, 166) Table 2.7. Plasmids used in this study. Plasmid pKK22 pKK22-tsuAB pKK22-tsuA pKK22-tsuB Description Derivative of the naturally occurring S. aureus plasmid LAC-p01. Maintains stability in S. aureus without antibiotics. Linearized for Gibson assembly using primers PK85 & PK86. pKK22 expressing tsuA (SAUSA300_RS10985) and tsuB (SAUSA300_RS10980) under native promoter expression. Insert generated with following primers: PK5 & PK8 pKK22 expressing tsuA (SAUSA300_RS10985) under native promoter expression. Generated insert with following primers: PK5 & PK10 pKK22 expressing tsuB (SAUSA300_RS10980) under native promoter expression. Insert generated as follows: PK5 & PK6 (promoter), PK7 & PK8 (tsuB); PK5 & PK8 used to sew promoter and tsuB fragments together before Gibson assembly. Reference (153) This study This study This study 64 Table 2.8. Primers utilized to generate strains in Table 1. Primer Description Sequence (5’ → 3’) PK85 PK86 NE Martn- ermR* NE Buster* HL246 PK110 HL267 PK5 PK6 PK7 PK8 PK10 pKK22 amplification/linearization for Gibson assembly pKK22 amplification/linearization for Gibson assembly Used to confirm Tn insertions on plus strand Used to confirm Tn insertions on minus strand Confirming Bursa Tn insertion in cymR Confirming Bursa Tn insertion in hrtA Confirming Bursa Tn insertion in tsuA Amplify tsuAB promoter and operon for Gibson assembly Amplify tsuAB promoter for Gibson assembly Amplify tsuB for Gibson assembly Amplify tsuB for Gibson assembly Amplify promoter and tsuA operon for Gibson assembly *Reference: (164, 166) GCGGCCGCTAGCCTAGGAGC ATCGCCTGTCACTTTGCTTGATATATGA CTCGATTCTATTAACAAGGG GCTTTTTCTAAATGTTTTTTAAGTAAATCA AGTAC CTAATAACAAGATAACTTGACCAGAC AGAACTTAATGTCCCAGCC TCCCATACATATGCCACC CAAGCAAAGTGACAGGCGATTAGATGTT GTGATTCTAACTAC CGTGTATCATAATCTTAACCTCTCATTTCC GGTTAAGATTATGATACACGAATTAGGTA C GCTCCTAGGCTAGCGGCCGCTTAAACTT TTTGAATTGTAATTGTC GCTCCTAGGCTAGCGGCCGCCTATACTA TTTGCGTTTGC 65 FIGURES Figure 2.1. S. aureus CymR responds to at least two different stimuli. A) Model of CymR function described by Soutourina et al (118). CymR senses the intracellular Cys pool through the product of serine transacetylase (CysE), O-acetylserine (OAS), Top Left: CysE forms a complex with OAS-thiol-lyase B (CysM, aka MccA) and, under low intracellular Cys levels, produces OAS which is then consumed by CysM for use in 66 Figure 2.1 (cont’d) inorganic sulfur assimilation. CymR is inactive in this condition and transcription of sulfur acquisition and metabolism systems proceed. Top Right: CysE is inhibited by Cys when the amino acid is present at threshold concentrations. In this situation, CysM is released and interacts with CymR. The CysM-CymR complex then binds target DNA and inhibits expression of the sulfur regulon (118, 134). B) Model of CymR activity as described by Ji et al. Bottom Left: CymR additionally senses the oxidation state of S. aureus via its sole Cys residue at position 25. When the Cys is in the reduced stated (i.e., has the -SH thiol group), CymR will bind to DNA. Bottom Right: When this residue is oxidized by H2O2, the thiol group will form a sulfinic acid (-SOH) intermediate. The low molecular weight thiol, Acetyl CoA, can then form a disulfide bond with CymR, decreasing the affinity of this regulator for DNA (right) (121). Made with BioRender. 67 Figure 2.2. Experimental design employed to define the CymR-dependent and – independent responses to sulfur starvation in S. aureus. WT or cymR::Tn S. aureus were sub-cultured from normalized overnights into CDM supplemented with 25 µM CSSC and grown to mid-exponential phase (4 h). Cultures were pelleted, washed, and resuspended to the same cell density in medium with 25 µM CSSC or no viable sulfur source. Cells were grown for an additional 2 h prior to RNA isolation. Image generated with BioRender.com. 68 Figure 2.3. Comparison of the S. aureus sulfur starvation transcriptional response to the CymR regulon. A-C) Venn diagrams comparing genes upregulated in sulfur starved WT (WT -S; purple), cymR::Tn cultured in CSSC (cymR::Tn +S, pink), and the previously published ∆cymR mutant sulfur Table (∆cymR published, grey). The previously defined ∆cymR transcriptome was generated from using Table 2 and Table 4 from (118) as well as Table 1 from (122). A) The total number of genes upregulated in all three Tables. B) Sulfur metabolism associated genes differentially regulated between the three Tables. Table 2 from Soutourina et al. (118) was used for this assessment. Functional annotation of genes identified in the current study were defined using eggNOG-mapper (168, 169). The COG Database was then used to identify COGs associated with the terms “sulf”, “methionine”, “cysteine”, “cystine”, “glutathione”, and “γ-glutamyl-transpeptidase” (172, 173); these genes were classified as sulfur metabolism related. *Note that the genes shared between ∆cymR published, WT -S, and cymR::Tn +S should be eight. One gene, (ABC-type amino acid SAUSA300_RS13025 transport/signal transduction system, periplasmic component/domain) and was thus not identified within the abovementioned parameters. (tcyA), was assigned COG0834 69 Figure 2.3 (cont’d) four: control regulon function (S) under CymR -S and ∆cymR published should be However, tcyA is known to participate in cyst(e)ine transport in S. aureus (90). Of the remaining two genes, SAUSA300_RS00910 is a hypothetical protein assigned the COG (91); category of unknown SAUSA300_RS00930 was designated COG “I” for lipid transport and metabolism but is part of the SfnB family of sulfur acquisition oxidoreductases (91). **Additionally, genes tcyC shared between WT (SAUSA300_RS13015) was assigned to COG1126, and tcyB (SAUSA300_RS13020) was assigned to COG0765; neither of these COGs were found with the search parameters for sulfur metabolism genes described above. However, these genes encode subunits of the TcyABC complex (90). C) Cell wall associated genes differentially regulated between the three Tables. Table 4 from Soutourina et al. (118) was used to generate this Venn diagram. Genes for the current study were defined as cell wall to COG category M (cell associated wall/membrane/envelope biogenesis). No genes were found to be common amongst all three Tables using this method. However, upon comparing Table 4 from Soutourina et al. to the total upregulated genes for sulfur starved WT (WT -S) and CSSC supplemented cymR::Tn (cymR::Tn +S) revealed two common genes, SAUSA300_RS01875, which was assigned as COG S (unknown function), and SAUSA300_RS15260 (no homolog found). D) Comparison of the Log2 ratio of genes upregulated in either cymR::Tn supplemented with CSSC (Table 2; pink) or sulfur depleted WT (Table 1; purple). *denotes genes that were shared with WT -S and cymR::Tn +S sulfur metabolism genes in B, including genes tcyA, SAUSA300_RS00910, and SAUSA300_RS00930. if eggNOG-mapper assigned the gene 70 Figure 2.4. The S. aureus sulfur starvation response is not fully dependent on CymR. A) WT sulfur starvation response loci from Table 1a (n=567; yellow) were 71 Fgiure 2.4 (cont’d) compared against the starvation response when CymR is absent (Table 1c; n=255; cyan). Genes that shared between WT and the cymR::Tn mutant are considered to CymR- independent (n=138). CymR independent genes are those genes unique to the WT sulfur starvation condition (n=429). Additionally, genes unqiue to the cymR::Tn condition (n=117) are likely differentially expressed to compensate for the loss of CymR during sulfur starvation. Figure created in Biorender. B-D) Genes that shared between the WT and cymR::Tn sulfur starvation (B) or are unique to WT or cymR::Tn sulfur starvation (C and D, respectively) conditions catagorized by COG assignment. COG assignments are as follows: A (RNA processing and modification), B (chromatin structure and dynamics), C (energy production and conversion), D (cell cycle control, cell division, chromosome partitioning), E (amino acid transport and metabolism), F (nucleotide transporter and metabolism), G (carbohydrate transport and metabolism), H (coenzyme transport and metabolism), I (lipid transport and metabolism), J (translation, ribosomal structure and biogenesis), K (transcription), L (replication, recombination and repair), M (cell wall/membrane/envelope biogenesis), N (cell motility), O (posttranslational modification, protein turnover, chaperones), P (inorgnaic ion trasnport and metabolism), Q (secondary metabolites biosynthesis, transport and catabolism), S (function unknown), T (signal trasnduction mechanisms), U (intracellular trafficking, secretion, and vesicular transport), V (defense mechanisms), Z (cytoskeleton) not classified (NC). In some instances no homologs were found (NHF). Values above each bar represents the number of genes for each COG category. 72 Figure 2.5. GSH alleviates oxidative stress in S. aureus. S. aureus WT and hrtA::Tn were cultured in chemically defined medium (CDM) in the absence of a sulfur source or supplemented with 50 µM or 750 µM reduced glutathione (GSH) (A and B, respectively) as well as 25 µM or 375 µM oxidized glutathione (GSSG) (C and D, respectively) in the presence or absence of 10 (cid:0)M hemin. Curves are the mean of three independent trials and the error bars represent ±1 standard error of the mean. E) WT preloaded with either 50 (cid:0)M GSH (squares), 750 (cid:0)M GSH (circles), or 375 (cid:0)M GSSG (triangles) were left untreated (open symbols) or exposed to 1 M H2O2 (closed symbols) before enumeration for CFU. Presented are the average of three independent trials ±1 standard error of the mean. Statistical significance represents Brown-Forsythe and Welch ANOVA tests. * Represents P-values <0.05 while ** are for P-values <0.005. “Not significant” indicates a P-value >0.05. Any comparisons not represented are not significant. 73 Figure 2.6. SAUSA300_RS04580 does not contribute to S. aureus proliferation on CSSC as a source of nutritional sulfur. WT and SAUSA300_RS04580::Tn (denoted 4580::Tn) were cultured in chemically defined medium (CDM) supplemented with either 25 µM cystine (CSSC; closed symbols) or 50 µM cysteine (Cys; open symbols). Curves are the mean of three independent trials and the error bars depict ±1 standard error of the mean. 74 Figure 2.7. Supplementation with sodium thiosulfate induces considerable transcriptional changes in S. aureus. Differential expression in WT cells was determined by comparing cells cultured in chemically defined medium (CDM) supplemented with cysteine (Cys; black), oxidized glutathione (GSSG; green), reduced glutathione (GSH; orange), or sodium thiosulfate (sTS; purple) to the CDM supplemented with cystine (CSSC). Only a single gene is differential expressed (downregulated) in S. aureus grown in Cys compared to CSSC and is therefore not presented in these comparisons. A) Total number of genes that are differentially expressed in response to distinct sources of nutrient sulfur. B) Venn diagram comparing genes upregulated when S. aureus is cultured in GSSG-, GSH- or sTS-supplemented medium. *The four upregulated (SAUSA300_RS15735, SAUSA300_RS15740, SAUSA300_15090, SAUSA300_RS15730) were not shared with GSSG, GSH, or sTS and were thus excluded from the Venn Diagram. C) Genes that are downregulated between the GSSG, GSH, and sTS. *The two downregulated genes in CSSC (SAUSA300_04580 and SAUSA300_06690) were both shared with GSSG, GSH, or sTS and are represented within the total shared genes in the Venn Diagram. D) Transcriptional regulators differentially expressed when S. aureus is grown on GSH, GSSG or sTS. in CSSC genes 75 Figure 2.8. The S. aureus transcriptome is influenced by different metabolites utilized to meet the nutritional sulfur requirement. Heat map of the shared 76 Figure 2.8 (cont’d) upregulated genes in S. aureus cultured in medium supplemented with 50 µM sodium thiosulfate (sTS), 25 µM oxidized glutathione (GSSG), and 50 µM reduced glutathione (GSH) compared medium supplemented with cystine (CSSC). Genes included are at least 2-fold upregulated with an adjusted P-value <0.05. The color of the boxes indicates the log2 expression ratio. Genes labeled in bold text were also found to be upregulated in the published CymR regulon (118). A “+” indicates the gene encodes a putative transporter or a protein with a transporter -associated function. 77 Figure 2.9. Staphylococcus aureus employs the TsuAB thiosulfate import system to utilize thiosulfate as a source of nutrient sulfur. A) An illustration of the 985 (SAUSA300_RS10985) and 980 (SAUSA300_RS10980) operon and location of the Tn insertion (position 2,156,268, bold) (164, 166). P985 (position 2,156,611-2,157,110) denotes the genetic region used in complementation studied that includes the native promoter region. Image generated using BioRender. B-C) Resulting growth kinetics of strains cultured in chemically defined medium (CDM) supplemented with either 50 µM sodium thiosulfate (sTS) (B) or 25 µM cystine (CSSC) (C) or as the sole sulfur source. WT S. aureus harboring an empty pKK22 vector (EV) cultured in medium lacking a viable sulfur source represents the no sulfur control. The mean of three independent trials is presented and the error bars represent ±1 standard error of the mean. 78 CHAPTER 3: Staphylococcus aureus DtpT supports nutrient sulfur acquisition of γ-glutamyl cycle intermediates 79 ABSTRACT Staphylococcus aureus is an opportunistic pathogen that incites a range of infections such as osteomyelitis, pneumonia, or bacteremia. Proliferation within the host, however, demands the ability to procure local nutrients. Sulfur is one such nutrient S. aureus must acquire but the metabolites obtained, and the machinery employed to do so remain incomplete areas of study. Glutathione is the most abundant non-protein thiol in mammals and previous work has demonstrated that S. aureus engages the glutathione import system and at least one other importer to sustain growth on this nutrient as a sulfur source. The current study establishes that the di-tripeptide transporter, DtpT, supports S. aureus proliferation on glutathione. Furthermore, we identify that S. aureus utilization of cysteinyl-glycine (the glutathione breakdown product) as a sulfur source involves DtpT activity. A systemic infection study underscores the relevance of dtpT, as harboring this gene drives maximal colonization of S. aureus in the murine liver. Our investigations additionally illustrate the exploitable nature of sulfur metabolism pathways by characterizing the DtpT-dependent susceptibility of S. aureus to the peptide antibiotic, bialaphos. Bialaphos was also used to ascertain the Staphylococcus epidermidis DtpT homolog, B4U56_10070. The contributions of this gene to S. epidermidis propagation on both glutathione and cysteinyl-glycine is additionally highlighted. In summary, this investigation expands our knowledge regarding staphylococcal sulfur acquisition systems by accentuating the strategic redundancy engaged by S. aureus and S. epidermidis to acquire sulfur sources. 80 INTRODUCTION The opportunistic pathogen Staphylococcus aureus represents a major cause of nosocomial infections and its burden on society is exacerbated by the rise in community- acquired methicillin-resistant S. aureus (MRSA) (86, 125, 174). Infections range from infective endocarditis to skin lesions and even sepsis, making S. aureus the most common cause of fatally acquired invasive infections (86, 175–181). As MRSA becomes increasingly treatment-recalcitrant, a better understanding of its essential pathways is needed. The host-pathogen interface for macronutrients such as iron are well defined for this pathogen (3, 182–186). In comparison, how S. aureus engages sulfur metabolism to acquire the equally important nutrient sulfur is not fully defined (14, 89, 90, 134). Within all cells, sulfur is stored in carbon-containing organosulfur compounds that contain reactive sulfhydryl groups, called thiols (R-SH). This element partakes in numerous critical cellular reactions, demanding high prioritization to meet the sulfur requirement. For example, proteins harbor cysteine (Cys) residues that coordinate ions and form disulfide bonds to maintain tertiary structure (4, 5). Furthermore, Cys is the precursor to several cellular cofactors. Fe-S clusters are involved in redox reactions while coenzyme A and biotin contribute to general metabolism via the citric acid cycle or fatty acid biosynthesis; therefore, proper cofactor maintenance influences proliferation of a pathogen during infection (6–8, 129, 130). Redox balance is also managed by organosulfur compounds such as Cys and glutathione (GSH) that contain thiol groups (4, 131, 132, 187). Thus, there is potential to expand S. aureus therapeutic strategies by targeting its sulfur acquisition and utilization pathways. 81 GSH is the most abundant non-protein thiol in mammals (42). This tripeptide is composed of glutamate, Cys, and glycine. Glutamate forms a unique γ-peptide bond with Cys through interaction of its γ-carboxyl group and the Cys amide while Cys and glycine are linked via a typical α-carboxyl peptide bond. The γ-peptide bond in GSH is considered stable as it can only be cleaved by specialized enzymes such as γ-glutamyl transpeptidase (aka γ-glutamyl transferase). Given its stability and reactive thiol group, GSH is vital to the maintenance of host physiology—it acts as a major antioxidant by reducing reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), a process that results in the formation of oxidized glutathione (GSSG) (12). Additionally, GSH acts as a Cys reservoir through the γ-glutamyl cycle (12, 22, 23, 188, 189). The γ-glutamyl cycle begins when transporters export intracellular GSH from the cell. The γ-peptide bond is then cleaved by γ-glutamyl transpeptidase 1 (GGT1), which is peripherally anchored to the cell membrane. GGT1 produces γ-glutamate and the dipeptide cysteinyl-glycine (CysGly). From there, the CysGly peptide bond is cleaved by dipeptidases, allowing Cys to be transported into cells for incorporation into various cellular components or stored once again in GSH. Given the abundance of host GSH, it is unsurprising that pathogens have evolved mechanisms to exploit the metabolite as a source of nutrient sulfur. Indeed, Francisella tularensis, Haemophilus influenzae, Streptococcus mutans, and Streptococcus pneumonia are known to consume host GSH to meet the sulfur requirement (100, 144, 190, 191). F. tularensis, interestingly, imports GSH by first cleaving its γ-peptide bond in the periplasm with Ggt and then importing CysGly through a di-tripeptide transporter, DptA (101). 82 Recently, we have shown that S. aureus encodes a specialized glutathione import system (GisABCD) to import GSH and GSSG into the cytoplasm where Ggt then begins catabolism of these metabolites for use as nutritional sulfur (89). However, the complexity of S. aureus GSH acquisition was discovered when deletion of the gisABCD-ggt system failed to abolish S. aureus propagation on physiologically relevant concentrations of GSH in vitro. Furthermore, a DgisABCD-ggt mutant does not impact the ability of S. aureus to colonize during systemic infection. In the current study, we expand upon our previous work by demonstrating that the di-tripeptide transporter, DtpT, supports S. aureus growth on GSH. Our investigations also reveal that CysGly is a source of nutritional sulfur for S. aureus in a DtpT-mediated manner. Phylogenetic studies indicate that homologs for this protein are found across a number of other Gram-positive bacteria. Assessing colonization of a MRSA strain during systemic infection, we further corroborate earlier findings that DtpT promotes maximal S. aureus proliferation in vivo. Finally, we determine that S. aureus sulfur acquisition machinery can be taken advantage of by delivering a toxic tripeptide, bialaphos, via DtpT. This proof-of-concept is extended into another clinically relevant species, Staphylococcus epidermidis. Collectively, these data expand our knowledge regarding Staphylococcus sulfur acquisition strategies and provide evidence that S. aureus sulfur metabolism pathways can yield new avenues to combat this threat to societal health. RESULTS Staphylococcus aureus employs redundant strategies to support utilization of GSH and CysGly as sources of nutritional sulfur. Prior investigations in our lab demonstrate the presence of at least two GSH transporters (89). Given that GisABCD was defined as 83 a putative nickel-peptide ABC transporter, we surmised that another oligopeptide transporter encoded by S. aureus would associate with this metabolite. These potential systems include the NiKA & NikBCDF importer, CntABCD, Opp3BCDFA, Opp4ADFB, and OppACME-ABCDF, and DtpT (192, 193). Within this list, the proton-dependent oligopeptide transporter (POT) family protein DtpT has been implicated in the uptake of both di- and tripeptides in S. aureus (91, 192). Given that GSH is a tripeptide, we hypothesized that DtpT would play a role in use of this nutrient as a sulfur source. To test this, a clean dtpT deletion was generated in the WT and isogenic ΔgisABCD-ggt backgrounds (denoted ΔdtpT and Δgis ΔdtpT, respectively). The growth profile of these strains on CDM supplemented with GSH was assessed (Fig. 3.1). When low a concentration (i.e., 50 µM) of GSH is supplied as the sulfur source, the Δgis EV mutant exhibits drastically reduced optical density at 600 nm (OD600) values compared to WT (Fig. 3.1A). This is in accordance with previous observations (89). A ΔdtpT EV propagates like WT while the Δgis ΔdtpT EV mutant phenocopies growth kinetics of the Δgis EV strain. Complementation of Δgis ΔdtpT with the WT dtpT allele in trans allows for marginally improved growth compared to the EV counterpart. While 50 µM GSH is sufficient for S. aureus to meet the sulfur requirement in vitro, this concentration does not reflect what the pathogen might encounter in vivo. Indeed, GSH is the most abundant non-protein thiol in mammals and can reach millimolar concentrations (42); thus, the strains were cultured in CDM containing 750 µM GSH (Fig. 3.1B). As seen previously, this condition supports WT-like proliferation of the Δgis EV mutant (89). In fact, the only strain with an impairment is the Δgis ΔdtpT EV mutant whose proliferation is restored upon complementation with WT dtpT. However, the appreciable 84 growth of Δgis ΔdtpT EV on 750 µM GSH suggests the presence of at least one more active transporter in this condition. Furthermore, it is important to note that these phenotypes are sulfur specific given all strains propagate like WT when provided with a control sulfur source such as cystine (CSSC) or in the rich medium, tryptic soy broth (TSB; Fig. 3.1C and 3.1D, respectively). GisABCD is an ABC-transporter while DtpT is a POT family member (89, 192). With such a difference in protein families, we predicted that there would be subtle variations in how each transporter correlates with GSH. Thus, we sought to investigate which properties of GSH influence the ability of DtpT and GisABCD to support S. aureus proliferation on this metabolite. To do so, a GSH analog was supplemented in the CDM (Fig. 3.2). In this analog the γ-peptide bond shared between the glutamyl and cysteinyl residues of GSH was replaced with an α-peptide bond; this glutamyl-cysteinyl-glycine peptide is denoted as ECG for short. Notably, ECG is a sulfur source for WT and the Δgis mutant displays no growth defect regardless of the concentration supplied (Fig. 3.2A-C). However, deleting dtpT severely impacts growth compared to WT at 50 µM ECG (Fig. 3.2A). The severity of this phenotype is not compounded upon inactivation of both transporters. Interestingly, increasing ECG concentration in the CDM restores the ability of the ΔdtpT and Δgis ΔdtpT strains to proliferate (Fig. 3.2B and 3.2C). These observations stand in stark contrast compared to what is observed with 50 µM GSH where strains harboring a Δgis mutation struggle to propagate and the ΔdtpT has WT-like growth (Fig. 3.2D). As expected, no growth defects are observed when CSSC is the sulfur source (Fig. 3.2E). These data imply that the γ-peptide bond of GSH is important for GisABCD 85 and while its tripeptide nature is what DtpT relates to when assisting S. aureus proliferation on this nutrient. Given that DtpT also imports dipeptides we postulated that this transporter would also sustain the use of CysGly (the GSH breakdown product) as a sulfur source for S. aureus. Importantly, WT EV is capable of robust proliferation in CDM supplemented with 25 µM CysGly, demonstrating the sufficiency of this dipeptide for meeting the S. aureus nutritional sulfur requirement (Fig. 3.3A). Deletion of DtpT reduces, but does not abolish, the ability of S. aureus to grow in this condition. Notably, this phenotype can be complemented. GisABCD does not contribute to propagation on CysGly as the Δgis ΔdtpT EV strain growth kinetics mimic those of ΔdtpT EV. However, these are the assessments of a laboratory strain. To evaluate whether clinical S. aureus isolates retain the ability to utilize CysGly for nutritional sulfur needs, six isolates from cystic fibrosis (CF) patients were cultured in the presence of CysGly. After 24 h each strain showed at least a 225% increase in OD600 when compared to being cultured the absence of a viable sulfur source (Fig. 3.3B). Collectively, these data indirectly suggest that DtpT is both a GSH and CysGly transporter in S. aureus and that at least one additional importer is contributing to propagation on these nutrients. DtpT is broadly conserved among Gram-positive bacteria. Phylogenetic analyses were employed to look at the conservation of DtpT across bacteria. First homologs that retained ³80% coverage and 40% identity were isolated. All returned strains belonged to the Staphylococcaceae family, with the majority being S. aureus isolates and the second most represented species S. epidermidis. Considering that some hits were only classified as Staphylococcus, we sought to expand the selection of bacteria 86 that might contain a protein with lower homology to S. aureus DtpT. Thus, a tree was generated for homologs that had a least ³50% coverage and 40% identity (Fig. 3.4). There are 14 families containing at least 50 representatives that harbor a DtpT homolog. The top three most abundant families are Bacillaeceae, Staphylococcaceae (including S. aureus and Staphylococcus epidermidis), and Lactobacillaceae. When looking at all the families, four clades are observed (Fig. 3.4B, upper left). Clade 1 largely constitutes the Staphylococcaceae, but also contains Listeriaceae. Bacillaceae, Caryophanaceae, and Paenibacillaceae. Clade 2, in addition to those families in Clade 1, represent the homologs with the closest relation to S. aureus DtpT given they share the same common ancestor. Found within Clade 3 are Leuconostocaceae, Micrococcaceae, Pseudonocardiaceae, Streptomycetaceae, and bacteria that had <50 representatives (i.e., Others) while Clade 4 constitutes Enterococcaceae, Lactobacillaceae, and Streptococcaceae. This information reveals that DtpT-like proteins are selectively found within a Gram-positive bacterial group that includes pathogens, commensals, and environmental isolates. Maximal S. aureus colonization of the murine liver requires functional DtpT. In addition to previously defined di-tripeptides (192), in vitro work above demonstrates two new, sulfur containing peptides associated with DtpT. Given this, we sought to identify the impact this transporter has on S. aureus proliferation during infection. Earlier studies employing signature tagged transposon (Tn) mutant pools suggested that a dtpT::Tn mutant negatively impacts S. aureus strain RN6390 in murine abscess, wound, and systemic bacteremia infection models (194). Individual assessment of a dtpT::Tn mutant in rabbit endocarditis and murine abscess or wound models demonstrate attenuated 87 mutant phenotypes (194). Furthermore, inactivating dtpT led to increased survival of mice in a median lethal dose (LD50) challenge. However, RN6390 is a derivative of the methicillin sensitive S. aureus (MSSA) laboratory strain NCTC8325 and contains a deletion in the critical stress response regulator, rsbU (195). To better understand the impact of dtpT on a strain that represents the current epidemic S. aureus lineage, we utilized the methicillin resistant USA300 LAC derivative, JE2. Using a systemic model of infection, mice were inoculated with either WT or a dtpT::Tn mutant. Mice infected with dtpT::Tn tend to exhibit lower bacterial burdens in the heart or kidneys, along with having significantly reduced CFUs in the liver (Fig. 3.5). This data therefore extends the biological impact of DtpT to include both MRSA and MSSA isolates. DtpT is a relevant model to assess exploitation of sulfur metabolism for delivery of a lethal substance into S. aureus. Given the demonstrated impact of DtpT on S. aureus proliferation in vivo, this transporter was employed to investigate the potential for inhibiting pathogen proliferation by exploiting sulfur transportation pathways. Bialaphos is a tripeptide produced by Streptomyces viridochromogenes as well as Streptomyces hydroscopius and is used commercially as a non-selective herbicide (196, 197). This peptide antibiotic consists of glufosinate-alanyl-alanyl residues. Glufosinate (aka phosphoinothricin) is a toxic glutamate analog harboring a phosphinate moiety that mimics the tetrahedral intermediate of glutamine synthetase (196, 197). Characterizing this peptide with Escherichia coli and Bacillus subtilis indicates that, upon import, glufosinate is cleaved from the alanyl residues before inhibiting glutamine synthetase. If the cell cannot import exogenous glutamine to compensate for the loss of glutamine synthetase then cellular death ensues. Interestingly, oligopeptide transporters in E. coli 88 (Opp; (198)), B. subtilis (OppABCD; (199, 200)), Salmonella typhimurium (Opp, Tpp, Dpp; (201)) and Listeria monocytogenes (OppA; (202)) are associated with bialaphos import. However, bialaphos sensitivity in staphylococcal species has not yet been explored. We hypothesized that S. aureus would be sensitive to bialaphos in both a glutamine and transportation-dependent manner. Conducting a bialaphos Kirby Bauer on a rich medium (trypic soy agar, TSA) did not result in a zone of inhibition (Fig. 3.6A and B). However, plating cells on a CDM agar plate that lacks glutamine (Gln) resulted in WT EV sensitivity to bialaphos which could be mitigated by adding 500 µM Gln to the CDM. Most importantly, the deletion of dtpT confers complete resistance to bialaphos regardless of the presence of Gln in the medium, and sensitivity can be reintroduced through complementation (Fig. 3.6A and B). It was noted that the WT and DdtpT pdtpT strains develop bialaphos resistant colonies (RCs) in the CDM + 0 mM Gln condition (Fig. 3.6A and C). Nine of these RCs were isolated from the WT plates and were challenged with bialaphos (Fig. 3.7). Six out of nine RC strains were completely resistant to the peptide antibiotic and the remaining three displayed significantly increased resistance (Fig. 3.7A and B). These strains displaying reduced zones of inhibition also developed resistant colonies like WT (Fig. 3.7C). Whole genome sequencing of the nine RCs reveals several genes being mutated (Table 3.1). Importantly, there is only one gene, dtpT, that is uniquely mutated in all the bialaphos RC strains (Table 3.1, Fig. 3.7D). As such, it is concluded that DtpT is both sufficient and necessary for S. aureus import of bialaphos. Bialaphos is a tool to identify sulfur metabolism machinery in S. epidermidis. While S. aureus is the most notorious of the Staphylococcal species, S. epidermidis is another clinically relevant organism known for causing skin, mucosa membrane, and 89 prosthetic join infections (203, 204). Thus, it is equally pertinent to understand what sulfur metabolism pathways help this organism meet the nutritional sulfur requirement. Our work has indicated that S. epidermidis utilizes GSH as a source of nutritional sulfur but does not encode GisB nor GisD homologs of the GisABCD-Ggt system (89). Since DtpT is associated with both GSH and bialaphos import in S. aureus, we hypothesized that bialaphos could serve as an effective means to discern the GSH transporter in S. epidermidis. Kirby Bauer assays reveal that, like S. aureus, WT S. epidermidis 1457 is only sensitive to bialaphos when plated on CDM that does not contain Gln and develops resistant colonies (Fig. 3.8A-C). Selecting nine RCs and challenging them against bialaphos demonstrates that each strain has either complete or near complete resistance to the peptide antibiotic (Fig. 3.8A-C). Strikingly, these bialaphos RCs only share mutations in one common gene, B4U56_10070, which encodes for a peptide MFS peptide (Table 3.2, Fig. 3.8D). BlastP alignment of B4U56_10070 and DtpT from S. aureus results in 100% coverage and 82.83% identity. This observation is consistent with the phylogenetic studies where many S. epidermidis isolates were found to harbor a DtpT homolog (Fig. 3.4). To further assess B4U56_10070 as a DtpT homolog, S. epidermidis 1457 bialaphos RCs were cultured in a modified CDM that does not contain Met or sulfate (CDMmod), given they support the nutritional sulfur requirement in this species (89). In comparison to WT, each of the B4U56_10070 mutated strains display reduced growth in CDMmod supplemented with 750 µM GSH (Fig. 3.8E). We next tested the ability of S. epidermidis 1457 to utilize CysGly as a sulfur source (Fig. 3.8F). Here the WT strain 90 shows robust proliferation on CDMmod supplied with 75 µM CysGly while the bialaphos resistant colonies demonstrate poor propagation. These phenotypes are sulfur specific given the ability of all strains to grow to WT-like levels in the presence of CSSC (Fig. 3.8G). Collectively, these data strongly suggest that B4U56_10070 is a DtpT homolog and represents the major transporter associated with S. epidermidis 1457 growth on both GSH and CysGly as sources of nutrient sulfur. DISCUSSION This investigation increases our knowledge of GSH acquisition strategies employed by S. aureus in addition to identifying CysGly, the breakdown product of GSH, as a nutrient that satisfies the nutritional sulfur requirement for this pathogen. Data for both peptides highlight the fact that S. aureus employs redundant strategies to procure select nutritional sulfur sources. Previous work with S. aureus has revealed the involvement of multiple transporters (i.e., TcyABC and TcyP) in the utilization of Cys and CSSC (90). In contrast, only one transporter associates with S. aureus proliferation on thiosulfate (see Chapter 2). It would seem, then, that there is an evolutionary strategy for S. aureus to encode multiple acquisition systems for what might be considered preferred sulfur sources. The current study underscores this notion by demonstrating that presence of at least three importers driving S. aureus proliferation on physiologically relevant concentrations of GSH in vitro (Fig. 1B). To that end, our work is the first to associate the di-tripeptide transporter, DtpT, with GSH import. Indeed, results from Fig. 3.1A and 3.1B suggest that DtpT contributes to S. aureus growth on physiologically relevant concentrations of GSH. This comes as little surprise given that DtpT has been associated with the import of glutamate or glycine containing di-tripeptides as sources of nitrogen (192). However, the 91 contribution of DtpT toward GSH uptake is not to the same extent as the GisABCD-Ggt system given its WT-like growth at 50 µM (Fig. 3.1A). This may be because GisD harbors a solute-binding protein family 5 domain, known for its high substrate specificity; in contrast, transporters from the POT family such as DtpT identify substrates through specificity pockets that establish substrate affinity based on peptide charge (91, 205– 208). Our work additionally investigates the properties involved with GSH substrate identification for both DtpT and GisABCD using an ECG analog (Fig. 3.2). Employing ECG has indirectly implied that DtpT recognizes GSH due to its tripeptide nature given the growth defect of a dtpT mutant on low concentrations of this metabolite (Fig. 3.3A). This stands in contrast to the GisABCD-Ggt system which did not display any growth impairments in ECG (Fig. 3.2A-C) or the dipeptide CysGly (Fig. 3.3A); this signifies that GisABCD requires the γ-peptide bond of GSH and perhaps the tripeptide aspect as well in order to support growth of S. aureus on this nutrient. However, to solidify whether GisABCD identifies with dipeptides, growth kinetics using the GSH precursor, γ-glutamyl- cysteine would be needed. Furthermore, binding assays with purified protein or uptake of radiolabeled GSH and/or CysGly will provide direct insight into substrate binding for these two transporters. Future investigations are also necessary to elucidate the complete set of transporters involved with growth of CDM supplemented with GSH. Given that both GisABCD and DtpT are peptide importers, we predicted that another oligopeptide transporter is responsible for the growth of the Δgis ΔdtpT on physiologically relevant concentrations of tripeptides such as GSH or ECG. Opp3BCDFA is associated with import of tripeptides and represents an ideal candidate for future studies (192, 193). 92 CysGly has also been uncovered as a sulfur source for S. aureus (Fig. 3.3). Interestingly, other groups have shown the capacity of bacteria to utilize oligopeptide transporters to bring CysGly into the cytoplasm. One study demonstrates that another opportunistic pathogen, Campylobacter jejuni, employs an oligopeptide transporter family protein (OPT), CptA, to import CysGly as a source of nutrient sulfur (209). The F. tularensis di-tripeptide transporter DptA is also known to import a CysGly into the cytosol; DtpA also strongly impacts the survival of F. tularensis during macrophage infection (101). Perhaps even more significant are the structural studies that confirm a Staphylococcus hominis POT family protein, PepT, as an importer for the malodor precursor called cysteinyl-glycine 3-methyl-3-sulfanylhexanol (S-CysGly-3M3SH) (103). PepT and other proteins denoted as S-CysGly-3M3SH transporters were found within the Staphylococcaceae family when conducting phylogenetic analyses on DtpT (Fig. 3.4). This reinforces the notion that S. aureus DtpT is likely a CysGly transporter. In total, the DtpT-mediated utilization of CysGly to meet S. aureus nutritional sulfur demands adds to our knowledge surrounding the use of CysGly by microbial residents of the mammalian host. This study not only expands our understanding of S. aureus sulfur metabolism but defines the biological relevance of the involved pathways. This was done by confirming the ability of several S. aureus CF isolates to proliferate on CysGly as a source of nutrient sulfur (Fig. 3.3B). Though some strains have a higher capacity to growth on CysGly than others, this overall suggests that the pathways needed for import of this dipeptide are actively maintained in the CF lung environment. Furthermore, we find that DtpT helps drive successful colonization of S. aureus JE2 to in the murine liver (Fig 3.5). Whether 93 the biological relevance of DtpT during infection lies within the import of GSH and/or CysGly will need to be assessed. How S. aureus sulfur metabolism pathways can be exploited to inhibit proliferation of this prevalent threat to societal health is also explored. Some prior investigations in this have focused on the O-acetylserine sulfhydrylase isomers, which are involved in thiosulfate and sulfate assimilation to Cys (14). Inhibitors for O-acetylserine sulfhydrylase in Salmonella typhimurium, Haemophilus influenzae, and the protozoan Entamoeba histolytica have been identified (210–212), illustrating that inhibition of sulfur metabolism could be an effective treatment strategy across multiple phyla. Another aspect to consider for therapeutics is sulfur transportation. However, the data presented here suggest that sulfur transporters are not ideal candidates for inhibition because S. aureus can acquire multiple sulfur sources, and there is extensive transporter redundancy for some of these metabolites. While targeting the transporter may not effectively inhibit S. aureus proliferation, these proteins can still be utilized in a Trojan horse approach for delivering toxic substances through the system of interest. Here we conduct a proof-of-concept investigation, where the sensitivity of S. aureus to bialaphos is DtpT-dependent. Directed genetics by deleting the transporter and indirectly through mutations occurring within this gene in bialaphos RCs demonstrate this (Figs. 3.7 and 3.8). Together this suggests that, for S. aureus, DtpT is the route of bialaphos import. Bialaphos sensitivity of another clinically relevant Staphylococcal species, S. epidermidis, which depends on the activity of a DtpT homolog, B4U56_10070 reinforces the S. aureus data (Fig. 3.8). This protein is also involved in S. epidermidis sulfur metabolism—B4U56_10070 is established as a main factor in S. epidermidis growth on the previously characterized sulfur source, GSH. 94 CysGly also supports nutritional sulfur needs in S. epidermidis, a process that largely hinges on B4U56_10070. Because of this, we propose to rename this gene as DtpT (di- tripeptide transporter). It is worth noting that the consistent correlation between DtpT and the growth of staphylococcal species on GSH and CysGly suggests that this transporter likely recognizes these metabolites as substrates. However, the absence of direct experiments is a limitation to that assertion. Collectively, this study reveals new GSH and CysGly import routes for S. aureus and S. epidermidis, highlighting an evolutionary strategy of sulfur acquisition redundancy that enables these staphylococcal species to access multiple host sulfur sources. ACKNOWLEDGEMENTS Transposon mutants were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH, and the Nebraska Transposon Mutant Library (NTML) Screening Array NR-48501. We thank Vinai Chittezham Thomas for donating the WT S. epidermidis 1457 strain. Our gratitude to the Hammer laboratory members for their insights into this manuscript. This work is funded by the National Institutes of Health R01 AI139074 and R21 AI142517. MATERIALS AND METHODS Strains and Primers. All strains, plasmids, and primers used throughout the course of this study can be found in Tables 3.3, 3.4, and 3.5, respectively. JE2, a derivative of the community acquired USA300 LAC, is the WT S. aureus strain used in these studies (164). Generation of Bursa aurealis transposon (Tn) inactivated strains were done by transducing the inactivated gene from the Nebraska Transposon Mutant Library into JE2 (164, 165). Correct Tn location and deletions were determined using PCR. The pKK22 95 plasmid was generated via Gibson assembly in the Escherichia coli DH5a strain before being transferred to S. aureus RN4220 to obtain appropriate vector methylation patterns that would permit transformation into the appropriate JE2 background strain. The pKOR1 plasmid was generated as follows using restriction digestion: first the 1kb upstream and 1kb downstream of dtpT was amplified. Then these fragments were PCR sewn before blunt end cloning into pJET1.2 (ThermoFisher). From there, the 2kb up/downstream fragment was removed from pJET1.2 using NotI and KpnI restriction digestion enzymes for cloning into pKOR1. The resulting pKOR1-dtpT plasmid was transformed into E. coli DH5a before being transferred into S. aureus RN4220 and then JE2. Note that all plasmids were confirmed using Plasmidsaurus whole plasmid sequencing. Complementation vectors were generated using the pKK22 expression vector (153). Deletion of the dtpT gene was performed following a well-defined allelic exchange protocol using the pKOR1-mcs plasmid donated from the laboratory of Taeok Bae (213). Media and growth conditions. Each strain was cultured overnight in 5 mL tryptic soy broth (TSB) at 37°C, shaking at 225 rpm. Strains harboring a pKOR1-based plasmid were supplemented with 10 µg/mL of chloramphenicol unless otherwise noted during the allelic exchange protocol (213). pKK22-based plasmids were supplemented with 10 µg/mL of trimethoprim except for during growth curve assays. A base chemically defined medium (CDM) was prepared using a previously described recipe with slight modifications (149, 150). Please refer to Chapter 2 Materials and Methods for the complete CDM recipe. 5 mg mL-1 of freshly prepared glucose was supplemented into the base medium and, depending on the condition, either no sulfur source, 25 mM of CSSC, 50 mM of GSH, or 25 mM of CysGly was added into the CDM prior to inoculation. Note that this medium 96 lacks the amino acids glutamine and asparagine. S. epidermidis growth curves utilized a modified CDM (CDMmod) where Met was removed, and sulfates were also taken out by replacing MgSO4 with MgCl and (NH4)2SO4 with NH4Cl. The CSSC stock was dissolved in 1N HCl while all other sulfur sources (and glucose) were dissolved in deionized water before filter sterilization. CDM agar plates were made by mixing a 1:1 ratio of 2x CDM and 2x Select Agar before plating in 20 mL aliquots. All plates were supplemented with 25 mM of CSSC as the sulfur source and lacked glutamine unless otherwise noted. 96-well plate growth curves. Overnight cultures were centrifuged at 4°C, 4000 rpm, washed in 5 mL phosphate buffered saline (PBS), and then normalized to an optical density at 600 nm (OD600) equal to 1. From there the cells were sub-cultured 1:100 into the desired growth medium (i.e., starting OD600 is 0.01). The OD600 was monitored every hour for 24 h in a 96-well round bottom plate in an Epoc2 Biotek microplate spectrophotometer at 37°C, with continuous linear shaking. To remove background, blank measurements were subtracted from growth measurements. For each strain the growth condition was conducted in technical triplicate, which were blanked and then averaged. Averaged triplicates were considered as one biological replicate. All growth curves were conducted three independent times. End point OD600 measurements of Cystic Fibrosis S. aureus isolates. Cells were prepared and 96-well round bottom plates containing CDM harboring no viable sulfur source or 25 mM CysGly were set up as described in the previous section. Inoculated plates were then incubated at 37°C, static, for 24 h. Wells were then gently resuspended before taking an endpoint OD600 using an Epoc2 Biotek microplate spectrophotometer. Averages of technical triplicates were acquired after being blanked. Percent growth of the 97 averaged value was then achieved by applying the endpoint OD600 to the following equation: ("#$%&#’() $+&,+-) () $+&,+- each strain. . This experiment was performed four independent times for Identification of DtpT homologs across bacteria. The USA300_FPR3757 (NC_007793.1) S. aureus DtpT protein sequence (SAUSA300_RS03825) was used as the query for homolog evaluation using Blastp (214), using refseq database, an e-value of 1e-05, and allowing 10,000 hits. Proteins that demonstrated at least 50% coverage and 40% identity were selected for downstream analysis. Protein sequences were then retrieved from NCBI and aligned using ClustalW2 (215). A phylogenetic tree was constructed using IQ-TREE (216) selecting LG+F+I+G4 as the substitution model. Tree visualization was conducted using Iroki (217). The computational analyses were provided by the Institute for Cyber Enabled Research at Michigan State University. Murine model of systemic infection. TSB overnights of WT and dtpT::Tn were sub- cultured 1:100 into 5 mL TSB and grown for 3 h. Cultures were centrifuged at 4°C, 4000 rpm, before being washed into 12 mL of Dulbecco's Phosphate Buffered Saline (DPBS; no CaCl2 or MgCl2) and normalized to an OD600 = 0.4. Eight-week-old Balb/cJ female mice (Jackson Laboratories) were then anesthetized with avertin before being retro- orbitally infected with 107 CFU of either WT or the dtpT::Tn. After 96 hpi, mice were euthanized via CO2 inhalation and the heart, liver, and kidneys were harvested. The heart and kidneys were homogenized in 500 µL DPBS using an Air-cooling Bullet Blender Storm 24 (Next Advance, Inc.) using Navy, 1.5 mL RINO lysis beads (Next Advance, Inc.). Once homogenized, 500 µL DPBS was added to bring the total volume to 1 mL DPBS for the heart and kidneys. Livers, once harvested, were placed into a Whirl-Pak (Nasco) and 98 had 1 mL DBPS added before being homogenized by rolling a 1 L glass Pyrex bottle over the organ 20 times. Liver homogenate was then transferred to a 1.5 mL Eppendorf tube. Bacterial burden in each tissue was enumerated through serial dilution onto TSA plates. Infections were performed at Michigan State University under the principles and guidelines described in the Guide for the Care and Use of Laboratory Animals (218). Executed animal work followed the protocol approved by Michigan State University Institutional Animal Care and Use Committee (IACUC): PROTO202200474. Bialaphos Kirby Bauer assays. Overnight Staphylococcus cultures were centrifuged at 4°C (4000 rpm), washed once with an equal volume of PBS, and normalized to an OD600 of 1. For S. aureus, cells were then swabbed onto appropriate CDM agar plates or tryptic soy agar (TSA). S. epidermidis strains had 100 µL of normalized cells added to 1.5x CDM agar plates before being spread plated to ensure consistent growth on this medium. Sterilized, 6 mm Whatman disks were placed into an empty petri dish and had 10 µL of 10 mM bialaphos applied. Herbicide-soaked disks were applied to the center of the TSA or CDM agar plates. CDM agar plates had either 0 µM, 500 µM, or 2 mM Gln added with or without TMP. Only strains containing a pKK22-based plasmid were plated onto CDM agar plates harboring TMP to ensure maintenance of the pKK22-dtpT vector in the presence of bialaphos. Plates were then put at 37°C for 24 h before quantifying the zone of inhibition (ZOI) and resistant colonies (RC). For each condition, three technical replicates were averaged for ZOI and RC with the average being considered a biological replicate. Three biological replicates were conducted for each condition. Pictures were taken with an iPhone XR, using the same distance from lens and lens zoom. 99 Isolation of Genomic DNA and whole genome sequencing. Nine RC from the WT bialaphos Kirby Bauer assays were picked and struck for isolation onto TSA. Five mL TSB was inoculated and cultured at 37°C, 225 rpm, overnight. The cell wall was digested by resuspending the overnight in 485 µL water buffered with TSM (50 mM Tris, 500 mM sucrose, and 21 mM MgCl2; pH 7.5) and 15 µL of 2 mg mL-1 lysostaphin (ABMI; Lawrence, NY) before incubation at 37°C for 30 min. Afterwards, the protoplast was obtained by spinning at 9000 rpm for 5 min and decanting the supernatant. Genomes were isolated using a Promega kit (Madison, WI) following manufacturer’s instructions. Briefly, protoplast was resuspended in 600 µL nuclei lysis buffer and heated at 80°C for 10 min. After cooling to room temp, 10 µL of RNase was added to lysate and incubated at 37°C for 45 min. Then, 200 µL protein precipitation solution was added, and tubes were incubated on ice for 10 min before being centrifuged at top speed for 10 min. The DNA- containing fraction was then added to 600 µL isopropanol and gently rocked until the DNA precipitated. After pelleting the DNA (top speed, 5 min), the pellet was first washed with 600 µL of ice-cold 70% ethanol and then 90% ethanol. After completely drying, the pellet was resuspended in HPLC grade water. Following Qbit quantification, genomes were sent to SeqCoast Genomics (Newington, New Hampshire) for Illumina short-read whole genome sequencing using 150 bp paired end reads. For whole genome sequencing analysis, reads for both strands were uploaded into Geneious Prime 2022.0.2 (Dotmanics, Boston, MA). Adaptors and low-quality reads were trimmed using BBDuk default settings. Trimmed sequences were mapped to the S. aureus USA300_FRP3757 (NC_007793.1) or S. epidermidis 1457 (CP020463.1) reference genome. Variants were then called in the mapped genomes using a 0.9 minimum variant frequency. Mutations 100 not found in the WT strain and located within open reading frames (ORF) were then identified. The abovementioned reference genomes were also used to extract protein sequences of DtpT (S. aureus) and B4U56_10070 (S. epidermidis) for NCBI Blastp studies. Data visualization. Growth curves and bar graphs were generated using GraphPad Prism software version 10.0.0 (153). All finalized figures were generated using the open source Inkscape 1.2.2 (b0a8486, 2022-12-01). Available at: https://inkscape.org. 101 RC01 RC02 RC03 RC04 RC04 RC05 RC06 RC07 RC08 RC09 RC03 RC07 RC08 RC09 RC06 RC04 RC05 RC06 Table 3.1. S. aureus bialaphos resistant colony mutations. TABLES Genome Locus Gene Product SAUSA300 _RS03825 dtpT peptide MFS transporter Deletion Amino acid change Nucleotide change AACAAAAC C G -> V C -> A G -> D C -> T A -> ANT TATTAG CAA -> TCT VV -> EI A -> T G -> T G -> A G -> T C -> T Transversion A -> D P -> L Q -> K G -> D SAUSA300 _RS04175 emp extracellular matrix protein-binding adhesin Emp A -> C Transversion SAUSA300 _RS10835 int3 site-specific integrase T -> A L -> I SAUSA300 _RS13025 tcyA transporter substrate- binding domain- containing protein A -> T Transversion SAUSA300 _RS13270 pnbA carboxylesterase/lipa se family protein C -> T Transition 102 Table 3.2. S. epidermidis bialaphos resistant colony mutations. Genome Locus Product RC7 B4U56_01010 RC1 B4U56_04105 RC3 B4U56_09585 ABC transporter substrate-binding protein RNA polymerase sigma factor SigB methionine import ATP- binding protein MetN 1 Nucleotide change Amino acid change C -> G A -> G G -> T Transversion G -> A A -> V RC1 RC2 RC3 RC4 RC5 RC6 RC7 RC8 RC9 B4U56_10070 MFS transporter T -> A C -> A C -> G G -> A C -> A T -> A G -> A C -> G Truncation S -> R N -> K G -> D Deletion T -> N S -> R D -> N Truncation RC6 B4U56_11050 DNA-directed RNA polymerase subunit β C -> G R -> T RC5 RC5 B4U56_11965 hypothetical protein G -> T G -> A L -> F M -> I 103 Reference (164, 166) (89) This study This study This study This study This study This study This study This study This study This study This study This study (219) This study This study This study This study This study This study This study This study This study Table 3.3. Strains used in this study. Strain Staphylococcus aureus strains S. aureus WT Description DgisABCD-ggt DdtpT dtpT::Tn DgisABCD-ggt DdtpT HL1427 JE2, derivative of USA300 LAC Clean deletion of SAUSA300_0200-0404 genetic region Clean deletion of dtpT (SAUSA300_0712); sae sequenced & α hemolysin positive B. aurealis Tn insertion at genome position 2156268 Clean deletion of the dtpT locus in the DgisABCD-ggt background strain; sae sequenced and α hemolysin positive Resistant colony 1 (RC1) isolated from a bialaphos Kirby Bauer using WT S. aureus RC2; Same as HL1427 RC3; Same as HL1427 RC4; Same as HL1427 RC5; Same as HL1427 RC6; Same as HL1427 RC7; Same as HL1427 RC8; Same as HL1427 RC9; Same as HL1427 HL1419 HL1420 HL1421 HL1422 HL1423 HL1424 HL1425 HL1426 Staphylococcus epidermidis strains S. epidermidis 1457 WT Isolated from central venous catheter infection HL1406 HL1407 HL1408 HL1409 HL1010 HL1411 HL1412 HL1413 HL1414 Resistant colony 1 (RC1) isolated from a bialaphos Kirby Bauer using WT S. epidermis 1457 RC2; Same as HL1406 RC3; Same as HL1406 RC4; Same as HL1406 RC5; Same as HL1406 RC6; Same as HL1406 RC7; Same as HL1406 RC8; Same as HL1406 RC9; Same as HL1406 104 Table 3.4. Plasmids used in this study. Plasmid Description pKK22 pKK22- dtpT pKOR1 pKOR1- DdtpT Derivative of the naturally occurring S. aureus plasmid LAC- p01. Maintains stability in S. aureus without antibiotics. Linearized for Gibson assembly using primers PK85 & PK86. pKK22 expressing the WT S. aureus dtpT allele under native promoter conditions. Plasmid generated using primers PK27 and PK28. Temperature sensitive vector for generating clean deletions in S. aureus. pKOR1 harboring 1 Kb upstream & 1 Kb downstream of dtpT to generate a clean deletion. Reference (153) This study (213) This study 105 Table 3.5. Primers used in this study. Primer NE Martn-ermR* NE Buster* HL267 PK126 PK125 PK85 PK86 PK27 PK28 PK102 PK103 PK104 Description Used to confirm Tn insertions on plus strand Used to confirm Tn insertions on minus strand Used to confirm Tn insertion in dtpT (SAUSA300_RS03825) Confirming gisABCD-ggt deletion Confirming gisABCD-ggt deletion pKK22 amplification for Gibson assembly pKK22 amplification for Gibson assembly Amplification of dtpT from JE2 for Gibson assembly with pKK22 Amplification of dtpT from JE2 for Gibson assembly with pKK22 To amplify 1kb upstream of dtpT to generate pKOR1-DdtpT using NotI restriction cut site. Pair with PK105 to PCR sew To amplify 1kb upstream of dtpT to generate pJET1.2-DdtpT. Has homology with PK104 for PCR sewing before putting into pJET1.2 To amplify 1kb downstream of dtpT to generate pJET1.2-DdtpT using KpnI restriction cut site. Has homology with PK103 for PCR sewing before cloning into pJET1 106 Sequence (5’ → 3’) CTCGATTCTATTAACAAGGG GCTTTTTCTAAATGTTTTTTAAGTAA ATCAAGTAC CAACACTTCCGATAATAAGACC TCAAAGCTGGCGATGATGG TCAGTTGTTGGATCAGATGAGC GCGGCCGCTAGCCTAGGAGC ATCGCCTGTCACTTTGCTTGATATA TGA CAAGCAAAGTGACAGGCGATTTTTC CTAACTTATTGGTGTG GCTCCTAGGCTAGCGGCCGCTTAA CGTATACCTTTCATCG GCGGCCGCTTTTCACAGCAATACTT GG GCCAACAATAGTATACATCCCATCC TTTC GGATGTATACTATTGTTGGCCTAAT TCAAAAAAC Table 3.5 (cont’d) To amplify 1kb downstream of dtpT to generate pJET1.2-DdtpT using KpnI restriction cut site. Pair with PK102 for PCR sewing. To amplify the deleted dtpT region for PCR sequence confirmation To amplify the deleted dtpT region for PCR sequence confirmation saeS amplification saeS amplification GGTACCTCCAATTCATGCTATCACG CACATTATATTGAAGTCTGG AACACCGTTTATAAGTTCG GCTTTACAACATATACCATCACAAC TG AGCCCTCATTAATGGGAGCTTC PK105 PK116 PK117 TB96 TB97 107 FIGURES Figure 3.1. The di-tripeptide transporter, DtpT, supports Staphylococcus aureus growth on GSH as a source of nutrient sulfur. WT, ΔgisABCD-ggt (Δgis), ΔdtpT, ΔgisABCD-ggt ΔdtpT, and their respective dtpT complements were cultured in chemically defined medium (CDM) supplemented with either 50 µM or 750 µM GSH (A and B, respectively), 25 µM cystine (CSSC; C), or tryptic soy broth (TSB; D). WT S. aureus maintaining an empty pKK22 vector (EV) cultured in CDM lacking a viable sulfur source denotes the no sulfur control. Each complementation strains express, under native promoter conditions, the WT dtpT allele from the pKK22 vector (pdtpT). The mean and ± 1 standard error of three biological replicates is depicted. 108 Figure 3.2. The γ-peptide confers specificity for GisABCD-supported growth on GSH. Growth assessments in CDM of WT, ΔgisABCD-ggt, ΔdtpT and the ΔgisABCD-ggt, ΔdtpT double transport mutant on glutamate-cysteine-glycine (ECG) tripeptide as the provided sulfur source. This GSH analog harbors and α-peptide bond between Glu and Cys rather than the γ-peptide found in GSH. Strains were cultured in a chemically defined medium supplemented with either 25 µM CSSC (A), 50 µM GSH (B), 50 µM, 150 µM, or 300 µM ECG (C, D, E, respectively). WT cultured in CDM lacking a sulfur source acts as the no sulfur control. Curves are the mean of three biological replicates with error bars denoting the ± 1 standard error. 109 Figure 3.3. Cysteinyl-glycine (CysGly) is a viable sulfur source for Staphylococcus aureus in a DtpT-mediated fashion. A) growth kinetics of WT, ΔgisABCD-ggt (Δgis), ΔdtpT, ΔgisABCD-ggt ΔdtpT, and the dtpT complement strains in CDM supplemented with 25 µM CysGly. pKK22 is the empty vector (EV) and pKK22-dtpT, expressing WT dtpT allele under native promoter conditions, is denoted as pdtpT. Each curve represents the means of three biological replicates with error bars indicating the standard error. WT EV cultured in CDM lacking a viable sulfur source designates the no sulfur control. B) Percent growth using endpoint OD600 values of clinical CF isolates grown in CDM supplemented with 25 µM CysGly. The average of four biological replicates are represented with error bars representing ± 1 standard error of the mean. 110 (orange), Caryophanaceae Figure 3.4. Distribution of DtpT homologs across Gram-positive bacteria. Phylogenetic reconstruction of DtpT homologs (n=3,649). Homologs are defined as those proteins sharing ³50% coverage and ³40% when aligned against S. aureus DtpT. Host species are grouped into families with each family containing ≥50 representatives. Each family is designated with its own unique color depicted within the circular tree as follows: (brown), Bacillaceae Lactobacillaceae (salmon), Leuconostocaceae (light grey), Listeriaceae (light blue), Micrococcaceae (dark grey), Paenibacillaceae (yellow), Pseudonocardiaceae (light green), Streptococcaceae (royal blue), Streptomycetaceae (dark green). Families with <50 representatives were grouped together into one category: Others (black). The Staphylococcaceae (pink) and Staphylococcus epidermidis (lavender) split out as their own sub-families for emphasis. Clades were identified by displaying the DtpT homologs (n=3,649) as a radial tree (upper left). (purple) had Staphylococcus aureus Enterococcaceae family (red), 111 Figure 3.5. DtpT contributes to Staphylococcus aureus fitness during murine systemic infection. Bacterial burdens in the heart, liver, and kidneys of 8-week-old Balb/cJ female mice inoculated WT or dtpT::Tn. The limit of detection is 100 CFU and is represented with a dotted, horizontal line. Statistical significance within each organ was determined as follows: first the presence or absence of normal distribution was assessed with the Shaprio-Wilk test which returned the following P-values: WTheart = 0.02415, dtpTheart = 0.001623, WTliver = 0.6531, dtpTliver = 0.1715, WTkidney = 0.002334, dtpTkidney = 0.01517. From this it was determined that the WT and dtpT::Tn liver Tables were normally distributed and thus an unpaired t-test with Welch’s correction was conducted (see figure for P-value). All other Tables were determined as not normally distributed and the Mann- Whitney t-test was employed (see figure for P-values). Solid, horizontal lines represent the mean with error bars being ± 1 standard error of the mean. 112 Figure 3.6. Staphylococcus aureus sensitivity to the tripeptide antibiotic, bialaphos, is glutamine- and DtpT dependent. The Kirby Bauer assay was preformed utilizing JE2 WT and the ΔdtpT mutant. WT harbors the empty pKK22 vector (EV) while ΔdtpT contains either EV or the pdtpT complement in which dtpT is expressed under native promoter conditions. A) Representative photo of each bialaphos Kirby Bauer assay 113 Figure 3.6 (cont’d) using cells plated on CDM-trimethoprim agar plates (CDM-TMP) containing either 0 µM (left) or 500 µM Gln as well as tryptic soy agar (TSA). B) Quantification of the Kirby Bauer zone on inhibition. The limit of detection (6 mm) is defined by a horizontal, dashed line. Statistical analysis was conducted using a 2-way ANOVA using Tukey’s multiple comparison test. P-values are represented as follows: * = 0.0409, **** = <0.0001. C) Enumeration of the Kirby Bauer resistant colonies. An unpaired t-test returned a non- significant P-value of 0.3061. B and C present the mean of three biological replicates ± 1 standard error of the mean. 114 Figure 3.7. Staphylococcus aureus develops varying resistance to bialaphos due to dtpT mutation. A) Pictural representation of S. aureus WT and the nine bialaphos resistant colonies (RC1-9) when challenged against bialaphos in a Kriby Bauer assay. B) Quantification of the Kirby Bauer zone of inhibition. The limit of detection (6 mm) is denoted with a horizontal, dashed line. C) Enumeration of resistant colonies in the Kirby Bauer assays. For B and C, the mean of three biological replicates ± 1 standard error of the mean is shown. D) To-sale depiction of dtpT mutations in the bialaphos RCs as defined by whole genome sequencing. Red is to denote that RC9 has the same mutation as RC3. The blue line indicates a second mutation in dtpT for RC4. Please see Table 3.1 for specific information regarding mutations. Image created with BioRender.com. 115 Figure 3.8. Staphylococcus epidermidis 1457 harbors a dtpT homolog, B4U56_10070, that supports growth on both GSH and the newly identified sulfur 116 Figure 3.8 (cont’d) source, CysGly. A) Representative pictures of bialaphos Kirby Bauer on TSA (specified) and CDM with 2 mM Gln (specified) or no Gln (all other pictures, not specified). In the top row, the first three pictures (starting from the left) are WT S. epidermidis 1457. Bialaphos resistant colonies (RC) 1-9 are indicated in the appropriate picture. Bialaphos Kirby Bauer zones of inhibition (ZOI; B) and RCs that arose in the ZOI (C) were quantified. The limit of detection (6 mm) is defined by a horizontal, dashed line (B). D) Position specific location of the mutations in B4U56_10070 in each RC as defined by whole genome sequencing. Refer to Table 3.2 for mutation information. Image created with BioRender.com. E-G) Growth kinetics of S. epidermidis 1457 WT and RC1-9 on CDMmod supplemented with either 75 µM CSSC (C), 75 µM CysGly (D), or 750 µM GSH (E). Both the Kirby Bauer and growth curve assays (B, C, E-F) were performed in biological triplicate with the mean and ± 1 standard error of the mean being presented. 117 CHAPTER 4: Employing MALDI-IMS to visualize the host-pathogen nutritional sulfur interface during Staphylococcus aureus systemic infection 118 ABSTRACT The opportunistic pathogen Staphylococcus aureus persistently colonizes 20% of the population and is a leading cause of infections such as bacteremia and infective endocarditis. Successful proliferation of S. aureus throughout numerous host tissue depends on an ability to develop antibiotic resistance and procure essential nutrients from diverse metabolite forms within the local environment. With increasing knowledge regarding S. aureus host-sulfur acquisition, confirming the in vivo relevance of these pathways is important. This pioneering study examines the host-pathogen nutritional sulfur interface using Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) on systemically infected murine kidneys. In doing so, we have confirmed that three S. aureus sulfur sources—reduced and oxidized glutathione as well as cysteinyl-glycine—are present in this organ during infection. Additionally, the capacity of MALDI-IMS to identify biologically relevant sulfur sources is demonstrated with the mixed cysteine-glutathione disulfide, a metabolite particularly abundant within healthy or S. aureus colonized kidneys. Growth of S. aureus on cysteine-glutathione disulfide in vitro is supported by the cysteine/cystine transporters TcyABC and TcyP as well as the GisABCD-Ggt reduced/oxidized glutathione import and catabolism system. Lastly, the ability of GisABCD-Ggt to support S. aureus proliferation on GSH derivatives S- nitrosoglutathione and S-lactoylglutathione is established. In total, this investigation harnesses the unique capabilities of MALDI-IMS to better comprehend how this formidable pathogen impacts host sulfur metabolism during infection. 119 INTRODUCTION Methicillin Resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and leading cause of hospital acquired infections in the United States (86, 87). This is due to the propensity of S. aureus to colonize numerous host organs and rapidly develop antibiotic resistance. Successful proliferation within tissue requires S. aureus acquisition of essential nutrients such as sulfur that are found within host metabolites (134). Though our understanding of S. aureus host-sulfur procurement is increasing, we lack insight regarding the distribution and abundance of these sulfur containing compounds during infection. Targeted metabolomics using mass spectrometry (MS) is a powerful tool commonly used to detect and quantify metabolites within a sample (e.g., tissue) (220). For assessing host-pathogen interfaces, though, MS alone is limiting when both quantity and distribution of a metabolite is also desired. With recent advances in technology, descriptive spatial localization and relative quantification of a compound is possible with matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) (221). Using this technique, spectra are obtained at discrete locations across a sample. From these spectra an image is generated using the intensity signal of a metabolite. In S. aureus, MALDI-IMS has provided invaluable data regarding host redistribution of nutrients such as calcium, manganese, and zinc during systemic infection (185). Additionally, MALDI-IMS can assess the abundance of either host (e.g., calprotectin) or S. aureus (e.g., siderophores) proteins, reinforcing the notion that this tool is well equipped for examining both sides of a host- pathogen interaction (185, 222). 120 Here, MALDI-IMS was utilized to gain preliminary insights on host sulfur distribution during S. aureus systemic infection of the murine kidney. We discover that several sulfur sources identified for this pathogen in vitro are present within healthy tissue and experience an increased relative abundance and/or redistribution upon infection. Furthermore, the efficacy of MALDI-IMS is exemplified by its discovery of a novel sulfur source in S. aureus, known as cysteine-glutathione mixed disulfide (CSSG). Growth of S. aureus on CSSG requires the collective presence of the GSH importer and γ-glutamyl transpeptidase (GisABCD-Ggt) machinery (89) as well as the cysteine (Cys)/cystine (CSSC) transporters, TcyABC and TcyP (90). Lastly, we demonstrate that two other GSH derivatives, S-nitrosoglutathione (GSNO) and S-lactoylglutathione (SLG) support the S. aureus nutritional sulfur requirement in a GisABCD-Ggt mediated fashion. Collectively, this body of work broadens our knowledge regarding the levels of redundancy employed by S. aureus—from the acquisition of structurally similar compounds to the use of multiple transporters for a single metabolite—to ensure that the nutritional sulfur status is maintained. RESULTS Matrix assisted laser desorption/ionization imaging mass spectrometry reveals the presence of known and putative S. aureus sulfur sources in the systemically infected murine kidney. Prior studies have revealed several host-derived sulfur sources reinforcing the S. aureus nutritional sulfur requirement in vitro (88–90). Our work has additionally observed several sulfur associated importers sustaining maximal propagation of S. aureus JE2 in the heart and/or liver (TcyP (89) and DtpT, see Chapter 3) during murine systemic infection. Nevertheless, the distribution of sulfur metabolites associated 121 with S. aureus within healthy and infected host tissue remains unknown. To begin addressing this, matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was conducted on both mock infected mice and those inoculated with the WT USA300 LAC derivative, JE2 (Fig. 4.1). Kidney samples were used for downstream analysis given that WT colonization in this environment consistently results in abscess formation, a hallmark of S. aureus infection; this allows for easy discernment between mock and infected tissues using hematoxylin and eosin (H&E) staining (Fig. 4.1A). MALDI-IMS on kidney samples assessed the distribution of three known sulfur sources: reduced and oxidized glutathione (GSH and GSSG, respectively) (88, 89) as well as its breakdown product, cysteinyl-glycine (CysGly) (see Chapter 3). In a healthy kidney, both GSH and GSSG are present—here each metabolite has the highest abundance in the medulla but shows moderate levels throughout the tissue during S. aureus infection (Fig. 4.1B and 4.1C, respectively). However, during systemic infection the kidney experiences an increase in abundance throughout the kidney (Fig. 4.1B and 4.1C, respectively). Interestingly, the presence of a mixed GSH disulfide species, cysteine-glutathione disulfide (CSSG), displayed high abundance in the renal cortex (Fig. 4.1D). During S. aureus infection, CSSG increased its presence by spreading throughout the tissue sample (Fig. 4.1D). Ionization of CysGly, which is a breakdown product of GSH in the γ- glutamyl cycle, exhibited improved results when a different matrix was used compared to the one employed for GSH, GSSG, and CSSG (details in Materials and Methods). Consequently, distinct tissue samples from both mock and infected kidneys were used for H&E staining (Fig. 4.1E) and for the MALDI-IMS visualization of this metabolite. In the mock-infected murine kidney, CysGly is observed within the renal cortex with a slight 122 redistribution occurring towards the center during S. aureus infection (Fig. 4.1F). Collectively, these data indicate that sulfur sources identified in vitro for this pathogen can be found in at least one tissue environment during systemic infection. S. aureus GisABCD, TcyABC, and TcyP support the use of CSSG as a source of nutritional sulfur. Given that all GSH derivates tested thus far (i.e., GSSG and CysGly) are S. aureus sulfur sources, we predicted that CSSG would also support nutritional demands for this pathogen. Indeed, supplementation of a chemically defined medium (CDM) with 12.5 µM CSSG as the only viable sulfur source promotes robust growth of WT (Fig. 4.2A); we next ascertained the S. aureus machinery supporting this phenotype. Due to the mixed disulfide nature of CSSG, we hypothesized that several transporters are involved with growth on this nutrient. We first investigated the gisABCD- ggt genetic region, where gisABCD encodes a GSH/GSSG ABC transporter and ggt produces a g-glutamyl transpeptidase cleaves the g-peptide bond of these metabolites (89). Proliferation of a gisABCD-ggt clean deletion mutant (denoted as Δgis) on CSSG is not impaired, potentially implying the presence of redundant systems that support growth on this nutrient (Fig. 4.2A). Considering the Cys residue of CSSG, the Cys/CSSC transporters TcyABC and TcyP (90) were next assessed. Transposon (Tn) inactivating mutations in either transporter (tcyA::Tn or tcyP::Tn) fails to impact S. aureus propagation on CSSG (Fig. 4.2A). Given that the single transporter mutants all displayed WT-like growth, we investigated the growth kinetics of strains deficient for two of the three importers. A Δgis tcyA::Tn mutant displayed in a modest growth defect compared to WT while the Δgis tcyP::Tn strain did not (Fig. 4.2A). Interestingly, the tcyA::Tn tcyP::Tn mutant exhibited the growth 123 greatest impairment of all the double mutants tested (Fig. 4.2A). These observations indicate that all three transporters support, at different levels, S. aureus growth on CSSG. This is further indicated by the fact that a Δgis ΔtcyA tcyP::Tn triple transport mutant fails to proliferate in CSSG (Fig. 4.2A). Furthermore, all phenotypes are sulfur specific as each mutant strain proliferates to WT-like levels when cultured in CDM harboring sodium thiosulfate (sTS) as the sulfur source (Fig. 4.2B). S. aureus GisABCD drives the use of GSH-derived species to sustain the nutritional sulfur requirement in vitro. Host GSH derivatives are not limited to GSSG, CysGly, and CSSG. S-nitrosogluathione (GSNO) forms when GSH indirectly reacts with nitric oxide (NO) (223). GSNO acts as a signaling molecule by transferring the NO to target proteins, effectively altering their function, and is found in the high picomole to low nanomole range human plasma (224, 225). Consistent with other GSH related metabolites evaluated, supplementation of CDM with 75 µM GSNO promotes WT proliferation if a functional gisABCD-ggt system is present (Fig. 4.3A). S- lactoylglutathione (SLG) is also found within the host environment as an intermediate in the methylglyoxyl detoxification pathway and is present at low micromolar concentrations (~12-16 µM) in human blood (226). As expected, SLG supports growth of S. aureus when provided at low micromolar concentrations (i.e., 50 µM) in CDM; use of this nutritional sulfur source largely depends on the GisABCD-Ggt system (Fig. 4.3B). Collectively, GSNO and SLG provide further evidence supporting the notion that GSH derivatives can be targeted as nutritional sulfur sources for S. aureus during infection. 124 DISCUSSION This study of MALDI-IMS on the host-pathogen nutritional interface visualizes the distribution and relative abundance of three established sulfur sources (GSH, GSSG, and CysGly) during systemic S. aureus infection. Interestingly, both GSH and GSSG increased in relative abundance during infection (Figure 4.1B and C). This could result from the host immune response to S. aureus. Neutrophils combat S. aureus by delivering reactive oxygen species (ROS) to the site of infection and resulting have high intracellular levels of GSH to modulate internal ROS (227). As these neutrophils die, this GSH pool is likely released to the extracellular environment, becoming accessible to S. aureus. One might predict that this relative abundance of GSH and GSSG in the kidney might increase even more in mice infected with the Δgis mutant; this would suggest that S. aureus is importing and catabolizing these nutrients during infection. From the GSH and GSSG MALDI-IMS data alone, it is also tempting to speculate on the state of the host sulfur pool in environments where S. aureus sulfur associated transporters contribute to maximal proliferation (e.g., heart (90) and liver; Chapter 3). Will MALDI-IMS reveal depletion of the metabolites associated with TcyP (i.e., Cys/CSSC) or DtpT (GSH, CysGly)? As discussed previously, would inactivating these transporters replenish the respective sulfur pools during systemic infection? Overall, MALDI-IMS has provided further support for the comprehensive characterization of sulfur acquisition systems in S. aureus in order to establish a clear understanding of the host-pathogen nutritional sulfur interface. This investigation additionally highlights the robust utility of MALDI-IMS as a tool for unveiling novel sulfur sources for pathogens. Indeed, CSSG is highly abundant in both the mock and systemically infected kidney. Very little is known about the contributions of 125 CSSG to mammalian physiology, but it is ubiquitous within cells and can protect mice against acetaminophen-induced hepatotoxicity (228). Here CSSG is given a new function as a potent source of nutrient sulfur for S. aureus, as particularly low micromolar concentrations of this disulfide elicits robust proliferation of the bacterium. This could be related to the fact that, once reduced, Cys—the crux of sulfur distribution within cells—is immediately accessible to donate its sulfur atom to other biological processes (e.g., Fe-S cluster assembly). Interestingly, two distinct types of sulfur transporters are associated with S. aureus proliferation on CSSG as a nutritional sulfur source: GSH/GSSG (GisABCD) and Cys/CSSC (TcyABC and TcyP). Except for CSSG, all substrates implicated in the GisABCD system (GSH/GSSG, GSNO, SLG) share common tripeptide and γ-peptide bond features, as discussed in Chapter 3. CSSG deviates from this pattern as it involves an amino acid, Cys, forming a disulfide bond with GSH. This suggests that the substrate binding protein (SBP), GisA, may possess an active site primarily recognizing a single molecule of GSH, with limited or no involvement in recognizing the molecule disulfide-bonded to GSH. A similar concept could be applicable to TcyA, the substrate binding protein (SBP) of the TcyABC complex, and TcyP—here recognition of Cys in CSSG takes precedence, with GSH playing a minimal or negligible role in substrate recognition. This inference is drawn from the understanding that sulfur sources related to both TcyABC and TcyP primarily involve Cys and its close derivatives (such as CSSC, N- acetylcysteine, and homocystine) (90). The above scenario only applies to CSSG if this metabolite is transported into the cell as a disulfide, implying that the reductase is intracellular (Fig. 4.4A). Supporting this model is the knowledge that Ggt, a cytoplasmic protein, can cleave the g-peptide bond of 126 GSSG, suggesting that GSSG is reduced in this space (89). Indeed, most reductases are found within the cytoplasm, helping to drive the cytoplasmic redox status of the cell (229). However, in Gram-negative bacteria the Dsb system functions to introduce (DsbA, DsbB) and correct (DsbC, DsbD) protein disulfide bonds in the periplasm; S. aureus only encodes DsbA, a protein whose ability to regain the oxidative state is predicated to rely upon extracellular oxidants (230). One might wonder whether CSSG is involved in that process. With such a model, reduction of CSSG would occur before transportation through GisABCD, TcyABC, and TcyP (Fig. 4.4B). Promoting this model is the notion that neither TcyABC nor TcyP are associated with tripeptide import in bacteria, including S. aureus (90, 93, 231). There is one study in Streptococcus mutans where a GSH SBP (GhsT) acts in concert with the TcyBC component to import the metabolite into the cell (232). It is noteworthy to point out that, in the current study, the TcyBC components are intact in the Δgis ΔtcyA tcyP::Tn mutant, suggesting that there is not an additional SBP that binds CSSG for import through the TcyBC complex. Future studies looking at the growth of a dsbA mutant on CSSG as the only sulfur source will shed light on whether S. aureus utilizes an extracytoplasmic protein to reduce sulfur sources before transportation into the cytoplasm. Lastly, the implication of CSSG as a GisABCD substrate led us to hypothesize that other GSH derivatives are viable S. aureus sulfur sources. GSNO and SLG reinforce this notion as both can be utilized by S. aureus to meet the nutritional sulfur requirement. Importantly, growth is supported by the GisABCD-Ggt system when either metabolite is provided at a concentration above values reported for human blood (224–226). Determining whether GSNO and SLG are immediate Ggt substrates or if the respective 127 NO and D-lactate groups need to be removed before Ggt catalysis represents areas of future investigation. Overall, this pioneering work provides insight into the host's sulfur status during systemic infection and emphasizes the valuable role of MALDI-IMS in expanding our understanding of the sulfur metabolism strategies employed by this formidable pathogen. ACKNOWLEDGMENTS We sincerely thank Boone Prentice and Justin Ellenburg for conducting the MALDI-IMS and for their thoughtful insights on this work. Each transposon mutant was obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH, and the Nebraska Transposon Mutant Library (NTML) Screening Array NR-48501. Funding for this work was provided by the National Institutes of Health R01 AI139074 and R21 AI142517. MATERIALS AND METHODS Strains, media, and growth conditions. All strains, plasmids, and primers used throughout the course of this study can be found in Tables 4.1, 4.2 and 4.3, respectively. JE2, a derivative of community acquired USA300 LAC, is the WT S. aureus strain for this body of work (164). Correct Tn location and deletions were determined using PCR. Plasmids were generated via Gibson assembly in the Escherichia coli DH5a strain before being transferred to S. aureus RN4220 to obtain appropriate vector methylation patterns that would permit transformation into the appropriate JE2 background strain. Note that all plasmids were confirmed using Plasmidsaurus whole plasmid sequencing. Deletion of the tcyA gene was performed following a well-defined allelic exchange protocol using the pKOR1-mcs plasmid donated from the laboratory of Taeok Bae (213). Each strain was 128 cultured in 5 mL tryptic soy broth (TSB) overnight at 37°C, 225 rpm. The base chemically defined medium (CDM) was prepared as previously described with slight modifications (149, 150). For the full recipe, please refer to Chapter 2 Materials and Methods. CDM harboring 5 mg mL-1 of freshly prepared glucose was supplemented with either no sulfur source, 12.5 mM cysteine-glutathione disulfide (CSSG), 50 mM sodium thiosulfate (sTS), 75 mM S-nitrosoglutathione (GSNO), or 50 mM S-lactoylglutathione (SLG). For the stock sulfur sources CSSG, sTS, GSNO, SLG, and glucose were dissolved in deionized water before filter sterilization. Murine model of systemic infection. A WT TSB overnight was sub-cultured 1:100 into 5 mL TSB and grown for an additional 3 h. The culture was pelleted at 4°C, 4000 rpm, before being washed into 12 mL of Dulbecco's Phosphate Buffered Saline (DPBS; no CaCl2 or MgCl2) and normalized to an OD600 = 0.4. Eight-week-old Balb/cJ female mice (Jackson Laboratories) were then anesthetized with avertin before being retro-orbitally infected with 107 CFU. After 96 hpi, mice were euthanized via CO2 inhalation. The heart, liver, and kidneys were snap-frozen on dry ice and sent to the Prentice laboratory at University of Florida for imaging. Infections were performed at Michigan State University under the principles and guidelines described in the Guide for the Care and Use of Laboratory Animals (218). All animal work followed the protocol approved by Michigan State University Institutional Animal Care and Use Committee (IACUC): PROTO202200474. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI- IMS). Frozen tissue samples were sectioned at 10 µm using a Leica CM3050S Cryostat. One section of PBS inoculated mouse kidney (control) and one section of S. aureus WT- 129 infected kidney were then thaw mounted onto an indium-tin oxide (ITO) coated slide. A matrix was then applied depending on the desired molecules to be ionized: 9- aminoacridine (9AA; prepared using 90% methanol) was used for GSH, GSSG, and CSSG tissue samples while those used for CysGly observation used dihydroxybenzoic acid (DHB). The designated matrix was applied using an M5 robotic sprayer (HTX Technologies) at 85°C with a 0.11 mL/min flow rate. Nozzle velocity was set to 700 mm/min with a 2 mm track spacing and crisscross pattern with the nitrogen pressure being set at 10 psi (2 L/min flow rate). IMS was conducted using a 7T solariX FT-ICR mass spectrometer (Bruker Daltonics) in negative ion mode. Continuous Accumulation of Selected Ions (CASI) was employed to improve ion signal over the mass window of m/z 280-620. All IMS experiments were performed at 150 µm spatial resolution. Mass resolution at m/z 400 is 67,000 measured by FWHM. Image processing was done utilizing flexImaging and SCiLS software (Buker Daltonics). Tissue staining with Hematoxylin and eosin (H&E). After MALDI-IMS, the matrix was removed using a continuous flow of 100% ethanol (20 sec). Samples were then exposed to 95% ethanol (30 sec), 70% ethanol (30 sec), and then washed in Mili-Q water (30 sec) before hematoxylin solution was applied for 2 min. From there the tissues were washed in Milli-Q water (20 sec), 70% ethanol (30 sec), 95% ethanol (30 sec), and then eosin solution was administered (1 min). Additional applications of 95% ethanol (30 sec) and 100% ethanol (30 sec) preceded Xylene application (2-2.5 min). Once completed, a cover slip was added, and the tissue were imaged using an Azio imager M2 optical microscope (Zeiss). 130 96-well plate growth curves. Overnights were spun at 4°C, 4000 rpm, washed in 5 mL phosphate buffered saline (PBS), and then normalized to an optical density at 600 nm (OD600) equal to 1. Normalized cells were sub-cultured at a 1:100 ratio into each growth condition. Using an Epoc2 Biotek microplate spectrophotometer (37°C, continuous shaking), the OD600 was observed every hour for 18 h in a 96-well round bottom plate. For each condition, a strain was inoculated into three wells (i.e., technical triplicate). After being blanked the technical triplicates were averaged, with the average representing a single biological replicate. All growth curves were performed independently three times. Data visualization. Growth curves were generated using GraphPad Prism software version 10.0.0 (153). All finalized figures were generated using the open source Inkscape 1.2.2 (b0a8486, 2022-12-01). Available at: https://inkscape.org. 131 Table 4.1. Strains used in this study. TABLES Description Strain Staphylococcus aureus strains S. aureus WT DgisABCD-ggt tcyA::Tn tcyP::Tn DgisABCD-ggt tcyA::Tn DgisABCD-ggt tcyP::Tn DgisABCD-ggt DtcyA tcyP::Tn JE2, derivative of USA300 LAC Clean deletion of SAUSA300_0200-0404 genetic region B. aurealis Tn insertion at genome position B. aurealis Tn insertion at genome position B. aurealis Tn insertion at genome position in the DgisABCD-ggt background strain B. aurealis Tn insertion at genome position in the DgisABCD-ggt background strain Clean deletion of the tcyA locus in the DgisABCD-ggt background strain; sae sequenced and α hemolysin positive Reference (164, 166) (89) (90) (90) This study This study This study Table 4.2. Plasmids used in this study. Plasmid Description pKOR1 pKOR1- DtcyA Temperature sensitive vector for generating clean deletions in S. aureus. pKOR1 harboring 1 Kb upstream & 1 Kb downstream of tcyA to generate a clean deletion. Reference (213) This study 132 Table 4.3. Primers used in this study. Primer NE Martn-ermR* NE Buster* PK126 PK125 HL130 HL211 JLD15 JL16 JLD25 JLD26 JLD32 JLD33 TB96 TB97 Description Used to confirm Tn insertions on plus strand Used to confirm Tn insertions on minus strand Confirming gisABCD-ggt deletion Confirming gisABCD-ggt deletion Confirming the Tn insertion in tcyA Confirming the Tn insertion in tcyP Amplifying 1kb downstream of tcyA for Gibson assembly into pKOR1 Amplifying 1kb downstream of tcyA for Gibson assembly into pKOR1 Amplifying 1kb upstream of tcyA for Gibson assembly into pKOR1 Amplifying 1kb upstream of tcyA for Gibson assembly into pKOR1 To amplify the deleted tcyA region for PCR sequence confirmation To amplify the deleted tcyA region for PCR sequence confirmation saeS amplification saeS amplification Sequence (5’ → 3’) CTCGATTCTATTAACAAGGG GCTTTTTCTAAATGTTTTTTAAGTAA ATCAAGTAC TCAAAGCTGGCGATGATGG TCAGTTGTTGGATCAGATGAGC CGTCAATAAATATAAGTTGCTAGC CGAAGCAAATATCACGACAGC CATATGATGAGTTCACAAAAAAAGA AAATTAGT ATAAAAATTAGATACGATGTTGCAT GGTTATC TTCTATTTGAAGAATATATCTCCTTA TTCTTATTATTCTAATC CGGAACCGGTACCAATGGATAACTA CAATTAAAGTACCTATTGATTTTATT TC ATGACCATTGTCATGCCTTCATT TCTTAGGTAATTACTCGGCGG GCTTTACAACATATACCATCACAAC TG AGCCCTCATTAATGGGAGCTTC 133 FIGURES Figure 4.1. MALDI-IMS of the systemically infected kidney reveals a potential nutrient sulfur source for Staphylococcus aureus, cysteine-glutathione mixed disulfide (CSSG). A) Hematoxylin and eosin (H&E) stain of PBS (mock) inoculated kidney (left) and S. aureus WT-infected (right) murine kidney. Sections from these kidney samples were used for identification of metabolites that harbor glutathione (GSH). MALDI- IMS for GSH (B), oxidized glutathione (GSSG; C), and CSSG (D) are depicted. E) H&E stain of mock-infected (left) and WT-infected (right) kidney—sections from these kidneys were used to assess cysteinyl-glycine relative abundance and distribution (CysGly; F). 134 Figure 4.1 (cont’d) For the H&E stains (A and E), representative S. aureus abscesses are denoted with black arrowheads. These images were produced in collaboration with the Prentice laboratory at the University of Florida. 135 Figure 4.2. Cysteine-glutathione disulfide is a sulfur source for Staphylococcus aureus. Strains were cultured in chemically defined medium (CDM) supplemented with either 12.5 µM cysteine-glutathione disulfide (CSSG) (A) or 50 µM sodium thiosulfate (sTS) (B) as the sulfur source. WT S. aureus cultured in CDM lacking a viable sulfur source represents the no sulfur control. Each line represents the mean of three independent trials while the vertical bars depict ±1 standard error of the mean. 136 Figure 4.3. Staphylococcus aureus can meet the nutritional sulfur requirement using S-nitrosoglutathione and S-lactoylglutathione as sources of nutritional sulfur. S. aureus WT and the DgisABCD-ggt mutant (denoted Dgis) were propagated in CDM harboring 75 µM S-nitrosoglutathione (GSNO) (A) or 50 µM S-lactoylglutathione (SLG) (B). Each line depicts the mean of three biological replicates ±1 standard error of the mean. SLG data courtesy of Joelis Lama Díaz. 137 Figure 4.4. Potential mechanisms of cysteine-glutathione disulfide transportation. GisABCD is a reduced and oxidized glutathione (GSH/GSSG) importer (89) while TcyP and TcyABC have been associated with cysteine and cystine transportation (Cys/CSSC) (90). This study now implicates all three transporters with S. aureus growth on cysteine- glutathione disulfide (CSSG). However, it is unknown whether CSSG is imported as a disulfide and reduced in the cytoplasm (A) or if an extracytoplasmic enzyme acts on CSSG, allowing reduced GSH and free Cys to be imported through their respective transporters (B). Image created with BioRender.com. 138 CHAPTER 5: Summary and Future Directions 139 SUMMARY In Chapter 1, we discuss the host sulfur pool, its influence on host well-being through sulfur metabolism, and the mechanisms employed by pathogens to acquire sulfur- containing metabolites. Cysteine (Cys) plays a pivotal role in the distribution of sulfur within cells, serving as the building block for numerous organic and inorganic compounds. These compounds collectively form the mammalian sulfur reservoir (134). The machinery responsible for synthesizing and breaking down these metabolites is present across various tissue types, each subject to intricate regulatory processes that govern metabolite levels and distribution (134). Consequently, one can view each tissue type as its own distinct sulfur pool. An illustrative example of this phenomenon is the γ-glutamyl cycle, characterized by its layered regulation that promotes the heightened expression of glutathione (GSH) synthesis enzymes in the liver, while GSH catabolism enzymes are less prevalent in this specific environment (134). Notably, imbalances in the γ-glutamyl cycle can lead to various health issues, such as an increased risk of cardiovascular diseases (related to γ-glutamyl transpeptidase and glutamate-cysteine ligase) or the development of hemolytic anemia (linked to GSH synthase) (134). Furthermore, pathogens like S. aureus have evolved mechanisms to import GSH into their cytoplasm (14, 89, 134, 232, 233). In fact, S. aureus possesses a characterized glutathione import system, GisABCD, as well as a γ-glutamyl transpeptidase (Ggt) system that transports GSH and initiates its breakdown for use as a sulfur source (89). Given the potential for altering sulfur metabolism to result in adverse outcomes within a normally healthy host, it becomes imperative to gain a comprehensive understanding of when, where, and how S. aureus impacts the host sulfur pool during infection. 140 As S. aureus encounters diverse sulfur environments during infection, Chapter 2 delves into how S. aureus physiology is altered when exposed to varying states of sulfur supplementation. Using RNAseq, we characterize the S. aureus transcriptional profile during sulfur starvation, a process predominantly governed by CymR-dependent genes, although a CymR-independent response is also observed. Strikingly, iron metabolism and oxidative stress genes were differentially expressed during sulfur starvation, prompting an investigation into the relationship between sulfur and these two pathways. In doing so, an additional role for GSH in S. aureus was discovered, where the free thiol of GSH provides protection against inhibitory concentrations of heme and hydrogen peroxide (H2O2) stress. Furthermore, we explore the S. aureus transcriptional response when comparing growth on various sulfur sources—such as organic Cys, GSH, oxidized GSH (GSSG), or inorganic thiosulfate (TS)—to cystine (CSSC). We demonstrate that two genes upregulated in the presence of TS, tsuA and tsuB, play a role in the ability of S. aureus to utilize TS as a source of nutritional sulfur, confirming that sulfur associated genes with altered expression under these conditions are indeed involved in sulfur metabolism. Chapter 3 explores the machinery responsible for ensuring sulfur acquisition in S. aureus, building upon our improved understanding of how S. aureus responds to extracellular sulfur. DtpT is a di- and tripeptide transporter that is controlled by the stationary phase regulatory protein CodY (91). A direct mutagenesis approach unveiled the significance of the DtpT in utilizing not only physiologically relevant concentrations of GSH but also a newly identified sulfur source, cysteinyl-glycine (CysGly). Moreover, our studies demonstrate that this transporter plays a role in maximizing the fitness of S. 141 aureus within the systemically infected murine liver. These findings position DtpT as an ideal model protein for investigating how sulfur transporters can be hijacked to inhibit staphylococcal proliferation. To this end, we observed that the toxic peptide antibiotic, bialaphos, is delivered into S. aureus through DtpT, a mechanism also noted in another clinically relevant species, Staphylococcus epidermidis. Further investigations revealed the contributions of the S. epidermidis DtpT homolog to the proliferation of this organism when utilizing GSH and CysGly as nutrient sulfur sources, providing additional evidence that these two metabolites are substrates of DtpT. We also investigated conservation of DtpT and found broad conservation among Gram-positive bacteria, including cysteinyl- glycine 3-methyl-3-sulfanylhexanol (S-CysGly-3M3SH) transporters like PepT found in Staphylococcus hominis (103). These findings highlight the complexity surrounding S. aureus sulfur metabolism wherein broadly conserved, promiscuous proteins can reinforce the nutritional status of a cell. Having investigated the properties surrounding acquisition of GSH and its derivative CysGly, Chapter 4 extends this narrative by delving into the host sulfur status during infection. MALDI-IMS emerged as an invaluable tool, shedding light on the presence of GSH, GSSG, and CysGly in both healthy and S. aureus-infected murine kidneys. Additionally, this technique unveils the substantial existence of a previously unrecognized sulfur source, cysteine-glutathione disulfide (CSSG), in the kidney. The growth of S. aureus on CSSG in vitro is intricately tied to the presence of GisABCD-Ggt, TcyABC, and TcyP. Lastly, this chapter drives home the importance of S. aureus GSH acquisition through the GisABCD system by identifying two other GSH derivatives that 142 support the S. aureus nutritional sulfur requirement: S-nitrosoglutathione (GSNO) and S- lactoylglutathione (SLG). FUTURE DIRECTIONS Chapters 2, 3, and 4 collectively represent significant progress in our understanding of S. aureus sulfur metabolism. These chapters also underscore the need for future experiments to comprehensively evaluate the impact of S. aureus redundancy during infection. Although the complete set of sulfur sources for S. aureus remains to be fully determined, we have gained substantial insights into the core sulfur transportation systems. This knowledge sets the stage for a more detailed examination of their roles during host colonization. Indeed, previous studies on TcyABC, TcyP, and GisABCD-Ggt (89, 90) have highlighted the challenges in elucidating the contributions of sulfur associated transporters to S. aureus colonization during systemic infection studies. In cases like TcyABC and TcyP, it required competition experiments against the wild type (WT) strain to reveal significant impairments, while the ΔgisABCD-ggt mutant exhibited no difference in bacterial burden compared to WT during mono-infection. Furthermore, even though DtpT impacts the ability of methicillin resistant S. aureus to achieve maximum proliferation within the liver during mono-infection, whether this is attributed to decreased import of GSH and/or CysGly rather than other di- and tripeptides remains to be determined. From these infection studies one might question the actual impact sulfur transporters have on S. aureus proliferation in vivo. However, it is essential to bear in mind that the host sulfur pool is highly diverse, as discussed in Chapter 1. Additionally, S. aureus employs multiple transporters to acquire various metabolites, as highlighted in 143 previous studies (89, 90) along with Chapters 3 and 4. In some instances, this pathogen even possesses redundant systems dedicated to obtaining just one of these sulfur sources. This evolutionary strategy essentially safeguards S. aureus by compensating for the loss of one transporter or sulfur source with the ability to access a range of other sulfur-containing compounds. Consequently, when bacterial burdens are evaluated (i.e., at 96 hours post-infection), any defects observed in a sulfur transportation mutant might be concealed by the activity of alternative acquisition routes. Therefore, it would be valuable to conduct studies where systemic infections are terminated at earlier time points, such as 24 hpi or 48 hpi, to assess whether sulfur transporters like TycABC, TcyP, or GisABCD impact the initial colonization of the host. Another avenue for investigation is to understand the collective impact of sulfur transporters during S. aureus systemic infection. Building on the findings from published work and Chapter 3, it would be intriguing to create a quadruple transport mutant (i.e., ΔgisABCD-ggt ΔdtpT ΔtcyA ΔtcyP) and evaluate the colonization abilities of this strain during a mono-systemic infection. We know from previous research that a tcyP::Tn mutant shows a competitive disadvantage compared to wild type (WT) JE2 in heart colonization (90). Therefore, any increased severity in the heart defect using the ΔgisABCD-ggt ΔdtpT ΔtcyA ΔtcyP would strongly implicate the importance of the TcyABC, GisABCD-Ggt, and DtpT systems for effective propagation in this organ. Increased severity in this context would likely manifest as a colonization defect during mono-infection. Similar outcomes would be anticipated in the liver, as the deletion of dtpT alone has been shown to impair growth in this environment. However, although a dtpT mutant results in significantly decreased colonization, the distribution of bacterial burdens 144 within individual mice is variable. For the anticipated decreased colonization in the ΔgisABCD-ggt ΔdtpT ΔtcyA ΔtcyP mutant, a narrower range of bacterial burdens is expected. This would suggest a more pronounced colonization defect due to the contributions of GisABCD-Ggt, TcyABC, and TcyP. Furthermore, we predict significantly reduced bacterial burdens in the kidneys. This expectation arises from the loss of all known transporters contributing to the acquisition of GSH, GSSG, CSSG, and CysGly, all of which are abundant during S. aureus infection in this organ. Conversely, if we observe WT-like colonization of the kidneys with the quadruple transport mutant during both mono-infection and competition against WT, it may suggest the utilization of inorganic sulfur within this environment. Indeed, thiosulfate production in the kidney can result from the actions of Cysteinesulfinate Decarboxylase (Figure 1.1 and Table 1.1). Thus, it would be of interest to investigate the impact of deleting both inorganic sulfur transporters—the known thiosulfate transporter, tsuA, and the predicted sulfonate importer ssuABC—in the ΔgisABCD-ggt ΔdtpT ΔtcyA ΔtcyP background. This particular strain is likely to exhibit unique growth profiles in vitro due to the loss of so many sulfur importers. However, it is worth noting that growth can still be supported by the unknown di- and tripeptide transporter identified in Chapter 3. Therefore, supplementing the culture medium with higher concentrations (e.g., >100 µM) of ECG, GSH, or CysGly as the nutritional sulfur source should stimulate the proliferation of the ΔgisABCD-ggt ΔdtpT ΔtcyA ΔtcyP ΔtsuA ΔssuABC strain in vitro. During systemic infection, we speculate that this sextuple transport mutant may result in bacterial burdens that are close to, if not below, the limit of detection in the heart and liver. At the very least, we expect significantly reduced colonization in the kidneys. These experiments would help confirm the key 145 players in S. aureus sulfur acquisition during systemic infection and contribute to identifying a hierarchy of contributors. In essence, by characterizing the colonization profiles of strains deficient in one to six transporters, we aim to elucidate which systems have a more substantial impact on S. aureus propagation in a host environment. As described in Chapters 3 and 4, the redundancy of S. aureus sulfur transporters serves as an evolutionary strategy to ensure the acquisition of various related metabolites derived from the host. With such a nuanced system, it is vital to describe the nutritional sulfur interface from the host perspective as well. How does the host response to infection impact the sulfur composition at the infection site? Do tissues infected with a sulfur transportation mutant (e.g., a ΔgisABCD-ggt ΔdtpT ΔtcyA ΔtcyP strain) exhibit differences in sulfur distribution, abundance, or composition compared to tissues infected with WT S. aureus? These questions can be addressed through the continued use of MALDI-IMS, especially once we establish a baseline for how WT S. aureus influences the host sulfur pool during systemic infection. Indeed, examining the infection site from this perspective will undoubtedly yield a more comprehensive understanding of our systemic infection data. For instance, in Chapter 4, we illustrate the presence of at least four known sulfur sources (i.e., GSH, GSSG, CysGly, and CSSG) in the S. aureus-infected kidney, and we identify four transporters associated with the import of these metabolites (i.e., GisABCD- Ggt, DtpT, TcyABC, and TcyP). These insights help explain why kidney defects have never been observed in strains with single or double mutations in sulfur transporters of interest due to the presence of multiple viable sulfur sources and transporters to access them. Using the host perspective, MALDI-IMS has provided a rational basis for utilizing strains deficient in quadruple or even sextuple transporters during systemic infection. 146 To further expand our understanding of how the host responds to S. aureus infection from an elemental sulfur standpoint, MALDI-IMS can also be employed to look at the distribution of host sulfur metabolism proteins throughout infection as well. This line of investigation would be advantageous considering that there are several proteins such as GGT, CSB or CSE that inflict host disease when not abundance in regular concentrations (please see Chapter 1 for more information). Thus, a full picture of S. aureus infection would include an understanding of how acquisition of sulfur sources impacts the abundance and function of host enzymes whose downstream repercussions contribute to the clinical manifestations of S. aureus infection. Chapter 2 also highlights the ability of S. aureus to utilize the tripeptide GSH, not for meeting its nutritional sulfur requirement, but as a resource to counter heme- and H2O2- induced oxidative stress. It is somewhat unsurprising that a metabolite with diverse functions within the host (as discussed in Chapter 1) also serves multiple roles within S. aureus. Consequently, future investigations into GSH in this organism are likely to unveil additional, unrecognized contributions. Akin to sulfur, the preference of compounds contributing to the nutritional nitrogen requirement in S. aureus remains underexplored compared to other bacteria such as E. coli or Bacillus subtilis (234, 235). In terms of nitrogen flux within a cell, imported nitrogen sources are assimilated to glutamate (Glu) and glutamine (Gln) which serve as the internal nitrogen donors for various biological processes (235). For E. coli, ammonium is the preferred nitrogen source although several other compounds such as Glu, Gln, and Glu precursors (e.g., proline, arginine, or glycine) can also drive proliferation when provided as nutritional nitrogen (235). Though ammonium also supports the B. subtilis nitrogen requirements, Gln is considered the preferred 147 nitrogen source. (234). The nitrogen sources for S. aureus have been studied to some extent. For instance, researchers have investigated the ability of S. aureus to utilize ammonium (NH4), glutamine (Gln), or glutamate (Glu) as nutritional nitrogen sources (236). In this study, CDM devoid of Gln, Glu, and NH4 (denoted CDM-Glu/Gln,-NH4) was used. The supplementation of CDM-Glu/Gln,-NH4 with NH4 or Gln stimulated the growth of S. aureus strain LAC, while Glu did not have the same effect. As a result, the study concluded that NH4 and Gln are preferred nitrogen sources over Glu in S. aureus (236). However, it's important to note that only one concentration was tested for each potential nitrogen source—a crucial consideration given that Glu is the sole precursor to Gln. In the tested conditions, Glu was required both as a nitrogen source and as a source of Gln. Therefore, it remains undetermined whether Glu truly cannot meet the nitrogen requirement or if it requires higher concentrations due to the experimental design. To explore this, the experimental setup used one used by Zeden et al. (236) can be employed to assess the growth of S. aureus on CDM-Glu/Gln,-NH4 with increasing Glu concentrations. It is predicted that higher concentrations of this amino acid will fulfill the nitrogen requirement in this organism. Once an environment is established where Glu is vital for robust growth, the CDM-Glu/Gln,-NH4 can be supplemented with GSH to assess whether this tripeptide can act as a source of Glu. This is of particular interest given previous research indicating that tripeptides can serve as a source of Glu in S. aureus (192). It is predicted that S. aureus will proliferate in this medium when sufficient GSH is provided. This, in conjunction with the oxidative stress assays in Chapter 2, would underscore the necessity for researchers in this field to consider how S. aureus sulfur sources contribute to bacterial physiology 148 beyond meeting the nutritional sulfur requirement. CONCLUDING REMARKS My thesis has played a significant role in establishing the groundwork for understanding how S. aureus acquires sulfur sources from the host, offering both regulatory and mechanistic insights. 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APPENDIX A: Chapter 2 sulfur starvation Tables Genes upregulated in WT sulfur starvation when compared to WT cystine (CSSC) Share d with cymR: :Tn -S (Table 3) COG Locus Old locus Gene Product TPM. 1 Starv TPM. 2 Starv TPM. 1 CSSC TPM. 2 CSSC DE Log2 FC Yes O SAUSA300 _RS10980 SAUSA3 00_1997 sulfurtransferase TusA family protein 719.6 8 710.6 9 2.67 2.08 Yes S SAUSA300 _RS10985 SAUSA3 00_1998 YeeE/YedE family protein 170.7 7 130.2 0.74 0.43 Yes P SAUSA300 _RS11525 SAUSA3 00_2092 Dps family protein 24049 29749 .5 209.5 9 66.25 Yes I SAUSA300 _RS00930 SAUSA3 00_0177 No S SAUSA300 _RS00910 SAUSA3 00_0173 Yes P SAUSA300 _RS02335 SAUSA3 00_0436 Yes S SAUSA300 _RS04570 SAUSA3 00_0846 547.1 5 115.0 3 2.77 1.16 694.9 626.5 7 4.54 4.88 638.3 3 220.4 4 2.72 2.91 171.0 8 76 1.42 0.65 acyl-CoA/acyl- ACP dehydrogenase DUF4242 domain- containing protein methionine ABC transporter permease Na+/H+ antiporter family protein 172 6.524 50659 6 6.339 19187 4 6.104 01684 8 5.543 08137 9 5.396 13928 7 5.339 35652 2 5.237 16145 8 DE Adj. P- value 1.568 27E- 48 1.132 21E- 52 4.153 46E- 38 1.585 52E- 27 6.367 63E- 33 4.850 64E- 30 1.956 86E- 36 Table A-1 (cont’d) Yes P SAUSA300 _RS02340 SAUSA3 00_0437 gmpC Yes no homol og found SAUSA300 _RS16040 hisF dipeptide ABC transporter glycylmethionine -binding lipoprotein imidazole glycerol phosphate synthase subunit HisF 1906. 11 1201. 65 10.16 14.16 5.138 08140 8 1.410 43E- 27 43.09 28.36 0.34 0.37 Yes P SAUSA300 _RS10250 SAUSA3 00_1874 ftnA H-type ferritin FtnA 6482. 34 4756. 04 77.14 50.27 Yes P SAUSA300 _RS01055 SAUSA3 00_0200 Yes P SAUSA300 _RS02330 SAUSA3 00_0435 Yes S SAUSA300 _RS14570 SAUSA3 00_2622 ABC transporter ATP-binding protein methionine ABC transporter ATP- binding protein rhodanese- related sulfurtransferase 182.9 5 207.4 5 343.6 9 145.1 8 371.0 8 274.7 6 1.44 2.45 2.36 2.49 5.35 2.72 SAUSA300 _RS00925 SAUSA3 00_0176 ABC transporter permease 118.6 2 84.08 1.5 0.99 No No P P SAUSA300 _RS00915 SAUSA3 00_0174 Yes E SAUSA300 _RS14090 SAUSA3 00_2539 ABC transporter ATP-binding protein aspartate aminotransferas e family protein 125.4 9 118.7 5 72.45 2.15 0.58 123 1.2 2.18 173 4.889 66808 4 4.858 96820 4 4.815 28742 7 4.815 08731 4 4.768 75260 4 4.712 41826 9 4.636 14025 4.329 46737 9 7.386 96E- 27 8.351 49E- 36 1.636 82E- 19 9.670 75E- 27 1.249 25E- 33 1.466 65E- 30 5.562 76E- 24 7.908 82E- 16 Table A-1 (cont’d) Yes no homol og found SAUSA300 _RS07125 hypothetical protein 2859. 37 3129. 02 89.03 22.95 No J SAUSA300 _RS01820 SAUSA3 00_0343 GNAT family protein 160.3 9 159.3 8 3.32 2.77 Yes S SAUSA300 _RS12540 SAUSA3 00_2268 Yes F SAUSA300 _RS00725 SAUSA3 00_0138 deoD bile acid:sodium symporter family protein purine- nucleoside phosphorylase 63.15 76.14 1.59 1.16 32.2 29.05 0.56 0.58 No Yes no homol og found no homol og found SAUSA300 _RS15260 SAUSA3 00_0937 hypothetical protein 1230. 36 1900. 55 43.62 23.66 SAUSA300 _RS10255 hypothetical protein 139.2 4 87.37 3.88 1.33 4.316 47520 8 4.103 08227 7 4.086 91557 2 4.061 06887 4 4.060 78001 2 3.834 81737 4 3.829 58655 8 1.890 65E- 18 5.372 53E- 20 2.217 42E- 18 3.420 94E- 17 6.515 46E- 17 9.775 67E- 15 1.038 68E- 20 Yes C SAUSA300 _RS07500 SAUSA3 00_1373 No E SAUSA300 _RS09195 SAUSA3 00_1683 ferredoxin bifunctional 3- deoxy-7- phosphoheptulon ate synthase/choris mate mutase 174 3199. 55 2852. 95 98.5 51.47 1457. 39 1317. 56 52.47 23.75 3.710 63807 9 6.544 52E- 19 Table A-1 (cont’d) Yes E SAUSA300 _RS02320 SAUSA3 00_0433 mccA Yes P SAUSA300 _RS12340 SAUSA3 00_2235 No no homol og found SAUSA300 _RS05130 cysteine synthase family protein ABC transporter substrate-binding protein hypothetical protein 28.3 17.42 0.52 0.56 289.0 3 81.16 6.61 2.65 28.16 66.88 1.65 0.97 Yes NOU SAUSA300 _RS08765 SAUSA3 00_1609 A24 family peptidase 15.85 22.63 0.83 0.33 Yes C SAUSA300 _RS05570 SAUSA3 00_1035 isdG No P SAUSA300 _RS00920 SAUSA3 00_0175 Yes S SAUSA300 _RS03075 SAUSA3 00_0575 No S SAUSA300 _RS09315 SAUSA3 00_1706 staphylobilin- forming heme oxygenase IsdG ABC transporter substrate-binding protein DUF1450 domain- containing protein TIGR01212 family radical SAM protein 43.52 46.29 1.76 0.88 75.98 41.15 2.57 0.88 333.1 5 267.1 2 9.41 7.44 87.53 59.39 2.18 1.85 Yes K SAUSA300 _RS14655 SAUSA3 00_2639 cold-shock protein 22322 .8 27330 .69 1319. 58 382.5 6 3.663 96345 7 3.639 70547 8 3.601 05142 2 3.579 02953 3 3.569 19932 1 3.557 11047 9 3.552 41454 2 3.542 61522 8 3.484 23540 8 6.023 26E- 15 3.635 52E- 14 4.095 84E- 08 2.162 9E-11 2.570 49E- 13 5.033 63E- 16 4.479 57E- 17 5.942 19E- 17 2.289 16E- 12 175 Table A-1 (cont’d) Yes S SAUSA300 _RS14550 SAUSA3 00_2618 Yes H SAUSA300 _RS03735 SAUSA3 00_0696 queD ECF-type riboflavin transporter substrate-binding protein 6- carboxytetrahydr opterin synthase QueD 41.15 45.27 1.11 1.36 74.16 31.83 1.57 1.37 No S SAUSA300 _RS05050 SAUSA3 00_0940 DoxX family protein 191.3 7 208.3 7 10.54 3.61 Yes E SAUSA300 _RS01075 SAUSA3 00_0204 ggt γ- glutamyltransfera se 119.2 1 118.0 8 2.76 4.15 Yes S SAUSA300 _RS00890 SAUSA3 00_0169 YbaN family protein 72.45 46.03 3.25 0.88 Yes C SAUSA300 _RS04255 SAUSA3 00_0788 No M SAUSA300 _RS04310 SAUSA3 00_0798 No S SAUSA300 _RS01875 SAUSA3 00_0354 nitroreductase MetQ/NlpA family ABC transporter substrate-binding protein low temperature requirement protein A 671.4 9 488.3 1 27.67 14.18 217.5 8 157.3 3 10.05 4.1 45.08 29.37 1.85 1.02 3.441 47019 8 2.170 06E- 11 3.424 76944 5 3.404 61594 8 3.365 08471 1 3.347 78883 3 3.278 61252 3 3.247 84510 7 3.144 51161 3 1.986 47E- 13 6.416 37E- 13 7.158 16E- 11 2.289 16E- 12 1.887 99E- 16 5.822 3E-15 1.971 62E- 14 176 Table A-1 (cont’d) No E SAUSA300 _RS02855 SAUSA3 00_0534 M20 family metallopeptidase 82.32 42.25 3.57 1.5 Yes H SAUSA300 _RS03730 SAUSA3 00_0695 queE Yes S SAUSA300 _RS10920 SAUSA3 00_1986 Yes C SAUSA300 _RS06395 SAUSA3 00_1183 No No S S SAUSA300 _RS13940 SAUSA3 00_2511 SAUSA300 _RS01485 SAUSA3 00_0277 Yes S SAUSA300 _RS07495 SAUSA3 00_1372 Yes K SAUSA300 _RS14555 SAUSA3 00_2619 107.1 2 33.76 1.97 2.64 37.79 40.11 1.78 1.44 201.3 6 129.7 4 8.87 4.97 186.1 8 230.0 1 11.9 6.79 18.43 20.75 1.18 0.62 96.9 111.1 5 6.3 3.4 19.02 14.91 0.83 0.62 7-carboxy-7- deazaguanine synthase QueE nitroreductase family protein 2- oxoacid:ferredoxi n oxidoreductase subunit β DUF896 domain- containing protein CHAP domain- containing protein helix-turn-helix domain- containing protein S-adenosyl-l- methionine hydroxide adenosyltransfer ase family protein 177 3.079 04484 9 3.037 59705 3.035 97010 7 3.035 33978 7 3.009 53177 6 2.969 24929 1 2.963 27268 1 2.311 18E- 13 8.168 54E- 09 1.553 84E- 10 1.973 14E- 14 1.634 66E- 10 6.336 13E- 10 6.665 8E-11 2.958 93782 9 1.023 43E- 10 Table A-1 (cont’d) Yes D SAUSA300 _RS07900 SAUSA3 00_1447 xerD Yes H SAUSA300 _RS00040 SAUSA3 00_0007 No not classif ied SAUSA300 _RS06580 SAUSA3 00_1213 site-specific tyrosine recombinase XerD NAD(P)H- hydrate dehydratase hypothetical protein 254.3 7 128.3 9 11.84 5.09 148.8 9 134.1 7.95 4.96 12.05 17.92 1.29 0.14 No Q SAUSA300 _RS04575 SAUSA3 00_0847 PaaI family thioesterase 515.2 1 437.9 9 29.12 15.76 No E SAUSA300 _RS02325 SAUSA3 00_0434 mccB No E SAUSA300 _RS05115 SAUSA3 00_0952 Yes O SAUSA300 _RS02020 SAUSA3 00_0379 ahpF bifunctional cystathionine γ- lyase/homocystei ne desulfhydrase aminotransferas e class I/II-fold pyridoxal phosphate- dependent enzyme alkyl hydroperoxide reductase subunit F 46.14 33.39 1.09 1.95 208.9 4 179.3 8 12.99 5.91 2994. 44 1970. 33 103.4 106.2 2.957 97855 9 2.941 73211 8 2.923 01785 2.917 56117 6 2.906 85451 7 9.179 36E- 13 3.373 04E- 12 2.604 38E- 05 1.826 18E- 12 5.072 23E- 08 2.902 52660 7 5.438 6E-12 2.889 93674 4 6.578 98E- 11 178 Table A-1 (cont’d) Yes F SAUSA300 _RS03850 SAUSA3 00_0717 nrdF No E SAUSA300 _RS00055 SAUSA3 00_0010 Yes O SAUSA300 _RS02025 SAUSA3 00_0380 ahpC Yes HP SAUSA300 _RS03395 SAUSA3 00_0633 Yes C SAUSA300 _RS02385 SAUSA3 00_0446 Yes K SAUSA300 _RS00650 SAUSA3 00_0126 sbnI No K SAUSA300 _RS04190 SAUSA3 00_0777 Yes E SAUSA300 _RS13265 SAUSA3 00_2395 1920. 34 866.8 2 94.33 35.28 48.26 38.96 1.96 1.87 3967. 13 3243. 81 215.8 6 133.7 2 144.2 58.29 7.25 2.39 483.9 8 241.2 1 25.93 10.25 43.44 26.75 2.35 1.17 7723. 34 10264 .53 757.1 7 261.1 9 42.04 29.12 2.34 1.36 2.888 01193 3 2.882 35841 5 2.846 85053 2 2.842 1453 2.801 06218 5 2.786 62502 6 2.780 15390 8 2.735 06481 2.419 88E- 11 2.983 3E-10 2.870 44E- 12 7.909 95E- 10 2.903 06E- 11 4.360 95E- 11 2.159 34E- 08 2.138 42E- 11 class 1b ribonucleoside- diphosphate reductase subunit β AzlC family ABC transporter permease alkyl hydroperoxide reductase subunit C ABC transporter ATP-binding protein glutamate synthase subunit β bifunctional transcriptional regulator/O- phospho-L-serine synthase SbnI cold-shock protein APC family permease 179 Table A-1 (cont’d) No D SAUSA300 _RS09440 SAUSA3 00_1726 CrcB family protein 30.4 32.26 1.79 1.43 Yes C SAUSA300 _RS01085 SAUSA3 00_0206 FMN-dependent NADH- azoreductase 48.05 65.73 2.77 2.98 No K SAUSA300 _RS04840 SAUSA3 00_0898 spxA transcriptional regulator SpxA 55513 .94 65522 .85 4499. 83 2337. 74 Yes C SAUSA300 _RS00885 SAUSA3 00_0168 isdI No C SAUSA300 _RS01170 SAUSA3 00_0222 193.9 4 172 15.34 5.57 18.07 10.31 0.96 0.52 staphylobilin- forming heme oxygenase IsdI glycerophosphor yl diester phosphodiestera se membrane domain- containing protein No O SAUSA300 _RS04535 SAUSA3 00_0839 NifU family protein 4814. 96 5336. 37 417.5 8 174.0 5 No F SAUSA300 _RS03845 SAUSA3 00_0716 nrdE Yes P SAUSA300 _RS07905 SAUSA3 00_1448 1116. 49 521.7 7 57.24 28.15 935.7 7 709.5 6 64.75 27.78 class 1b ribonucleoside- diphosphate reductase subunit α Fur family transcriptional regulator 180 2.730 50255 2 2.720 02560 4 2.709 96514 5 2.707 97401 5 2.701 69258 2.695 71536 8 2.694 65430 6 2.691 93921 9 4.391 93E- 08 2.238 04E- 07 2.553 31E- 09 1.950 23E- 09 1.671 74E- 10 3.631 2E-09 4.450 16E- 11 9.201 53E- 11 Table A-1 (cont’d) No No U K SAUSA300 _RS02035 SAUSA3 00_0382 L-cystine transporter 1423. 29 1354. 04 45.72 82.63 2.678 55695 SAUSA300 _RS09185 SAUSA3 00_1682 ccpA catabolite control protein A 373.6 8 401.8 2 28.23 16.05 No G SAUSA300 _RS01135 SAUSA3 00_0216 uhpT No S SAUSA300 _RS00060 SAUSA3 00_0011 No T SAUSA300 _RS03705 SAUSA3 00_0690 saeS hexose-6- phosphate:phosp hate antiporter AzlD domain- containing protein two-component system sensor histidine kinase SaeS 14.3 20.83 0.63 1.08 94.05 74.4 5.7 3.55 2407. 99 1402. 87 159.5 60.93 Yes E SAUSA300 _RS14085 SAUSA3 00_2538 amino acid permease 358.5 7 405.9 9 17.23 22.37 No S SAUSA300 _RS12610 SAUSA3 00_2282 sdpC Yes E SAUSA300 _RS11075 SAUSA3 00_2014 ilvA No S SAUSA300 _RS01480 SAUSA3 00_0276 160.7 2 131.7 7 96.58 10.9 4.13 80 7.82 4.12 20.16 22.76 2.11 0.6 CPBP family intramembrane glutamic endopeptidaseS dpC threonine ammonia-lyase IlvA DUF5080 family protein 181 1.023 79E- 06 1.621 38E- 09 5.568 09E- 06 6.275 63E- 10 3.878 29E- 10 3.817 64E- 07 6.336 13E- 10 6.066 2E-11 7.737 3E-07 2.667 45616 3 2.666 43000 7 2.656 31562 7 2.627 22530 8 2.623 20008 5 2.618 37110 3 2.618 18560 8 2.605 73898 9 Table A-1 (cont’d) Yes F SAUSA300 _RS03840 SAUSA3 00_0715 nrdI class Ib ribonucleoside- diphosphate reductase assembly flavoprotein NrdI 194.3 5 97.21 10.72 5.56 2.594 78534 1 3.769 57E- 10 Yes F SAUSA300 _RS05655 SAUSA3 00_1050 XTP/dITP diphosphatase 174.0 7 62.9 9.64 3.72 No S SAUSA300 _RS06575 SAUSA3 00_1212 No no homol og found SAUSA300 _RS09505 SAUSA3 00_1738 polymorphic toxin type 50 domain- containing protein DUF4909 domain- containing protein 26.85 47.17 3.59 1.33 25.49 35.7 3.5 0.82 No S SAUSA300 _RS05145 SAUSA3 00_0957 osmotic stress response protein 511.2 1 609.6 9 56.93 20.16 Yes P SAUSA300 _RS03880 SAUSA3 00_0721 No S SAUSA300 _RS04485 SAUSA3 00_0831 siderophore ABC transporter substrate-binding protein DUF3055 domain- containing protein 63.71 27.31 3.76 1.68 123.1 7 176.1 3 17.41 4.1 2.565 23973 3 2.540 68494 2.497 77330 6 2.495 28233 5 2.494 17382 5 2.490 08913 2 2.957 42E- 08 6.612 55E- 06 1.111 06E- 05 2.591 17E- 07 1.216 52E- 08 7.881 04E- 06 182 Table A-1 (cont’d) No EP SAUSA300 _RS01060 SAUSA3 00_0201 No K SAUSA300 _RS05125 SAUSA3 00_0954 ABC transporter permease MarR family transcriptional regulator 27.04 25.9 0.74 1.91 28.82 51.17 3.79 1.69 Yes CH SAUSA300 _RS13660 SAUSA3 00_2463 D-lactate dehydrogenase 184.4 No not classif ied SAUSA300 _RS09430 hypothetical protein 77.65 176.0 7 107.6 7 10.9 10.55 9.87 3.23 Yes C SAUSA300 _RS12320 SAUSA3 00_2231 fdhD No No no homol og found no homol og found SAUSA300 _RS10520 SAUSA300 _RS15420 formate dehydrogenase accessory sulfurtransferase FdhD 64.6 50.42 5.64 2.1 hypothetical protein 340.8 4 332.3 3 29.83 15.37 hypothetical protein 37.28 43.88 4.18 1.49 Yes L SAUSA300 _RS03790 SAUSA3 00_0705 recQ DNA helicase RecQ 86.19 30.45 4.75 2.28 2.489 75256 3 2.487 85914 8 2.480 58477 5 2.474 50835 5 2.459 94465 6 2.444 65067 2 2.439 60961 4 2.438 99684 6 4.236 08E- 05 6.488 21E- 06 9.520 5E-08 2.391 13E- 06 2.902 29E- 08 2.385 78E- 08 4.115 83E- 06 6.039 46E- 08 183 Table A-1 (cont’d) No G SAUSA300 _RS08910 SAUSA3 00_1633 gap No C SAUSA300 _RS14025 SAUSA3 00_2528 No V SAUSA300 _RS00290 SAUSA3 00_0056 No No P P SAUSA300 _RS05410 SAUSA3 00_1005 SAUSA300 _RS03400 SAUSA3 00_0634 type I glyceraldehyde- 3-phosphate dehydrogenase epoxyqueuosine reductase QueH DUF1643 domain- containing protein Nramp family divalent metal transporter iron ABC transporter permease 24.26 13.46 1.4 0.92 54.83 46.16 4.7 2.2 11 12.13 0.82 0.65 107.9 4 89.1 8.57 4.75 70.71 40.61 4.21 2.82 No O SAUSA300 _RS14575 SAUSA3 00_2623 pcp pyroglutamyl- peptidase I 38.31 36.65 2.78 2.14 No P SAUSA300 _RS10870 SAUSA3 00_1979 Yes S SAUSA300 _RS10695 SAUSA3 00_1949 Yes E SAUSA300 _RS11055 SAUSA3 00_2010 TrkH family potassium uptake protein dUTP pyrophosphatase 2- isopropylmalate synthase 39.46 43.09 3.98 1.93 68.32 40.19 2.35 3.76 58.74 40.37 3.86 2.67 2.431 11499 3 2.415 30322 8 2.410 36544 5 2.401 74594 2 2.400 77713 2.395 00032 1 2.377 60245 2 2.365 17681 9 2.364 63665 2 2.116 78E- 08 2.779 85E- 08 7.045 98E- 06 7.865 21E- 09 6.004 37E- 09 2.171 87E- 07 2.264 1E-07 6.961 58E- 06 1.081 91E- 08 184 Table A-1 (cont’d) No NOT SAUSA300 _RS04845 SAUSA3 00_0899 mecA adaptor protein MecA 1349. 11 1117. 69 136.0 1 46.21 No EG SAUSA300 _RS03830 SAUSA3 00_0713 queF preQ(1) synthase 132.1 8 207.4 4 15.06 9.52 No K SAUSA300 _RS08020 SAUSA3 00_1469 argR transcriptional regulator ArgR 131.1 122.4 1 13.47 5.25 Yes No no homol og found not classif ied SAUSA300 _RS10730 SAUSA3 00_1956 hypothetical protein 43.12 18.05 1.65 1.84 SAUSA300 _RS06570 SAUSA3 00_1211 hypothetical protein 84.09 108.9 6 9.19 4.88 Yes E SAUSA300 _RS01070 SAUSA3 00_0203 No not classif ied SAUSA300 _RS12240 SAUSA3 00_2218 sarV Yes L SAUSA300 _RS10735 SAUSA3 00_1957 No S SAUSA300 _RS03810 SAUSA3 00_0709 ABC transporter substrate-binding protein HTH-type transcriptional regulator SarV DnaD domain- containing protein 5'-3'- deoxyribonucleot idase 23.3 13.38 0.75 1.32 84.74 118.4 6 11.66 4.28 54.9 24.35 1.64 2.78 208.2 7 198.5 9 15 12.92 No O SAUSA300 _RS05300 SAUSA3 00_0985 nrdH glutaredoxin-like protein NrdH 130.8 136.8 7 12.78 7.02 2.355 84619 8 2.352 84193 2 2.348 22851 9 2.345 33538 7 2.344 42779 2.337 16317 2.318 62304 9 2.316 29998 2 2.308 37631 2.303 84259 6 1.723 18E- 07 3.899 42E- 06 2.264 1E-07 1.202 09E- 05 2.545 66E- 06 1.529 35E- 05 6.725 02E- 06 1.928 09E- 05 4.152 28E- 07 4.971 66E- 07 185 Table A-1 (cont’d) No L SAUSA300 _RS10260 SAUSA3 00_1875 3'-5' exonuclease 180.6 131.5 2 17.95 5.77 Yes U SAUSA300 _RS11755 SAUSA3 00_2135 No O SAUSA300 _RS06825 SAUSA3 00_1256 msrA No S SAUSA300 _RS01615 SAUSA3 00_0303 Yes EGP SAUSA300 _RS11720 SAUSA3 00_2128 sdrM iron ABC transporter permease peptide- methionine (S)- S-oxide reductase MsrA DUF4467 domain- containing protein multidrug efflux MFS transporter SdrM 50.33 30.96 3.9 1.93 292.8 8 266.3 7 27.9 14.22 19.94 10.29 1.79 0.43 88.69 42.39 5.65 3.47 No D SAUSA300 _RS05870 SAUSA3 00_1083 cell division protein SepF 453.7 4 262.4 35.79 17.19 No K SAUSA300 _RS03710 SAUSA3 00_0691 saeR No S SAUSA300 _RS04480 SAUSA3 00_0830 Yes E SAUSA300 _RS11070 SAUSA3 00_2013 leuD 1658. 72 546.8 4 114.6 8 41.91 124.8 9 116.8 7 13.56 5.71 54.54 20.11 3.65 1.65 response regulator transcription factor SaeR YutD family protein 3- isopropylmalate dehydratase small subunit 186 2.300 03317 1 2.287 54227 1 2.279 86174 9 2.276 81024 9 2.249 82656 1 2.244 90419 2 2.240 78712 2.232 49280 2 2.220 42757 4.381 69E- 07 3.617 E-08 1.077 96E- 07 3.500 29E- 05 6.372 22E- 08 3.290 04E- 08 2.563 22E- 06 7.327 22E- 07 2.307 85E- 06 Table A-1 (cont’d) Yes L SAUSA300 _RS08280 SAUSA3 00_1517 deoxyribonuclea se IV 132.6 4 76.96 11.13 4.85 No no homol og found SAUSA300 _RS04145 SAUSA3 00_0769 No M SAUSA300 _RS02875 SAUSA3 00_0538 DUF5067 domain- containing protein NAD-dependent epimerase/dehyd ratase family protein No O SAUSA300 _RS13000 SAUSA3 00_2354 DsbA family protein 14.1 11.83 1.7 0.43 167.9 8 149.2 1 52.98 7.28 6.75 78.06 13.23 4.45 No No No Yes S H no homol og found no homol og found SAUSA300 _RS10715 SAUSA3 00_1953 phi PVL orf 51- like protein 32.41 10.52 1.59 1.17 SAUSA300 _RS13380 SAUSA3 00_2417 AbgT family transporter 93.95 70.54 7.87 4.7 SAUSA300 _RS13590 hypothetical protein 43.71 58.72 6.27 2.28 SAUSA300 _RS10765 SAUSA3 00_1963 DUF2482 family protein 98.21 49.58 5.1 4.94 2.216 93092 3 2.215 25171 8 2.211 32300 6 2.205 10484 8 2.202 12062 2.201 07237 3 1.046 43E- 07 2.639 25E- 05 6.541 46E- 06 9.655 63E- 07 7.284 84E- 05 8.472 96E- 08 2.194 81224 0.000 13026 2.194 14883 7 2.862 25E- 06 187 Table A-1 (cont’d) No E SAUSA300 _RS07075 SAUSA3 00_1300 brnQ branched-chain amino acid transport system II carrier protein 40.33 33.11 4.13 1.79 No S SAUSA300 _RS13585 SAUSA3 00_2450 DedA family protein 64.71 60.55 7.02 3.26 Yes P SAUSA300 _RS06680 SAUSA3 00_1232 No E SAUSA300 _RS13700 SAUSA3 00_2469 sdaA A catalase L-serine ammonia-lyase, iron-sulfur- dependent, subunit α 539.8 5 377.2 2 43.86 27.02 43.54 41.45 3.34 3.04 No S SAUSA300 _RS05660 SAUSA3 00_1051 metallophosphoe sterase 235.4 7 162.5 2 23.34 9.38 No S SAUSA300 _RS10770 DUF1270 domain- containing protein 182.2 3 135.6 3 11.69 11.67 No CH SAUSA300 _RS12455 SAUSA3 00_2255 FAD-dependent monooxygenase 65.93 43.69 5.69 3.16 No S SAUSA300 _RS00935 SAUSA3 00_0178 1908. 25 1196. 98 184.7 5 75.82 DUF2294 domain- containing protein 188 2.194 12238 7 2.174 80999 7 2.170 19843 7 2.169 32606 5 2.153 53223 6 2.141 16198 2 2.118 40739 6 2.108 40937 1 4.781 11E- 07 1.157 01E- 06 7.710 06E- 08 3.911 26E- 06 4.617 05E- 07 4.427 01E- 06 2.442 62E- 07 4.827 12E- 07 Table A-1 (cont’d) No K SAUSA300 _RS11435 SAUSA3 00_2077 Yes C SAUSA300 _RS14195 SAUSA3 00_2554 No S SAUSA300 _RS04235 SAUSA3 00_0784 Yes L SAUSA300 _RS10740 SAUSA3 00_1958 ssb helix-turn-helix domain- containing protein assimilatory sulfite reductase (NADPH) flavoprotein subunit LysE/ArgO family amino acid transporter single-stranded DNA-binding protein 667.1 2 625.4 1 66.68 41.15 38.99 12.76 2.52 1.35 377.9 8 376.5 8 45.08 20.84 56.97 25.12 2.3 3.24 Yes no homol og found SAUSA300 _RS15665 SAUSA3 00_2604 hypothetical protein 143.3 2 151.5 7 21.54 5.79 No ET SAUSA300 _RS13025 SAUSA3 00_2359 transporter substrate-binding domain- containing protein 182.0 1 61.38 11.72 6.67 No no homol og found SAUSA300 _RS10745 SAUSA3 00_1959 MBL fold metallo- hydrolase 41.42 27.47 1.95 2.93 Yes EP SAUSA300 _RS01065 SAUSA3 00_0202 ABC transporter permease 35.16 28.03 1.1 3.06 2.105 10083 8 1.144 79E- 06 2.103 69981 5 2.101 66557 7 2.093 19813 9 2.092 30146 7 6.842 36E- 06 2.373 72E- 06 6.789 53E- 05 5.315 46E- 05 2.082 76138 5 5.379 96E- 06 2.080 39007 5 7.432 5E-05 2.078 40883 0.000 82141 189 SAUSA300 _RS05875 SAUSA3 00_1084 YggT family protein 483.8 9 615.5 7 60.84 36.34 Table A-1 (cont’d) No No S E SAUSA300 _RS13015 SAUSA3 00_2357 Yes C SAUSA300 _RS06390 SAUSA3 00_1182 No S SAUSA300 _RS03640 SAUSA3 00_0678 Yes M SAUSA300 _RS14270 SAUSA3 00_2565 clfB No S SAUSA300 _RS09900 SAUSA3 00_1809 Yes no homol og found SAUSA300 _RS14185 No S SAUSA300 _RS10775 SAUSA3 00_1964 Yes U SAUSA300 _RS04915 SAUSA3 00_0914 amino acid ABC transporter ATP- binding protein 2- oxoacid:acceptor oxidoreductase subunit α DUF1361 domain- containing protein MSCRAMM family adhesin clumping factor ClfB PTS transporter subunit IIC 696.0 3 354.0 7 37.73 39.69 28.17 10.25 1.6 1.25 91.9 44.78 8.99 2.81 50.07 27.8 4.24 2.28 60.73 49.38 6.38 3.33 hypothetical protein 286.0 9 209.6 5 26.66 15.83 194.1 153.0 2 12.37 14.71 65.79 68.24 6.08 5.26 DUF771 domain- containing protein alanine/glycine:c ation symporter family protein 190 2.067 34911 2 2.062 62947 2 2.059 82620 4 2.053 86324 5 2.045 84785 8 2.037 41538 8 2.031 42813 2.026 63681 9 2.025 87756 7 1.463 3E-05 7.411 13E- 06 1.120 55E- 05 1.161 33E- 05 5.333 69E- 07 1.631 32E- 06 2.300 27E- 06 3.242 39E- 05 1.595 05E- 05 Table A-1 (cont’d) No C SAUSA300 _RS14205 SAUSA3 00_2555 glutathione peroxidase 18.74 12.72 1.87 0.85 SAUSA300 _RS03975 SAUSA3 00_0738 prfB peptide chain release factor 2 159.5 4 115.5 6 14.02 9.4 No No No J P E SAUSA300 _RS13185 SAUSA3 00_2384 SAUSA300 _RS09890 SAUSA3 00_1807 Yes M SAUSA300 _RS13530 SAUSA3 00_2441 fnbA No no homol og found SAUSA300 _RS15465 No K SAUSA300 _RS13930 SAUSA3 00_2509 No S SAUSA300 _RS10725 SAUSA3 00_1955 No Q SAUSA300 _RS10395 SAUSA3 00_1899 52.71 25.91 4.39 2.25 18.92 10.72 1.66 0.86 46.33 20.75 3.52 2.03 36.02 24.35 3.73 1.59 27.57 19.52 2.89 1.32 21.45 11.32 0.84 1.47 146.3 7 80.05 13.36 6.34 sodium:proton antiporter amino acid ABC transporter ATP- binding protein fibronectin- binding protein FnbA IS5/IS1182 family transposase TetR/AcrR family transcriptional regulator RusA family crossover junction endodeoxyribonu clease isochorismatase family cysteine hydrolase 191 2.025 05763 1 2.022 67811 7 2.018 28401 9 2.015 08411 1 2.009 75835 7 2.009 41557 9 2.008 93044 7 2.008 20549 5 2.007 94065 1 1.969 32E- 05 1.023 79E- 06 1.366 16E- 06 9.036 49E- 06 1.994 91E- 06 4.400 03E- 05 8.284 41E- 06 0.000 52331 6 1.525 24E- 06 Table A-1 (cont’d) No No No L K S SAUSA300 _RS07325 SAUSA3 00_1343 SAUSA300 _RS03330 SAUSA3 00_0621 nth endonuclease III 44.97 19.52 4.35 1.33 metal-dependent transcriptional regulator 211.5 5 167 25.36 10.03 SAUSA300 _RS01850 SAUSA3 00_0349 DUF1398 family protein 20.26 13.22 1.74 1.09 No G SAUSA300 _RS01655 SAUSA3 00_0310 PTS sugar transporter subunit IIC 199.8 211.8 8 18.38 16.98 No F SAUSA300 _RS08440 SAUSA3 00_1548 ComE operon protein 2 230.3 4 236.8 9 32.44 12.64 No U SAUSA300 _RS01840 SAUSA3 00_0347 tatC No F SAUSA300 _RS04970 SAUSA3 00_0925 No C SAUSA300 _RS10400 SAUSA3 00_1900 No M SAUSA300 _RS09320 SAUSA3 00_1707 61.7 80.23 7.46 5.44 70.4 56.56 7.76 3.92 854.1 4 452.1 1 66.92 43.79 51.74 21.28 3.4 2.51 twin-arginine translocase subunit TatC bifunctional UDP-sugar hydrolase/5'- nucleotidase manganese- dependent inorganic pyrophosphatase class I SAM- dependent methyltransferas e 192 2.001 79183 2 1.998 15442 9 1.997 26080 4 1.993 85926 9 1.992 84589 6 1.988 03162 2 1.983 04419 1.981 24896 3 1.966 69127 6 4.174 84E- 05 4.804 13E- 06 3.391 29E- 05 2.980 69E- 05 1.841 3E-05 5.554 84E- 05 2.606 56E- 06 1.271 14E- 06 1.962 23E- 05 Table A-1 (cont’d) No not classif ied SAUSA300 _RS07840 No K SAUSA300 _RS12705 SAUSA3 00_2300 hypothetical protein TetR/AcrR family transcriptional regulator 72.01 53.27 8.65 3.28 50.55 52.78 7.6 2.72 SAUSA300 _RS10510 hypothetical protein 306.8 2 241.9 4 34.91 17.01 no homol og found E K No No No No No not classif ied not classif ied SAUSA300 _RS03805 SAUSA3 00_0708 hisC SAUSA300 _RS10810 SAUSA3 00_1969 No S SAUSA300 _RS10720 SAUSA3 00_1954 SAUSA300 _RS03655 SAUSA3 00_0681 histidinol- phosphate transaminase XRE family transcriptional regulator SA1788 family PVL leukocidin- associated protein hypothetical protein 26.35 26.04 2.54 2.17 87.88 65.87 8.1 5.72 30.01 14.97 1.56 1.94 59.26 52.14 9.23 2.23 SAUSA300 _RS10790 SAUSA3 00_1967 hypothetical protein 162.0 6 89.91 8.02 11.61 No S SAUSA300 _RS09830 SAUSA3 00_1796 DUF445 domain- containing protein 50.05 35.16 6.33 2.14 193 1.959 39362 4 1.953 72136 1 1.945 6827 1.937 37263 9 1.937 13986 5 1.936 12980 5 1.933 25251 1.927 50194 1.909 47471 3 1.450 55E- 05 5.729 42E- 05 5.372 06E- 06 4.400 03E- 05 5.507 02E- 06 0.000 23856 8 0.000 20353 1 0.000 19246 9 2.705 34E- 05 Table A-1 (cont’d) No No No No No No C K no homol og found no homol og found S K SAUSA300 _RS13655 SAUSA3 00_2462 SAUSA300 _RS13640 SAUSA3 00_2459 SAUSA300 _RS10690 SAUSA300 _RS04435 SAUSA300 _RS12415 SAUSA3 00_2249 NAD(P)H- dependent oxidoreductase MarR family transcriptional regulator hypothetical protein chorismate mutase CHAP domain- containing protein 59.66 47.8 5.09 4.51 54.31 68.16 6.7 5 27.4 11.26 1.36 1.64 16.94 14.48 2.25 0.78 278.5 3 305.0 2 40.58 19.23 SAUSA300 _RS12490 SAUSA3 00_2261 YafY family protein 183.2 6 148.1 9 22.38 10.7 Yes EH SAUSA300 _RS11940 SAUSA3 00_2166 alsS acetolactate synthase AlsS 15.3 12.12 1.39 1.14 No S SAUSA300 _RS11480 SAUSA3 00_2085 No no homol og found SAUSA300 _RS15825 DUF2750 domain- containing protein 188.8 9 109.3 1 21.61 7.96 transposase 21.6 19.82 2.97 1.22 1.908 98717 5 1.908 46815 6 1.899 76769 9 1.890 64755 8 1.886 81090 5 1.880 31699 2 1.880 18716 7 1.875 05992 6 1.874 95478 4 2.779 05E- 05 0.000 12736 7 0.000 81940 4 0.002 38163 5 4.095 2E-05 9.832 99E- 06 3.391 29E- 05 1.858 88E- 05 0.000 18818 9 194 Table A-1 (cont’d) No No No No No S P no homol og found S S SAUSA300 _RS03180 SAUSA3 00_0592 HD domain- containing protein 87.98 34.06 7.72 3.52 SAUSA300 _RS12660 SAUSA3 00_2291 gltS sodium/glutamat e symporter 13.27 14.67 1.4 1.25 SAUSA300 _RS13775 hypothetical protein 241.2 8 321.8 9 39.21 19.85 SAUSA300 _RS12535 SAUSA3 00_2267 HAD family hydrolase 74.01 62.21 6.97 5.85 SAUSA300 _RS02940 SAUSA3 00_0551 folE2 Yes I SAUSA300 _RS02535 SAUSA3 00_0472 ispE No no homol og found SAUSA300 _RS15405 GTP cyclohydrolase FolE2 4-(cytidine 5'- diphospho)-2-C- methyl-D- erythritol kinase 188.0 3 166.4 3 26.63 10.78 72.98 23.43 6.6 2.45 transposase 11.47 10.09 1.97 0.27 No M SAUSA300 _RS03450 SAUSA3 00_0643 GNAT family N- acetyltransferase 146.8 2 184.1 6 24.03 11.53 No no homol og found SAUSA300 _RS05725 hypothetical protein 12.01 23.82 2.28 1.46 195 1.872 09844 5 1.862 67898 9 1.862 51965 1.853 44532 3 1.842 28825 2 1.839 43461 2 1.834 80243 7 1.834 51461 4 1.814 10521 3.085 19E- 05 0.000 20641 7 0.000 19993 9 4.181 14E- 05 3.598 56E- 05 0.000 20557 9 0.008 34528 1 0.000 15266 0.006 63641 5 Table A-1 (cont’d) Yes S SAUSA300 _RS02775 SAUSA3 00_0518 Yes L SAUSA300 _RS02485 SAUSA3 00_0463 yabA Yes S SAUSA300 _RS10760 SAUSA3 00_1962 Yes P SAUSA300 _RS11750 SAUSA3 00_2134 No U SAUSA300 _RS04110 SAUSA3 00_0762 secG NYN domain- containing protein DNA replication initiation control protein YabA DUF1108 family protein iron chelate uptake ABC transporter family permease subunit preprotein translocase subunit SecG 94.09 45.85 7.92 5.27 67.87 30.67 6.61 3.03 84.77 40.92 5 5.87 70.34 52.01 7.99 4.49 1586. 21 2144. 2 307.2 2 117.3 9 No not classif ied SAUSA300 _RS03445 SAUSA3 00_0642 hypothetical protein 91.32 111.6 1 14.26 7.69 No S SAUSA300 _RS09490 SAUSA3 00_1736 yidD SAUSA300 _RS00590 SAUSA3 00_0114 sarS No No K J membrane protein insertion efficiency factor YidD HTH-type transcriptional regulator SarS 380.3 7 535.4 5 59.17 38.66 195.7 9 230.6 1 28.5 16.99 SAUSA300 _RS08660 SAUSA3 00_1589 dtd D-aminoacyl- tRNA deacylase 289.0 9 115.8 6 30.19 10.56 196 1.811 07936 9 1.808 70223 4 1.804 93507 7 1.804 70979 4 1.801 40828 5 1.799 94223 9 1.798 11070 4 1.797 79920 5 1.794 91439 4 2.635 34E- 05 7.184 61E- 05 0.000 34324 2 1.631 42E- 05 0.000 37516 0.000 19357 9 0.000 33496 1 0.000 13022 6 0.000 13212 2 Table A-1 (cont’d) No No No not classif ied not classif ied no homol og found SAUSA300 _RS05730 SAUSA300 _RS02080 SAUSA3 00_0390 SAUSA300 _RS09565 hypothetical protein hypothetical protein hypothetical protein 28.78 53.13 6.28 3.01 29.79 23.44 3.52 1.99 18.04 13.46 2.79 0.53 SAUSA300 _RS05325 SAUSA3 00_0989 ribonuclease J 663.6 4 360.3 9 72.69 33.59 No J No not classif ied SAUSA300 _RS15095 SAUSA3 00_0428 No P SAUSA300 _RS08160 SAUSA3 00_1495 hypothetical protein rhodanese-like domain- containing protein 14.83 29.95 3.6 1.61 427.1 8 610.3 6 90.88 32.37 No EGP SAUSA300 _RS13160 SAUSA3 00_2379 multidrug effflux MFS transporter 156.4 4 203.5 3 24.91 14.93 No S No not classif ied SAUSA300 _RS02905 SAUSA3 00_0544 Cof-type HAD-IIB family hydrolase 48.43 31.25 5.62 2.78 SAUSA300 _RS06935 SAUSA3 00_1277 hypothetical protein 67.91 81.45 10.89 5.74 197 1.784 87629 8 1.784 21963 7 1.778 64316 1 1.767 03865 6 1.763 58423 1.758 62165 4 1.758 02937 8 1.756 43502 2 1.756 15137 4 0.002 12483 1 7.183 71E- 05 0.011 12162 2 2.031 89E- 05 0.003 12662 5 0.000 76924 2 0.000 2983 3.308 69E- 05 0.000 31422 8 Table A-1 (cont’d) Yes H SAUSA300 _RS14190 SAUSA3 00_2553 NAD(P)-binding protein 203.8 3 96.11 18.93 11 No No No No no homol og found S S no homol og found No S No not classif ied SAUSA300 _RS15500 hypothetical protein 55.39 32.86 3 4.8 SAUSA300 _RS03715 SAUSA3 00_0692 DoxX family protein 1484. 7 458.6 7 124.7 2 63.56 SAUSA300 _RS10750 SAUSA3 00_1960 recombinase RecT 66.81 40.57 4.92 5.27 SAUSA300 _RS16000 minor capsid protein 17.86 28.08 3.59 1.78 SAUSA300 _RS10675 SAUSA3 00_1946 transcriptional activator RinB 62.75 25.43 3.59 4.26 SAUSA300 _RS10755 SAUSA3 00_1961 AAA family ATPase 48.5 27.36 3.34 3.81 No E SAUSA300 _RS00985 SAUSA3 00_0188 brnQ No X SAUSA300 _RS09695 SAUSA3 00_1771 97.47 106.1 1 13.61 8.9 48.56 53.25 8.13 3.69 branched-chain amino acid transport system II carrier protein DUF1828 domain- containing protein 198 1.753 13600 4 1.743 17271 4 1.743 07573 6 1.737 20398 3 1.727 09674 7 1.719 52293 7 1.719 37548 4 1.719 26991 3 1.716 46918 3 3.256 69E- 05 0.001 58401 6 0.000 23661 5 0.000 22483 7 0.001 45406 5 0.001 79340 4 0.000 33738 4 0.000 19993 9 0.000 29813 1 Table A-1 (cont’d) Yes JKL SAUSA300 _RS08285 SAUSA3 00_1518 DEAD/DEAH box helicase 39.64 11.31 3.57 1.51 No U SAUSA300 _RS01845 SAUSA3 00_0348 No P SAUSA300 _RS13050 SAUSA3 00_2363 No S SAUSA300 _RS13005 SAUSA3 00_2355 twin-arginine translocase TatA/TatE family subunit cation diffusion facilitator family transporter DUF4467 domain- containing protein 61.35 67.04 7.49 6.3 43.89 25.19 4.28 2.9 46.01 18.06 5.14 1.73 No no homol og found SAUSA300 _RS15635 SAUSA3 00_2510 hypothetical protein 101.3 126.9 4 15.17 10.59 Yes J SAUSA300 _RS06190 SAUSA3 00_1144 trmFO No not classif ied SAUSA300 _RS06490 No P SAUSA300 _RS11760 SAUSA3 00_2136 methylenetetrahy drofolate--tRNA- (uracil(54)- C(5))- methyltransferas e (FADH(2)- oxidizing) TrmFO hypothetical protein 172.3 69.25 15.26 9.33 47.73 33.75 7.87 1.84 Fe(3+) dicitrate ABC transporter substrate-binding protein 188.9 2 91.93 24.06 7.65 1.714 13593 6 1.704 74902 5 1.699 30836 4 1.696 73033 5 1.694 39063 6 0.000 73575 5 0.000 68447 6 7.184 61E- 05 0.000 83523 0.000 55602 2 1.687 27586 8 0.000 12027 9 1.684 68911 1 1.683 42459 9 0.001 93069 1 0.000 31422 8 199 Table A-1 (cont’d) No P SAUSA300 _RS13020 SAUSA3 00_2358 No I SAUSA300 _RS12345 SAUSA3 00_2236 No S SAUSA300 _RS11025 SAUSA3 00_2005 tsaE 220.0 4 59.36 11.75 13.04 97.08 73.56 5.7 10.29 20.13 12.47 2.89 0.87 amino acid ABC transporter permease acyl-CoA dehydrogenase family protein tRNA (adenosine(37)- N6)- threonylcarbamo yltransferase complex ATPase subunit type 1 TsaE SAUSA300 _RS11740 SAUSA3 00_2132 hypothetical protein 150.9 3 368.8 2 38.3 25.11 SAUSA300 _RS15975 SAUSA3 00_0768 hypothetical protein 39.53 26.78 5.21 2.27 No No No No not classif ied not classif ied no homol og found not classif ied SAUSA300 _RS15905 SAUSA300 _RS06930 No C SAUSA300 _RS12870 SAUSA3 00_2329 hypothetical protein 96.84 155.0 2 20.88 10.19 hypothetical protein 74.61 86.51 12.43 6.67 cation:dicarboxyl ase symporter family transporter 292.5 208.5 6 35.5 20.85 200 1.681 13863 5 1.676 21105 3 0.001 93244 6 0.002 72435 4 1.669 73262 8 0.001 09692 4 1.664 57851 2 1.662 98814 7 1.660 53691 9 1.657 17849 3 1.656 15384 8 0.005 55708 9 0.001 11993 6 0.002 27024 5 0.000 80940 9 5.827 62E- 05 Table A-1 (cont’d) No S SAUSA300 _RS05405 SAUSA3 00_1004 No S SAUSA300 _RS11450 SAUSA3 00_2080 DUF4064 domain- containing protein DUF2529 domain- containing protein 194.9 1 240.4 9 38.37 15.77 57.9 48.55 9.91 3.17 No no homol og found SAUSA300 _RS13480 scrA SaeRS system activator ScrA 34.34 33.12 6.55 1.89 No E SAUSA300 _RS13195 SAUSA3 00_2385 No No No No No not classif ied no homol og found T K S SAUSA300 _RS10680 SAUSA3 00_1947 SAUSA300 _RS12995 SAUSA300 _RS09390 SAUSA3 00_1719 arsC SAUSA300 _RS09835 SAUSA3 00_1797 SAUSA300 _RS03940 SAUSA3 00_0732 67.76 60.73 10.37 5.01 40.26 14.33 1.6 3.16 32.75 11.74 2.79 1.63 33.91 24.06 3.97 2.54 111.0 3 110.6 2 24.08 5.11 60.53 52.27 9.99 3.9 APC family permease hypothetical protein hypothetical protein arsenate reductase (thioredoxin) helix-turn-helix transcriptional regulator YigZ family protein 201 1.655 26972 7 1.648 93889 6 1.647 15243 2 1.639 35185 5 1.632 32548 6 1.632 13518 8 1.631 25639 9 1.627 42977 2 1.626 13142 3 0.000 80690 9 0.000 64581 9 0.001 97426 7 0.000 19507 8 0.007 85912 5 0.009 31050 1 0.000 33561 8 0.003 23772 2 0.000 40956 Table A-1 (cont’d) Yes I SAUSA300 _RS08990 SAUSA3 00_1647 accD No T SAUSA300 _RS11395 SAUSA3 00_2069 acetyl-CoA carboxylase, carboxyltransfera se subunit β low molecular weight protein arginine phosphatase 73.89 17.53 6.93 2.7 58.8 37.2 7.65 3.56 No S SAUSA300 _RS03185 SAUSA3 00_0593 YwhD family protein 270.6 5 186.9 2 38.43 16.36 Yes C SAUSA300 _RS00940 SAUSA3 00_0179 NAD-dependent formate dehydrogenase 25.32 13.8 1.88 2.09 No EG SAUSA300 _RS13720 SAUSA3 00_2472 DMT family transporter 28.16 27.58 3.54 2.84 No L SAUSA300 _RS11145 SAUSA3 00_2026 No No no homol og found not classif ied SAUSA300 _RS05670 SAUSA3 00_1052 ecb SAUSA300 _RS03725 SAUSA3 00_0694 type II toxin- antitoxin system PemK/MazF family toxin complement convertase inhibitor Ecb hypothetical protein 303.0 7 267.2 8 49.84 21.14 624.1 6 640.9 2 114.7 9 46.23 53.58 55.47 8.89 4.56 No S SAUSA300 _RS14600 SAUSA3 00_2628 rarD EamA family transporter RarD 14.96 16.43 2.48 1.38 1.623 18077 2 1.619 68459 3 1.619 22215 6 1.616 89403 3 1.610 33236 8 1.603 50724 4 1.603 23562 3 1.601 71711 5 1.595 71971 7 0.002 77458 9 0.000 22134 3 0.000 15636 6 0.001 01738 8 0.000 69870 3 0.000 34227 3 0.000 62243 9 0.000 59348 8 0.001 11013 6 202 Table A-1 (cont’d) No Znot classif ied SAUSA300 _RS07320 SAUSA3 00_1342 hypothetical protein 72.6 56.24 12.22 4.04 No O SAUSA300 _RS04430 SAUSA3 00_0822 sufB Fe-S cluster assembly protein SufB DNA damage- induced cell division inhibitor SosA 488.4 4 407.9 5 58.61 44.74 24.94 36.32 6.61 1.74 SAUSA300 _RS05685 SAUSA3 00_1054 hypothetical protein 61.87 109.7 9 15.22 7.48 No S SAUSA300 _RS05000 SAUSA3 00_0931 YkvS family protein 412.0 1 333.0 2 75.98 21.64 SAUSA300 _RS06715 No No Znot classif ied Znot classif ied Yes J SAUSA300 _RS08600 SAUSA3 00_1578 mnm A SAUSA300 _RS02480 SAUSA3 00_0462 SAUSA300 _RS10670 SAUSA3 00_1945 No No No S S E tRNA 2- thiouridine(34) synthase MnmA stage 0 sporulation family protein DUF1514 family protein 108.1 45.93 12.87 5.2 40.16 15.18 4.94 1.62 77.39 22.66 4.25 5.4 SAUSA300 _RS12515 SAUSA3 00_2265 amino acid permease 177.9 8 165.9 2 26.76 16.09 203 1.593 94340 7 1.592 29720 3 1.589 58277 7 1.579 69408 6 1.572 45449 6 1.571 14724 9 1.561 43048 7 1.549 10709 6 1.548 63797 9 0.000 84922 8 0.000 28978 0.007 85912 5 0.004 18745 1 0.001 41517 5 0.000 53436 8 0.001 91110 2 0.007 55492 2 0.000 44467 5 Table A-1 (cont’d) No S SAUSA300 _RS09795 SAUSA3 00_1789 DUF3267 domain- containing protein No No J S SAUSA300 _RS04835 SAUSA3 00_0897 trpS tryptophan-- tRNA ligase SAUSA300 _RS09125 SAUSA3 00_1671 HAD family hydrolase Yes I SAUSA300 _RS08985 SAUSA3 00_1646 No No S K SAUSA300 _RS10420 SAUSA3 00_1903 SAUSA300 _RS12900 SAUSA3 00_2336 acetyl-CoA carboxylase carboxyltransfera se subunit α YolD-like family protein MerR family transcriptional regulator 10.34 15.83 3 0.73 176.9 4 181.1 8 161.6 4 72.64 18.67 10.16 51.06 17.44 8.69 40.81 12.76 8.77 23.55 16.87 2.47 2.22 16.05 19.11 3.28 1.46 Yes E SAUSA300 _RS11035 SAUSA3 00_2006 ilvD dihydroxy-acid dehydratase 77.99 73.24 6.57 10.21 No G SAUSA300 _RS01790 SAUSA3 00_0337 glpT glycerol-3- phosphate transporter 19.07 18.53 2.83 1.92 1.544 51003 6 1.536 92679 2 1.522 79607 8 1.520 33318 9 1.517 51130 6 1.515 76496 1 1.508 12546 9 1.507 48102 1 0.011 68031 6 0.000 47404 1 0.002 2461 0.003 43958 9 0.002 11311 7 0.003 01818 5 0.006 30868 1 0.001 21541 204 Table A-1 (cont’d) No K SAUSA300 _RS12510 SAUSA3 00_2264 No KT SAUSA300 _RS08665 SAUSA3 00_1590 Yes S SAUSA300 _RS03690 SAUSA3 00_0687 MurR/RpiR family transcriptional regulator bifunctional (p)ppGpp synthetase/guan osine-3',5'- bis(diphosphate) 3'- pyrophosphohyd rolase hemolysin family protein 189.9 9 121.5 3 26.15 13.14 1.506 09562 3 0.000 29813 1 78.57 29.84 8.98 4.03 264.2 76.41 25.64 13.21 No E SAUSA300 _RS10305 SAUSA3 00_1883 putP sodium/proline symporter PutP 94.24 79.08 13 8.77 No M SAUSA300 _RS08655 SAUSA3 00_1588 N- acetylmuramoyl- L-alanine amidase 186.4 2 93.69 26.29 9.52 No no homol og found SAUSA300 _RS05040 SAUSA3 00_0938 hypothetical protein 24.21 13.03 4.33 0.65 205 1.503 34436 1 0.001 05211 9 1.502 65353 2 1.498 32046 4 1.497 89992 1 1.496 91738 9 0.002 27024 5 0.000 58324 8 0.000 93835 7 0.016 16133 3 Table A-1 (cont’d) No M SAUSA300 _RS03340 SAUSA3 00_0623 No M SAUSA300 _RS05045 SAUSA3 00_0939 Yes S SAUSA300 _RS13755 SAUSA3 00_2479 cidA No not classif ied SAUSA300 _RS03635 No S SAUSA300 _RS06050 SAUSA3 00_1118 No No K S SAUSA300 _RS01375 SAUSA3 00_0258 SAUSA300 _RS10665 SAUSA3 00_1944 Yes S SAUSA300 _RS00020 SAUSA3 00_0003 yaaA N- acetylglucosamin yldiphosphounde caprenol N- acetyl-β-D- mannosaminyltra nsferase TarA glycosyltransfera se holin-like murein hydrolase modulator CidA 51.13 36.6 8.93 2.93 47.35 29.22 7.63 2.56 13.21 14.08 2.19 1.36 hypothetical protein 181.7 1 149.0 3 36.03 10.23 Asp23/Gls24 family envelope stress response protein GntR family transcriptional regulator hypothetical protein S4 domain- containing protein YaaA 161.0 2 103.7 6 135.2 6 67.71 24.19 5.77 66.51 15.49 6.75 80.61 12.3 12.55 449.7 5 303.9 1 65.56 32.56 1.490 17036 0.001 61519 3 1.485 18234 2 1.483 83005 5 1.483 21901 8 1.481 78445 7 1.480 53195 4 1.476 37346 9 1.472 16923 0.001 28773 5 0.005 13774 1 0.002 88603 1 0.005 28626 9 0.000 59054 7 0.001 74690 8 0.000 46708 8 206 Table A-1 (cont’d) no homol og found K S No No No SAUSA300 _RS05780 SAUSA300 _RS08625 SAUSA3 00_1583 hypothetical protein Rrf2 family transcriptional regulator 23.86 11.79 3.42 1.14 182.3 6 158.8 7 34.91 12.67 SAUSA300 _RS13610 SAUSA3 00_2454 ABC transporter permease 136.9 240.3 3 33.92 19.38 No E SAUSA300 _RS09110 SAUSA3 00_1669 No DZ SAUSA300 _RS10915 SAUSA3 00_1985 alanine-- glyoxylate aminotransferas e family protein SdrH family protein 61.48 47.37 8.48 5.52 71.79 43.47 10.37 4.75 SAUSA300 _RS08250 SAUSA3 00_1511 rpmG 50S ribosomal protein L33 2136. 37 2911. 45 611.4 9 156.7 No Yes J L SAUSA300 _RS07130 SAUSA3 00_1309 tnpA No S SAUSA300 _RS10785 SAUSA3 00_1966 149.1 8 117.5 5 18.98 14.74 93.28 54.55 7.6 9.22 IS200/IS605 family transposase phage antirepressor KilAC domain- containing protein 207 1.470 86689 8 1.466 27102 8 1.461 44520 6 1.458 37528 6 1.456 18897 2 1.454 35994 3 1.450 02665 7 1.449 93738 1 0.009 84594 2 0.001 74379 6 0.007 55863 4 0.000 71171 6 0.000 62267 1 0.009 63741 5 0.001 12153 7 0.003 61269 5 Table A-1 (cont’d) Yes H SAUSA300 _RS11040 SAUSA3 00_2007 ilvB biosynthetic-type acetolactate synthase large subunit 32 28.37 3.48 3.85 No no homol og found SAUSA300 _RS16075 SAUSA3 00_1325 hypothetical protein 58.59 74.56 11.9 6.59 No S SAUSA300 _RS13650 SAUSA3 00_2461 SAUSA300 _RS04200 SAUSA3 00_0779 VOC family protein hypothetical protein 37.41 45.32 6.17 4.81 35.52 18.55 4.6 2.3 SAUSA300 _RS06460 SAUSA3 00_1196 hfq RNA chaperone Hfq 173.2 145.9 1 27.04 15.61 SAUSA300 _RS04405 SAUSA3 00_0817 DUF368 domain- containing protein 68.46 74 11.14 7.88 SAUSA300 _RS06510 SAUSA3 00_1204 hypothetical protein 13.37 15.09 2.66 1.27 SAUSA300 _RS13785 No G SAUSA300 _RS14335 SAUSA3 00_2577 manA 68.92 92.59 18.19 6 36.49 42.77 6.4 4.35 hypothetical protein mannose-6- phosphate isomerase, class I 208 No No No No No not classif ied J S not classif ied no homol og found 1.447 32409 7 1.444 8954 1.443 03479 4 1.440 99013 2 1.440 87066 5 1.439 57636 7 1.436 94966 4 1.435 81467 6 1.434 78019 5 0.003 80994 1 0.006 36126 3 0.004 14988 5 0.002 58444 1 0.001 10529 7 0.002 47351 1 0.016 68044 5 0.008 35562 6 0.003 40078 8 Table A-1 (cont’d) No No K J SAUSA300 _RS00420 SAUSA3 00_0082 No H SAUSA300 _RS14050 SAUSA3 00_2532 Yes C SAUSA300 _RS00280 SAUSA3 00_0055 SAUSA300 _RS08905 SAUSA3 00_1632 nrdR transcriptional regulator NrdR 121.1 8 54.72 15.69 6.99 tRNA- dihydrouridine synthase aspartate 1- decarboxylase zinc-dependent alcohol dehydrogenase family protein 36.49 14.62 3.37 2.66 55.14 49.81 9.29 5.09 129.5 5 78.69 8.73 14.51 Yes E SAUSA300 _RS02755 SAUSA3 00_0514 cysE serine O- acetyltransferase 43.79 18.54 5.02 2.82 No G SAUSA300 _RS14330 SAUSA3 00_2576 No No No S S SAUSA300 _RS12620 SAUSA3 00_2284 SAUSA300 _RS11745 SAUSA3 00_2133 not classif ied SAUSA300 _RS12530 PTS fructose transporter subunit IIABC MOSC domain- containing protein YjiH family protein 96.44 137.2 3 16.91 14.84 28.14 19.23 4.05 2.32 43.93 48.18 7.17 5.41 hypothetical protein 245.3 7 316.2 2 40.84 35.72 No M SAUSA300 _RS02345 SAUSA3 00_0438 aaa autolysin/adhesi n Aaa 171.6 7 92.28 23.93 11.67 209 1.431 59773 7 1.424 55196 8 1.423 53577 1.416 52421 7 1.414 36425 8 1.406 24172 8 1.399 18894 2 1.396 03756 1 1.388 84351 3 1.387 59625 6 0.001 42500 6 0.003 86172 9 0.001 96699 3 0.010 12381 3 0.002 03345 8 0.007 99770 6 0.001 67506 2 0.003 84165 3 0.007 56597 2 0.001 05315 Table A-1 (cont’d) No S SAUSA300 _RS05005 SAUSA3 00_0932 No S SAUSA300 _RS12650 SAUSA3 00_2289 No no homol og found SAUSA300 _RS09120 No P SAUSA300 _RS06475 SAUSA3 00_1199 No M SAUSA300 _RS09260 SAUSA3 00_1695 No S SAUSA300 _RS10150 SAUSA3 00_1858 yfkAB CPBP family glutamic-type intramembrane protease DUF805 domain- containing protein 285.9 4 286 70.5 18.18 42.42 44.88 7.92 4.54 hypothetical protein 213.7 3 78.28 26.86 11.41 aminotransferas e class I/II-fold pyridoxal phosphate- dependent enzyme phosphotransfer ase family protein radical SAM/CxCxxxxC motif protein YfkAB 24.91 20.2 3.62 2.52 284.6 127.6 9 42.69 14.61 35.64 15.8 4.29 2.44 No No S K SAUSA300 _RS12735 SAUSA3 00_2304 YdcF family protein 404.7 4 329.8 4 78.14 30.52 SAUSA300 _RS06710 SAUSA3 00_1237 lexA transcriptional repressor LexA 471.5 4 387.5 1 73.52 46.5 1.385 10091 3 1.383 11275 1 1.375 49677 0.008 77972 4 0.003 62052 2 0.005 73130 2 1.370 23067 8 0.002 29806 1 1.369 90349 4 1.365 22991 8 1.364 43482 4 1.359 65185 0.003 65029 5 0.002 27024 5 0.002 54971 5 0.001 70079 1 210 Table A-1 (cont’d) No J SAUSA300 _RS10095 SAUSA3 00_1848 No P SAUSA300 _RS05250 SAUSA3 00_0977 No M SAUSA300 _RS06755 SAUSA3 00_1244 mscL No S SAUSA300 _RS11535 SAUSA3 00_2094 Yes G SAUSA300 _RS11545 SAUSA3 00_2096 No S SAUSA300 _RS12440 SAUSA3 00_2253 Yes E SAUSA300 _RS02380 SAUSA3 00_0445 gltB No EGP SAUSA300 _RS13275 SAUSA3 00_2397 No F SAUSA300 _RS08670 SAUSA3 00_1591 DUF402 domain- containing protein energy-coupling factor transporter transmembranep rotein EcfT large conductance mechanosensitiv e channel protein MscL EVE domain- containing protein class I mannose- 6-phosphate isomerase CHAP domain- containing protein glutamate synthase large subunit 141.9 8 105.9 4 24.03 11.53 13.13 12.19 2.24 1.35 1290. 88 1343. 27 288.6 3 114.5 6 87.56 60.64 14.48 6.79 49.11 21.43 6.21 3.22 160.5 164.4 1 35.71 14.09 78.5 37.18 8.79 6.24 MFS transporter 30.51 24.01 3.1 3.82 adenine phosphoribosyltr ansferase 129.7 6 56.52 17.86 7.75 1.358 10847 5 1.354 1714 1.350 85586 3 1.350 40686 4 1.348 11544 5 1.346 51571 5 1.345 66566 6 1.342 95767 9 1.341 17588 9 0.001 71715 9 0.005 16210 3 0.004 80331 5 0.001 61872 3 0.002 67915 0.005 13774 1 0.002 01178 0.009 19007 2 0.003 21031 3 211 Table A-1 (cont’d) No H SAUSA300 _RS13220 SAUSA3 00_2388 Yes J SAUSA300 _RS02770 SAUSA3 00_0517 rlmB No M SAUSA300 _RS11485 SAUSA3 00_2086 Yes V SAUSA300 _RS09580 SAUSA3 00_1751 2- dehydropantoate 2-reductase 23S rRNA (guanosine(2251 )-2'-O)- methyltransferas e RlmB hypothetical protein restriction endonuclease subunit S 37.75 19.42 4.78 2.92 35.86 12.43 3.87 2.32 31.57 17.72 4.62 2.29 23.34 11.7 3.67 1.37 No J SAUSA300 _RS01950 SAUSA3 00_0368 rpsR 30S ribosomal protein S18 1193. 48 1377. 16 211.7 160.7 6 No G SAUSA300 _RS09395 SAUSA3 00_1720 No No not classif ied not classif ied SAUSA300 _RS10230 SAUSA3 00_1871 SAUSA300 _RS04135 SAUSA3 00_0767 N- acetylglucosamin idase hypothetical protein hypothetical protein 230.7 4 155.2 8 44.78 14.16 11.21 17.89 3.44 1.26 21.31 28.68 5.26 2.52 No K SAUSA300 _RS12325 SAUSA3 00_2232 GNAT family N- acetyltransferase 24.73 12.98 3.13 2 1.339 12120 4 1.338 19171 8 1.335 27242 9 1.323 03140 8 1.320 52840 3 1.319 49404 1.317 95591 1 1.316 63674 2 1.312 80371 1 0.002 24151 6 0.006 29746 5 0.002 13174 4 0.004 93153 7 0.006 93641 6 0.005 28551 7 0.027 98859 9 0.016 10815 5 0.003 77325 212 Table A-1 (cont’d) No F SAUSA300 _RS05970 SAUSA3 00_1102 gmk guanylate kinase 204.4 2 96.8 33.1 11.1 No no homol og found SAUSA300 _RS11885 No P SAUSA300 _RS04300 SAUSA3 00_0796 hypothetical protein methionine ABC transporter ATP- binding protein 26.46 27.79 5.32 2.82 38.65 25.81 4.9 3.94 No No No No No L L P F S SAUSA300 _RS08430 SAUSA3 00_1546 holA DNA polymerase III subunit delta 19.83 15.23 4.28 1.24 SAUSA300 _RS05345 SAUSA3 00_0992 YkyA family protein 196.7 6 166.2 7 45.06 13.18 SAUSA300 _RS04955 SAUSA3 00_0922 TerC family protein 173.3 7 172.3 5 34.48 18.67 SAUSA300 _RS08550 SAUSA3 00_1568 udk uridine kinase 149.7 6 58.64 23.67 7.14 SAUSA300 _RS11735 SAUSA3 00_2131 metal-dependent hydrolase 79.04 54.84 13.23 6.8 No G SAUSA300 _RS10415 SAUSA3 00_1902 beta-propeller fold lactonase family protein 152.9 6 54.17 16.85 10.86 1.305 89543 2 1.304 40647 1 1.300 35872 9 1.298 89407 5 1.297 23529 3 1.284 11193 6 1.276 60253 4 1.273 26640 5 1.272 69841 4 0.005 87962 2 0.016 55111 3 0.003 86172 9 0.010 57318 0.009 34947 6 0.005 16210 3 0.011 90017 0.003 36443 1 0.006 86438 2 213 Table A-1 (cont’d) Yes E SAUSA300 _RS11065 SAUSA3 00_2012 leuC No F SAUSA300 _RS02075 SAUSA3 00_0389 guaA No J SAUSA300 _RS06455 SAUSA3 00_1195 miaA No S SAUSA300 _RS05865 SAUSA3 00_1082 3- isopropylmalate dehydratase large subunit glutamine- hydrolyzing GMP synthase tRNA (adenosine(37)- N6)- dimethylallyltrans feraseMiaA YggS family pyridoxal phosphate- dependent enzyme 20.69 10.39 2.56 1.75 683.8 5 496.3 7 131.3 4 52.41 50.03 41.51 10.21 4.29 90.45 18.25 10.67 4.05 No No No No S S S A SAUSA300 _RS07195 SAUSA3 00_1321 bacilliredoxin BrxA 158.7 182.2 4 33.62 19.98 SAUSA300 _RS03545 SAUSA3 00_0660 SAUSA300 _RS01810 SAUSA3 00_0341 SAUSA300 _RS02045 SAUSA3 00_0383 DUF456 domain- containing protein YeiH family protein hypothetical protein 63.61 94.19 15.26 9.91 21.29 24.82 4.46 2.77 158.2 100.1 29.15 11.23 1.272 00293 1.271 32022 8 1.258 00725 1 0.004 75079 7 0.004 19132 8 0.006 03220 1 1.257 07792 8 1.254 26703 5 1.249 73368 8 1.248 74336 8 1.247 77718 9 0.032 28346 0.009 59696 8 0.018 93260 4 0.012 10829 1 0.005 28551 7 214 Table A-1 (cont’d) No S SAUSA300 _RS03610 SAUSA3 00_0673 No S SAUSA300 _RS13395 SAUSA3 00_2420 No F SAUSA300 _RS08180 SAUSA3 00_1499 GTP-binding protein DUF1433 domain- containing protein 71.89 81.92 14.82 9.38 12.45 10.01 2.93 0.79 shikimate kinase 84.65 84.64 16.47 10.07 No G SAUSA300 _RS06435 SAUSA3 00_1191 MIP/aquaporin family protein 61.75 77.99 15.24 7.79 No S SAUSA300 _RS05690 SAUSA3 00_1055 efb No O SAUSA300 _RS06465 SAUSA3 00_1197 No K SAUSA300 _RS03605 SAUSA3 00_0672 mgrA SAUSA300 _RS10805 No No no homol og found not classif ied complement convertase inhibitor Efb glutathione peroxidase HTH-type transcriptional regulator MgrA transcriptional regulator 157.6 4 51.02 21.7 8.61 257.3 7 246.9 4 51.13 28.85 10215 .53 6639. 23 2075. 02 663.1 2 51.87 51.53 7.93 7.37 SAUSA300 _RS03240 SAUSA3 00_0603 hypothetical protein 79.8 57.29 13.22 7.76 215 1.240 62916 9 1.233 58465 8 1.232 98450 8 1.229 98893 4 1.229 12856 2 1.227 60832 8 1.226 51415 5 1.224 01226 7 1.224 00905 5 0.010 46696 6 0.029 95832 2 0.008 39026 9 0.014 53771 6 0.014 77427 2 0.007 12503 6 0.009 41309 8 0.019 71775 5 0.004 62397 3 Table A-1 (cont’d) Yes L SAUSA300 _RS08900 SAUSA3 00_1631 replication initiation and membrane attachment family protein 38.67 10.3 4.78 2.11 No H SAUSA300 _RS12260 SAUSA3 00_2219 moaA GTP 3',8-cyclase MoaA 39.26 17.5 5.65 2.78 No V SAUSA300 _RS13605 SAUSA3 00_2453 No S SAUSA300 _RS03525 SAUSA3 00_0657 Yes H SAUSA300 _RS11050 SAUSA3 00_2009 ilvC no homol og found K S No No No SAUSA300 _RS08890 SAUSA300 _RS10800 ATP-binding cassette domain- containing protein DUF402 domain- containing protein ketol-acid reductoisomeras e 58.55 53.07 13.06 5.48 52.73 29.16 8.59 4.1 20.65 13.93 3.04 2.17 hypothetical protein 904.2 8 1279. 89 302.9 2 93.32 helix-turn-helix transcriptional regulator 67.8 50.72 8.01 9.05 SAUSA300 _RS05695 SAUSA3 00_1056 scb complement inhibitor SCIN-B 191.4 5 101.4 7 33.86 13.22 216 1.220 85193 5 1.215 72261 8 0.021 88066 0.007 55863 4 1.204 61912 0.010 41708 1.198 55853 1.196 36089 3 1.193 40428 6 1.178 65765 4 1.175 21708 1 0.006 87217 4 0.008 42999 2 0.032 61905 5 0.022 29163 9 0.009 95154 3 Table A-1 (cont’d) No no homol og found SAUSA300 _RS07845 No H SAUSA300 _RS09465 SAUSA3 00_1730 metK No not classif ied SAUSA300 _RS04215 Yes E SAUSA300 _RS06640 SAUSA3 00_1225 No S SAUSA300 _RS03995 No S SAUSA300 _RS13320 SAUSA3 00_2405 hypothetical protein 246.1 6 171.2 7 42.45 24.01 methionine adenosyltransfer ase hypothetical protein 222.5 5 164.9 9 40.92 21.89 50.22 48.82 12.3 4.8 aspartate kinase 36.67 21.13 6.66 2.77 CsbA family protein CPBP family lipoprotein N- acylation protein LnsB 23.37 25.11 6.84 1.88 33.91 42.42 9.76 3.9 SAUSA300 _RS07045 SAUSA3 00_1296 msaA regulatory protein MsaA 75.87 58.62 12.87 8.55 No No S S SAUSA300 _RS02910 SAUSA3 00_0545 No G SAUSA300 _RS12460 SAUSA3 00_2256 NADPH- dependent FMN reductase N- acetylglucosamin idase 152.8 2 129.6 3 31.42 15.85 48.97 24.28 8.33 3.5 217 1.168 13638 2 1.167 11073 5 1.164 37255 2 1.159 05377 4 1.158 73752 3 1.154 89972 7 1.153 77095 3 1.153 72675 8 1.142 08471 7 0.007 57869 0.006 59009 3 0.020 89623 0.009 89382 8 0.046 02923 6 0.027 67165 6 0.011 11118 1 0.009 95980 9 0.012 78423 7 Table A-1 (cont’d) No J SAUSA300 _RS10300 SAUSA3 00_1882 gatC No K SAUSA300 _RS10060 SAUSA3 00_1842 perR No L SAUSA300 _RS08675 SAUSA3 00_1592 recJ No No K S SAUSA300 _RS14275 SAUSA3 00_2566 SAUSA300 _RS00960 SAUSA3 00_0183 No S SAUSA300 _RS13310 SAUSA3 00_2403 No S SAUSA300 _RS10795 No T SAUSA300 _RS09085 SAUSA3 00_1665 Asp- tRNA(Asn)/Glu- tRNA(Gln) amidotransferas e subunit GatC peroxide- responsive transcriptional repressor PerR single-stranded- DNA-specific exonuclease RecJ Crp/Fnr family transcriptional regulator YagU family protein DUF1307 domain- containing protein DUF2829 domain- containing protein GAF domain- containing protein 218 31.63 21.33 7.42 1.88 1089. 67 1425. 44 309.6 7 142.5 2 40.63 12.69 6.23 2.16 18.07 16.91 3.88 2.07 669.0 7 851.0 3 107.1 6 130.9 2 328.5 6 390.1 6 88.57 40.57 125.1 6 72.27 12.11 15.99 96.91 75.87 17.75 10.9 1.137 43769 9 0.038 87032 6 1.133 66594 3 1.132 89977 8 1.131 04366 1 1.126 45836 2 1.122 00643 9 1.118 72981 6 1.115 69292 3 0.027 12680 7 0.031 54264 7 0.019 59857 7 0.045 75790 6 0.024 43578 6 0.036 35245 1 0.012 01021 6 Table A-1 (cont’d) No K SAUSA300 _RS12805 SAUSA3 00_2318 No S SAUSA300 _RS03785 SAUSA3 00_0704 GNAT family N- acetyltransferase ABC-F family ATP-binding cassette domain- containing protein 29.12 13.13 4.4 2.3 24.76 10.19 4.31 1.49 SAUSA300 _RS04195 SAUSA3 00_0778 hypothetical protein 49.17 23.9 7.52 4.14 SAUSA300 _RS05705 hypothetical protein 99.57 68.67 18.39 9.88 SAUSA300 _RS02070 SAUSA3 00_0388 guaB IMP dehydrogenase 170.9 95.65 29.65 14.65 No No No No not classif ied no homol og found F S SAUSA300 _RS13170 SAUSA3 00_2381 No S SAUSA300 _RS05400 SAUSA3 00_1003 No EGP SAUSA300 _RS12235 SAUSA3 00_2217 No H SAUSA300 _RS14065 SAUSA3 00_2535 C39 family peptidase DUF4064 domain- containing protein 53.1 56.51 11.8 7.28 817.4 399.2 6 125.9 5 70.47 MFS transporter 15.16 15.95 2.92 2.3 oxidoreductase 62.47 66.33 12.81 9.17 219 1.108 93827 6 1.103 68294 1.100 88045 4 1.100 08968 1 1.099 77313 8 1.094 42907 7 1.093 71469 5 1.093 29894 5 1.092 34415 6 0.023 72204 2 0.026 50557 7 0.023 49894 1 0.015 17820 2 0.010 46696 6 0.023 98781 0.011 34414 0.030 63703 0.025 00889 Table A-1 (cont’d) No M SAUSA300 _RS04935 SAUSA3 00_0918 No S SAUSA300 _RS11150 mazE No O SAUSA300 _RS08835 SAUSA3 00_1621 clpX No U SAUSA300 _RS11265 SAUSA3 00_2046 yidC No No S P SAUSA300 _RS02370 SAUSA3 00_0443 SAUSA300 _RS03405 SAUSA3 00_0635 No J SAUSA300 _RS09255 SAUSA3 00_1694 trmB No No E P SAUSA300 _RS06675 SAUSA3 00_1231 SAUSA300 _RS08270 SAUSA3 00_1515 34.8 14.53 5.05 2.79 333.3 4 355.5 1 89.76 37.35 247.4 1 346.2 5 76.03 33.69 16.79 176 61.76 26.6 21.74 21.15 5.18 2.52 148.0 2 109.3 9 25.24 17.57 192.6 6 112.8 6 41.54 13.17 92.52 65.14 14.47 11.31 29.89 18.53 4.3 3.46 1.090 02689 4 1.084 16903 8 1.082 92858 6 1.081 71912 9 1.079 12510 6 1.078 86147 4 1.073 04437 5 1.070 53652 2 1.068 11384 8 0.018 53083 3 0.028 34720 4 0.030 63703 0.015 04576 0.024 60168 6 0.014 25540 4 0.026 48225 1 0.016 92635 5 0.021 34235 5 diglucosyl diacylglycerol synthase type II toxin- antitoxin system antitoxin MazE ATP-dependent Clp protease ATP-binding subunit ClpX membrane protein insertase YidC YibE/F family protein iron ABC transporter permease tRNA (guanosine(46)- N7)- methyltransferas e TrmB amino acid permease metal ABC transporter permease 220 Table A-1 (cont’d) No No No No No No No H H S S E K S SAUSA300 _RS08815 SAUSA3 00_1617 hemC hydroxymethylbil ane synthase 69.66 38.64 10.69 7 SAUSA300 _RS04475 SAUSA3 00_0829 lipA lipoyl synthase 609.9 2 403.1 7 94.8 72.1 SAUSA300 _RS02950 SAUSA3 00_0553 YojF family protein 69.63 55.93 15.25 7.4 SAUSA300 _RS10240 SAUSA3 00_1872 SAUSA300 _RS07930 SAUSA3 00_1452 proC SAUSA300 _RS12730 SAUSA3 00_2303 type 1 glutamine amidotransferas e pyrroline-5- carboxylate reductase MarR family transcriptional regulator 84.42 34.78 13.93 6.27 70.76 53.81 12.44 8.94 1378. 09 1057. 11 334.3 4 122.6 4 SAUSA300 _RS02530 SAUSA3 00_0471 Veg family protein 1717. 12 2224. 68 498 260.6 1 No S SAUSA300 _RS08290 SAUSA3 00_1519 No L SAUSA300 _RS10815 SAUSA3 00_1970 58.94 29.78 10.56 5.04 39.35 29.05 9.36 3.58 Nif3-like dinuclear metal center hexameric protein exonuclease domain- containing protein 221 1.067 52749 1 1.055 54954 1 1.045 38281 3 1.034 75426 2 1.033 01388 2 1.032 20521 4 1.014 74517 5 1.007 20848 1 1.005 6324 0.014 65515 5 0.016 68044 5 0.023 36741 5 0.028 23519 1 0.022 28597 0.027 67165 6 0.047 95060 2 0.024 43578 6 0.033 43446 2 Table A-1 (cont’d) Genes downregulated in WT sulfur starvation when compared to WT CSSC Share d with cymR: :Tn -S (Table 3) TPM. 1 Starv Gene Product Old locus Locus COG TPM. 2 Starv TPM. 1 CSSC TPM. 2 CSSC DE Log2 FC No F SAUSA300 _RS05215 SAUSA3 00_0970 purQ No F SAUSA300 _RS05230 SAUSA3 00_0973 purM No F SAUSA300 _RS05220 SAUSA3 00_0971 purL Yes F SAUSA300 _RS05210 SAUSA3 00_0969 purS No F SAUSA300 _RS05235 SAUSA3 00_0974 purN phosphoribosylfo rmylglycinamidin e synthase I phosphoribosylfo rmylglycinamidin e cyclo-ligase phosphoribosylfo rmylglycinamidin e synthase subunit PurL phosphoribosylfo rmylglycinamidin e synthase subunit PurS phosphoribosylgl ycinamide formyltransferase 0.86 0.22 40.41 23.03 0.77 0.13 29.22 21.31 0.89 0.41 36.43 30.92 1.35 0.49 72.1 33.02 0.85 0.19 31.59 19.8 222 DE Adj. P- value 4.620 44E- 51 3.325 62E- 44 1.591 7E-61 - 7.111 86249 8 - 7.096 25442 8 - 7.036 78370 4 - 6.951 03635 4.968 82E- 48 - 6.891 35605 3 1.592 32E- 44 Table A-1 (cont’d) No F SAUSA300 _RS05225 SAUSA3 00_0972 purF amidophosphorib osyltransferase 1.31 0.57 38.02 34.89 No F SAUSA300 _RS05200 SAUSA3 00_0967 purK No F SAUSA300 _RS05240 SAUSA3 00_0975 purH No F SAUSA300 _RS05205 SAUSA3 00_0968 purC Yes F SAUSA300 _RS05245 SAUSA3 00_0976 purD Yes S SAUSA300 _RS04760 SAUSA3 00_0883 0.74 0.25 28.8 13.9 1.56 0.53 46.45 29.42 1 0.83 38.29 15.25 16.71 11.24 317.5 9 233.7 9 41.55 17.26 414.1 4 432.3 2 5- (carboxyamino)i midazole ribonucleotide synthase bifunctional phosphoribosyla minoimidazoleca rboxamide formyltransferase /IMP cyclohydrolase phosphoribosyla minoimidazolesu ccinocarboxamid e synthase phosphoribosyla mine--glycine ligase MAP domain- containing protein 223 - 6.655 96430 1 - 6.654 59084 2 - 6.515 50422 8 - 5.981 58317 1 - 5.644 00475 5 - 5.302 89579 8 5.458 43E- 52 8.323 01E- 50 1.998 21E- 51 1.239 56E- 41 3.705 64E- 47 1.583 78E- 32 Table A-1 (cont’d) No no homol og found SAUSA300 _RS11930 SAUSA3 00_2164 Yes S SAUSA300 _RS03005 MAP domain- containing protein C1q-binding complement inhibitor VraX 32.26 11.96 158.3 288.1 1 25631 .12 16475 .97 18299 6.97 23280 9.5 No S SAUSA300 _RS13975 SAUSA3 00_2518 alpha/beta hydrolase 9.82 6.47 43.36 106.5 4 Yes not classif ied SAUSA300 _RS03000 No S SAUSA300 _RS13280 SAUSA3 00_2398 hypothetical protein 637.5 3 308.7 4 4243. 07 3649. 74 iron export ABC transporter permease subunit FetB 19.01 15.86 78.94 208.8 8 No no homol og found SAUSA300 _RS15545 SAUSA3 00_2141 IS1182 family transposase 5.88 2.45 28.69 30 No S SAUSA300 _RS11805 SAUSA3 00_2142 asp23 Asp23/Gls24 family envelope stress response protein 594.6 8 188.2 8 2619. 07 2540. 32 - 4.847 76974 2 - 4.736 80252 5 - 4.645 38792 6 - 4.509 44012 9 - 4.455 02968 5 - 4.307 27829 3 - 4.231 06999 8 1.234 11E- 19 7.233 62E- 25 2.060 63E- 16 1.585 52E- 27 1.801 83E- 14 2.895 63E- 21 2.443 86E- 19 224 Table A-1 (cont’d) No GM SAUSA300 _RS11550 SAUSA3 00_2097 SDR family oxidoreductase 10.23 6 46.7 59.05 No G SAUSA300 _RS01005 SAUSA3 00_0191 ptsG Yes F SAUSA300 _RS05195 SAUSA3 00_0966 purE Yes S SAUSA300 _RS13155 SAUSA3 00_2378 Yes O SAUSA300 _RS09140 SAUSA3 00_1674 glucose-specific PTS transporter subunit IIBC 5- (carboxyamino)i midazole ribonucleotide mutase membrane protein trypsin-like peptidase domain- containing protein 6.6 4.59 36.3 35.28 1.66 2.67 19.08 13.89 52.31 22.37 212.3 8 198.0 4 41.35 23.5 184.6 7 173.5 8 Yes no homol og found SAUSA300 _RS09670 SAUSA3 00_1767 epiA gallidermin/nisin family lantibiotic 5.99 11.07 61.45 44.39 No Q SAUSA300 _RS00950 SAUSA3 00_0181 non-ribosomal peptide synthetase 11.5 4.61 39.43 35.55 - 4.169 36178 3 - 4.092 07745 2 - 4.089 78647 1 - 3.948 96652 1 - 3.921 11562 2 - 3.774 82775 1 - 3.720 75236 1 6.346 11E- 19 2.162 3E-21 7.690 67E- 16 1.910 46E- 19 2.103 26E- 20 1.140 84E- 12 3.134 34E- 17 225 Table A-1 (cont’d) Yes Yes no homol og found no homol og found SAUSA300 _RS15740 SAUSA300 _RS15735 Yes G SAUSA300 _RS02960 SAUSA3 00_0555 phenol-soluble modulin PSM- alpha-1 phenol-soluble modulin PSM- alpha-2 3-hexulose-6- phosphate synthase 24086 .72 14456 .95 80513 .32 90835 .34 32964 .12 19614 10066 3.79 12803 9.36 12.15 4.89 29.42 40.12 No M SAUSA300 _RS01750 SAUSA3 00_0329 aldehyde reductase 11.47 5.05 20.74 45.12 No no homol og found SAUSA300 _RS06215 No F SAUSA300 _RS14175 SAUSA3 00_2551 nrdD No G SAUSA300 _RS13740 SAUSA3 00_2476 ptsG hypothetical protein 30.93 27.32 128.6 6 128.8 5 anaerobic ribonucleoside- triphosphate reductase glucose-specific PTS transporter subunit IIBC 16.57 6.48 28.93 55.94 4.83 2.17 11.81 14.77 - 3.629 96614 1 - 3.612 45600 6 - 3.575 86678 2 - 3.563 95695 3 - 3.524 48483 3 - 3.467 18251 1 - 3.457 84172 2 6.553 75E- 16 1.137 77E- 14 1.019 85E- 12 2.425 18E- 10 3.071 67E- 14 2.932 64E- 10 7.116 11E- 13 226 Table A-1 (cont’d) No I SAUSA300 _RS13270 SAUSA3 00_2396 pnbA carboxylesterase /lipase family protein 19.06 8.99 38.39 66.14 No M SAUSA300 _RS13750 SAUSA3 00_2478 cidB LrgB family protein 18.38 13.8 44.89 79.26 No F SAUSA300 _RS09165 SAUSA3 00_1678 fhs No H SAUSA300 _RS00955 SAUSA3 00_0182 Yes no homol og found SAUSA300 _RS15090 No C SAUSA300 _RS11360 SAUSA3 00_2063 atpE formate-- tetrahydrofolate ligase 4'- phosphopanteth einyl transferase superfamily protein phenol-soluble modulin PSM- alpha-3 F0F1 ATP synthase subunit C 60.98 26.29 177.7 3 153.6 3 26.78 24.44 117.5 8 96.52 36859 .41 23267 .68 98132 .17 12580 6.27 32.97 17.66 90.41 96.99 No I SAUSA300 _RS00355 SAUSA3 00_0070 alpha/beta hydrolase 18.61 12.36 54.45 61.27 - 3.454 53656 3 - 3.427 31880 8 - 3.423 73895 5 - 3.421 76003 8 - 3.387 87570 3 - 3.379 19918 1 - 3.368 05660 6 4.360 95E- 11 8.019 27E- 11 2.171 7E-15 3.038 73E- 15 6.416 37E- 13 1.177 99E- 13 1.506 26E- 13 227 Table A-1 (cont’d) No not classif ied SAUSA300 _RS02995 SAUSA3 00_0561 protein VraC 6.12 2.31 14.5 14.94 Yes M SAUSA300 _RS02965 SAUSA3 00_0556 6-phospho-3- hexuloisomerase 30.97 21.99 68.49 115.7 8 Yes J SAUSA300 _RS02850 SAUSA3 00_0533 tuf elongation factor Tu 1122. 7 991.5 6 3207. 41 4132. 39 Yes H SAUSA300 _RS04995 SAUSA3 00_0930 lipoate--protein ligase 7.75 5.74 33.94 16.76 No S SAUSA300 _RS12470 SAUSA3 00_2257 No S SAUSA300 _RS11810 SAUSA3 00_2143 DUF1641 domain- containing protein DUF2273 domain- containing protein 13.01 7.36 32.33 35.22 234.9 2 76.71 547.9 3 459.1 1 No M SAUSA300 _RS07485 SAUSA3 00_1370 ebpS elastin-binding protein EbpS 78.5 57.76 222 No O SAUSA300 _RS04245 SAUSA3 00_0786 37.67 28.21 92.19 organic hydroperoxide resistance protein 228 241.3 6 128.0 5 - 3.316 34688 - 3.293 19601 4 - 3.226 53168 5 - 3.224 47977 3 - 3.219 98161 4 - 3.219 37747 1 - 3.216 09147 9 - 3.212 64378 8 1.581 93E- 11 2.372 57E- 10 3.215 03E- 11 2.558 79E- 15 2.305 72E- 12 3.210 76E- 12 1.088 08E- 12 6.578 98E- 11 Table A-1 (cont’d) No I SAUSA300 _RS03070 SAUSA3 00_0574 phosphomevalon ate kinase 5.16 2.82 10.06 15.37 No K SAUSA300 _RS06210 SAUSA3 00_1148 codY No P SAUSA300 _RS00900 SAUSA3 00_0171 No G SAUSA300 _RS14010 SAUSA3 00_2525 No S SAUSA300 _RS10180 SAUSA3 00_1864 Yes no homol og found SAUSA300 _RS15730 GTP-sensing pleiotropic transcriptional regulator CodY cation diffusion facilitator family transporter fructosamine kinase family protein YihY/virulence factor BrkB family protein phenol-soluble modulin PSM- alpha-4 67.53 37.35 156.1 4 178.6 8 8.63 4.65 17.97 23.15 14.48 11.97 27.03 55.83 20.06 12.9 37.38 57.47 28076 .72 19700 .69 67198 .08 73350 .18 No IQ SAUSA300 _RS12575 SAUSA3 00_2275 SDR family oxidoreductase 32.61 21.9 52.95 99.17 - 3.202 01967 3 - 3.182 82773 3 - 3.151 36336 7 - 3.133 04462 5 - 3.042 09501 8 - 3.023 94534 4 - 3.007 62496 4 3.828 96E- 10 3.373 04E- 12 6.665 8E-11 2.071 98E- 08 1.711 95E- 09 2.170 06E- 11 2.196 04E- 08 229 Table A-1 (cont’d) No G SAUSA300 _RS04090 SAUSA3 00_0758 tpiA triose-phosphate isomerase 15.05 5.81 21.08 34.31 No D SAUSA300 _RS01705 SAUSA3 00_0320 No O SAUSA300 _RS14170 SAUSA3 00_2550 nrdG No G SAUSA300 _RS04495 SAUSA3 00_0833 No G SAUSA300 _RS14100 SAUSA3 00_2540 YSIRK domain- containing triacylglycerol lipase Lip2/Geh anaerobic ribonucleoside- triphosphate reductase activating protein TIGR01457 family HAD-type hydrolase fructose bisphosphate aldolase 140.9 7 59.25 207.4 9 325.4 9 18.17 13.98 23.29 64.75 9.43 3.31 16.45 16.48 199.8 2 89.39 418.8 8 342.2 3 Yes E SAUSA300 _RS04565 SAUSA3 00_0845 ampA M17 family metallopeptidase 22.02 15.65 63.44 42.71 No F SAUSA300 _RS04085 SAUSA3 00_0757 pgk phosphoglycerat e kinase 35.94 20.82 53.97 89.46 - 3.005 65227 3 - 3.000 79235 3 - 2.974 52011 3 - 2.921 97974 6 - 2.899 28851 4 - 2.890 04652 9 - 2.887 3278 1.948 75E- 08 6.986 97E- 09 8.516 14E- 07 1.450 98E- 09 1.132 91E- 11 7.308 74E- 13 2.545 18E- 08 230 Table A-1 (cont’d) No no homol og found SAUSA300 _RS12420 hypothetical protein 319.1 5 307.0 9 1125. 37 722.8 No H SAUSA300 _RS10075 SAUSA3 00_1845 hemL glutamate-1- semialdehyde 2,1- aminomutase 10.28 6.22 23.73 20.27 No Q SAUSA300 _RS00990 SAUSA3 00_0189 entB isochorismatase family protein 4.71 5.33 15.04 13.73 No S SAUSA300 _RS09455 SAUSA3 00_1728 aldo/keto reductase 20.21 12.56 38.57 43.51 Yes H SAUSA300 _RS01160 SAUSA3 00_0221 pflA No C SAUSA300 _RS05170 SAUSA3 00_0962 qoxB No G SAUSA300 _RS03675 SAUSA3 00_0685 fruA pyruvate formate-lyase- activating protein cytochrome aa3 quinol oxidase subunit I fructose-specific PTS transporter subunit EIIC 9.12 6.73 10.15 27.96 69.9 32.65 87.12 148.1 5 16.67 15.01 19.71 57.27 - 2.883 80010 5 - 2.877 79601 6 - 2.858 09828 8 - 2.822 57080 4 - 2.803 28741 7 - 2.789 08636 3 - 2.787 50809 3 1.401 91E- 11 1.195 83E- 11 4.369 57E- 09 8.706 81E- 10 3.780 05E- 06 1.305 22E- 07 5.889 31E- 06 231 Table A-1 (cont’d) No F SAUSA300 _RS05190 SAUSA3 00_0965 folD bifunctional methylenetetrahy drofolate dehydrogenase/ methenyltetrahyd rofolate cyclohydrolase FolD 71.94 31.74 114.2 4 122.8 3 Yes S SAUSA300 _RS13645 SAUSA3 00_2460 GNAT family N- acetyltransferase 24.61 28.86 99.44 51.19 No C SAUSA300 _RS06765 SAUSA3 00_1246 acnA aconitate hydratase AcnA 51.23 22.95 65.18 95.79 No H SAUSA300 _RS01405 SAUSA3 00_0262 rbsK ribokinase 6.74 6.85 13.94 18.15 No not classif ied SAUSA300 _RS09275 SAUSA3 00_1698 No C SAUSA300 _RS05355 SAUSA3 00_0994 pdhB YtxH domain- containing protein transketolase C- terminal domain- containing protein 58.85 39.14 125.7 101.9 3 28.65 13.24 52.12 40.31 - 2.743 03919 7 - 2.731 65759 2 - 2.704 64100 5 - 2.662 07194 7 - 2.661 54169 7 - 2.638 28701 9 3.236 24E- 09 2.966 38E- 09 1.046 43E- 07 2.193 34E- 07 2.039 9E-10 5.044 02E- 10 232 Table A-1 (cont’d) No S SAUSA300 _RS05995 SAUSA3 00_1107 No C SAUSA300 _RS11365 SAUSA3 00_2064 atpB TM2 domain- containing protein F0F1 ATP synthase subunit A 1035. 35 1191. 02 2439. 26 2604. 9 28.1 13 42.65 39.51 No C SAUSA300 _RS01155 SAUSA3 00_0220 pflB formate C- acetyltransferase 39.54 24.31 38.44 84.3 Yes G SAUSA300 _RS04670 SAUSA3 00_0865 pgi glucose-6- phosphate isomerase 91.76 46.06 156.9 7 120.8 5 No S SAUSA300 _RS08360 SAUSA3 00_1533 flotillin-like protein FloA 123.5 9 67.69 180.4 9 194.3 5 No not classif ied SAUSA300 _RS08355 SAUSA3 00_1532 hypothetical protein 121.6 8 52.38 174.5 8 163.1 1 No C SAUSA300 _RS03190 SAUSA3 00_0594 adh alcohol dehydrogenase AdhP 159.2 5 78.73 184.0 5 250.8 - 2.557 12615 6 - 2.527 32706 3 - 2.523 75975 4 - 2.501 36028 7 - 2.498 33243 2 - 2.498 17972 3 - 2.445 44814 1 2.264 1E-07 1.564 47E- 08 8.494 84E- 06 1.950 23E- 09 4.016 57E- 08 2.779 85E- 08 7.984 98E- 07 233 Table A-1 (cont’d) No not classif ied SAUSA300 _RS04225 SAUSA3 00_0781 hypothetical protein 132.8 8 103.7 9 248.9 9 225.5 7 No S SAUSA300 _RS14620 SAUSA3 00_2632 No I SAUSA300 _RS13795 SAUSA3 00_2484 No K SAUSA300 _RS03665 SAUSA3 00_0683 HdeD family acid-resistance protein hydroxymethylgl utaryl-CoA synthase DeoR/GlpR family DNA- binding transcription regulator 51.71 37.15 64.51 102.9 9 84.76 62.34 164.8 9 131.1 7 108.2 2 53.2 152.7 7 145.0 6 No KOT SAUSA300 _RS10935 SAUSA3 00_1989 agrB accessory gene regulator AgrB 926.3 4 501.3 5 1575. 92 1121. 46 No P SAUSA300 _RS04605 SAUSA3 00_0853 mnhC No S SAUSA300 _RS04220 SAUSA3 00_0782 Na+/H+ antiporter Mnh1 subunit C sterile α motif- like domain- containing protein 34.2 17.04 54.14 41.16 248.0 1 192.6 1 383.7 3 439.2 - 2.442 93308 9 - 2.442 67942 1 - 2.440 14220 6 2.898 06E- 08 2.785 67E- 06 6.511 91E- 09 - 2.412 71013 5.295 21E- 08 - 2.386 48572 5 - 2.383 38330 7 - 2.378 10837 3 4.643 79E- 09 2.392 95E- 08 4.781 11E- 07 234 Table A-1 (cont’d) No C SAUSA300 _RS05360 SAUSA3 00_0995 Yes J SAUSA300 _RS03960 SAUSA3 00_0736 yfiA dihydrolipoamide acetyltransferase family protein ribosome- associated translation inhibitor RaiA 26.25 11.84 32.46 34.41 1334. 07 1434. 27 3386. 21 2297. 34 No G SAUSA300 _RS06725 SAUSA3 00_1239 tkt transketolase 83.27 51.86 117.5 1 123.9 8 No E SAUSA300 _RS03030 SAUSA3 00_0566 amino acid permease 30.32 20.78 39.1 50.82 Yes not classif ied SAUSA300 _RS13040 SAUSA3 00_2361 No P SAUSA300 _RS00595 SAUSA3 00_0115 sirC Yes J SAUSA300 _RS02575 SAUSA3 00_0479 putative metal homeostasis protein staphyloferrin B ABC transporter permease subunit SirC 50S ribosomal protein L25/general stress proteinCtc 749.1 1675. 86 1920. 36 3051. 79 29.97 24.63 44.59 53.55 698.2 1 446.9 2 1187. 14 887.4 2 - 2.369 85281 6 - 2.362 78535 7 - 2.346 32222 5 - 2.334 23180 4 - 2.333 27593 4 - 2.319 75872 4 - 2.308 26470 7 3.817 64E- 07 1.387 71E- 07 2.208 95E- 07 1.685 95E- 06 0.000 18971 5 1.636 74E- 06 1.896 93E- 08 235 Table A-1 (cont’d) No S SAUSA300 _RS11815 SAUSA3 00_2144 No K SAUSA300 _RS03250 SAUSA3 00_0605 sarA alkaline shock response membrane anchor protein AmaP global transcriptional regulator SarA 329 156.7 4 463.1 5 377.1 8 550.5 4 628.5 1240 1037. 55 No O SAUSA300 _RS10900 SAUSA3 00_1982 groEL chaperonin GroEL 74.38 38.88 95.38 86.68 No T SAUSA300 _RS09015 SAUSA3 00_1652 universal stress protein 315.7 260.7 3 601.5 9 401.9 1 No No Yes not classif ied no homol og found no homol og found SAUSA300 _RS12160 SAUSA3 00_2206 hypothetical protein 95.1 78.95 192.5 3 113.2 4 SAUSA300 _RS08615 SAUSA3 00_1581 SAS049 family protein 549.0 5 509.8 8 1111. 67 745.8 SAUSA300 _RS08745 hypothetical protein 144.2 5 117.9 4 318.8 9 146.6 8 - 2.303 96945 4 - 2.300 81455 - 2.204 73838 5 - 2.184 55110 2 - 2.172 23099 1 - 2.171 93099 9 - 2.141 16857 7.388 59E- 08 1.061 32E- 06 4.680 7E-07 1.895 01E- 07 2.051 21E- 07 5.079 68E- 07 5.300 06E- 07 236 Table A-1 (cont’d) No C SAUSA300 _RS11335 SAUSA3 00_2058 atpD No G SAUSA300 _RS04080 SAUSA3 00_0756 gap Yes S SAUSA300 _RS09825 SAUSA3 00_1795 No G SAUSA300 _RS05420 SAUSA3 00_1007 F0F1 ATP synthase subunit beta type I glyceraldehyde- 3-phosphate dehydrogenase YlbF/YmcA family competence regulator inositol monophosphatas e 47.87 24.09 63.18 48.62 1310. 2 855.8 6 881 2226. 45 388.1 6 375.7 7 977.9 5 392.6 6 198.7 4 144.3 1 276.3 247.6 9 No G SAUSA300 _RS00740 SAUSA3 00_0141 deoB phosphopentom utase 54.96 32.81 52.9 69.29 No no homol og found SAUSA300 _RS10940 SAUSA3 00_1990 agrD cyclic lactone autoinducer peptide 923.0 1 345.8 1 1059. 02 737.2 3 Yes S SAUSA300 _RS07160 SAUSA3 00_1314 YozE family protein 272.8 6 134.5 5 352.3 3 236.9 4 No M SAUSA300 _RS07315 SAUSA3 00_1341 pbp2 67.46 48.33 91.11 78.4 transglycosylase domain- containing protein 237 - 2.135 68903 6 - 2.124 84429 6 - 2.094 66604 1 - 2.075 75697 2 - 2.034 36517 7 - 2.026 64328 3 - 2.014 72078 - 2.012 42643 3 4.397 88E- 07 0.000 37335 4 3.342 79E- 06 2.121 78E- 06 3.501 49E- 05 5.166 29E- 06 1.263 39E- 06 3.342 79E- 06 Table A-1 (cont’d) No S SAUSA300 _RS12165 SAUSA3 00_2207 NCS2 family permease 90.94 73.55 138.3 3 109.0 2 Yes no homol og found SAUSA300 _RS15985 hypothetical protein 1448. 5 2256. 86 2477. 47 3277. 31 No G SAUSA300 _RS11445 SAUSA3 00_2079 fba Yes no homol og found SAUSA300 _RS13850 SAUSA3 00_2493 fructose- bisphosphate aldolase cell wall inhibition responsive protein CwrA 1472. 76 839.2 1540. 16 1574. 67 290.1 5 271.0 8 514.4 8 325.6 3 No T SAUSA300 _RS00340 SAUSA3 00_0067 universal stress protein 291.2 304.9 7 352.7 9 478.6 4 Yes C SAUSA300 _RS14075 SAUSA3 00_2537 L-lactate dehydrogenase 43.61 41.66 69.96 54.33 No M SAUSA300 _RS09205 SAUSA3 00_1684 Yes S SAUSA300 _RS13765 SAUSA3 00_2481 68.77 30.08 93.4 43.63 4957. 16 6293. 84 8840. 81 7590. 77 membrane protein sterile α motif- like domain- containing protein 238 - 2.012 31831 - 2.003 38213 9 - 1.962 91082 5 - 1.938 12754 9 - 1.937 70956 5 - 1.932 80898 3 - 1.901 90508 - 1.894 72036 4 3.115 05E- 06 0.000 40347 6 1.481 11E- 05 8.182 39E- 06 0.000 22882 5 1.701 4E-05 9.180 74E- 06 0.000 13022 6 Table A-1 (cont’d) No S SAUSA300 _RS01990 SAUSA3 00_0374 No G SAUSA300 _RS04230 SAUSA3 00_0783 GlsB/YeaQ/Ymg E family stress response membrane protein histidine phosphatase family protein 1269. 32 1302. 36 1739. 99 1802. 05 - 1.886 60073 0.000 10990 7 48.94 40.27 64.72 56.33 No S SAUSA300 _RS02055 SAUSA3 00_0385 general stress protein 282.4 5 309.0 5 401.3 4 416.9 8 No K SAUSA300 _RS13560 SAUSA3 00_2445 No S SAUSA300 _RS12400 SAUSA3 00_2246 Yes G SAUSA300 _RS07165 SAUSA3 00_1315 crr MerR family transcriptional regulator PH domain- containing protein PTS glucose transporter subunit IIA 44.52 36.46 55.51 52.21 81.81 60.2 106.8 1 80.57 306.2 5 144.1 1 347.8 4 219.6 No S SAUSA300 _RS08620 SAUSA3 00_1582 CsbD family protein 668.9 555.4 1 868.9 6 717.1 9 239 - 1.881 09355 8 - 1.880 19719 9 - 1.866 86995 4 - 1.839 01213 1 - 1.817 02368 2 - 1.806 68243 2.729 16E- 05 0.000 15069 2 4.347 83E- 05 1.655 75E- 05 1.383 01E- 05 4.248 47E- 05 Table A-1 (cont’d) No F SAUSA300 _RS08975 SAUSA3 00_1644 pyk pyruvate kinase 329.3 123.8 1 273.9 8 249.2 1 No S SAUSA300 _RS13385 SAUSA3 00_2418 carboxymuconol actone decarboxylase family protein 75.26 62.59 97.35 75.63 Yes J SAUSA300 _RS06310 SAUSA3 00_1166 rpsO 30S ribosomal protein S15 515.9 4 553.3 3 962.4 8 482.1 6 No G SAUSA300 _RS04100 SAUSA3 00_0760 eno surface- displayed α- enolase 368.4 2 166.6 238.8 5 325.7 7 No D SAUSA300 _RS02550 SAUSA3 00_0475 septation regulator SpoVG 611.8 4 344.5 3 528.7 9 535.0 2 No P SAUSA300 _RS07060 SAUSA3 00_1299 toxic anion resistance protein 99.85 40.68 79.45 73.06 No S SAUSA300 _RS05960 SAUSA3 00_1100 VOC family protein 135.4 8 94.36 158.5 8 112.6 6 No S SAUSA300 _RS01545 SAUSA3 00_0289 TIGR01741 family protein 60.83 50.32 85.5 50.93 - 1.788 19780 2 - 1.750 96406 5 - 1.722 18715 3 - 1.699 15174 7 - 1.695 68955 - 1.693 09711 9 - 1.682 59702 7 - 1.672 70061 9 0.000 14408 9 6.825 37E- 05 0.000 16530 3 0.000 94837 3 0.000 21307 5 0.000 28290 8 6.167 04E- 05 8.871 08E- 05 240 Table A-1 (cont’d) No O SAUSA300 _RS05460 SAUSA3 00_1015 ctaA heme A synthase 52.3 38.18 43.04 51.9 No not classif ied SAUSA300 _RS11230 SAUSA3 00_2041 Lmo0850 family protein 564.4 4 834.1 5 615.2 7 857.4 1 Yes C SAUSA300 _RS03045 SAUSA3 00_0569 heme-dependent peroxidase 174.2 3 122.9 6 200.1 6 110.8 8 No Yes no homol og found no homol og found SAUSA300 _RS10555 hypothetical protein 315.0 9 400.4 376.6 1 351.2 2 SAUSA300 _RS04395 SAUSA3 00_0815 ear DUF4888 domain- containing protein 273.4 262.3 2 451.3 3 144.3 9 No L SAUSA300 _RS07430 SAUSA3 00_1362 hup HU family DNA- binding protein 27446 .33 29697 .55 33446 .33 23578 .56 No S SAUSA300 _RS12820 SAUSA3 00_2320 76.41 81.38 63.99 72.04 DUF2871 domain- containing protein 241 - 1.595 46374 3 - 1.488 72026 6 - 1.465 51025 1 - 1.407 06884 8 - 1.385 16038 2 - 1.362 58825 3 - 1.242 29455 4 0.001 21776 8 0.010 59881 7 0.000 42298 0.006 81252 5 0.004 94624 8 0.003 87335 3 0.018 18165 9 Table A-1 (cont’d) No no homol og found SAUSA300 _RS15795 type I toxin- antitoxin system Fst family toxin PepA1 567.9 5 857.8 4 707.2 2 620.2 1 No D SAUSA300 _RS07290 SAUSA3 00_1337 cell division regulator GpsB 150.5 2 42 94.14 49.94 - 1.241 63196 4 - 1.118 77976 7 0.023 28664 8 0.030 34066 242 Table A-2. CymR regulon. Genes upregulated in cymR::Tn CSSC when compared to WT CSSC COG Locus Old locus Gene Product TPM. 1 cymR TPM. 2 cymR TPM. 1 WT TPM. 2 WT S S O P I M U S A SAUSA300 _RS10985 SAUSA3 00_1998 YeeE/YedE family protein 14.91 45.03 0.74 0.43 SAUSA300 _RS00910 SAUSA3 00_0173 DUF4242 domain- containing protein 112.9 3 431.8 1 4.54 4.88 SAUSA300 _RS10980 SAUSA3 00_1997 SAUSA300 _RS00925 SAUSA3 00_0176 SAUSA300 _RS00930 SAUSA3 00_0177 SAUSA300 _RS13915 SAUSA3 00_2506 SAUSA300 _RS02035 SAUSA3 00_0382 SAUSA300 _RS05050 SAUSA3 00_0940 SAUSA300 _RS02045 SAUSA3 00_0383 sulfurtransferase TusA family protein 61.56 165.7 8 2.67 2.08 ABC transporter permease acyl-CoA/acyl-ACP dehydrogenase 27.65 65.91 1.5 0.99 50.29 74.56 2.77 1.16 isaA lytic transglycosylase IsaA L-cystine transporter 11584 .71 594.8 1 1520. 78 2546. 04 DoxX family protein 89.95 169.4 7 hypothetical protein 404.4 9 285.2 1 189. 31 45.7 2 10.5 4 29.1 5 89.28 82.63 3.61 11.23 DE Log2 FC 3.435 17904 8 3.430 29289 5 3.324 84131 4 3.109 13158 3.059 22871 9 2.806 84014 5 2.699 31265 8 2.368 99952 6 2.290 27817 5 DE Adj. P- value 5.131 E-07 0.0000 01335 9.084 E-07 0.0000 02476 5.169 E-07 0.0012 55 0.0001 428 0.0006 815 0.0002 041 243 Table A-2 (cont’d) P P S P L E SAUSA300 _RS01055 SAUSA3 00_0200 ABC transporter ATP- binding protein 28.76 56.72 1.44 2.45 SAUSA300 _RS02340 SAUSA3 00_0437 gmpC SAUSA300 _RS12610 SAUSA3 00_2282 sdpC dipeptide ABC transporter glycylmethionine-binding lipoprotein CPBP family intramembrane glutamic endopeptidaseSdpC 123.3 4 372.0 9 10.1 6 14.16 86.59 133.0 2 10.9 4.13 SAUSA300 _RS00915 SAUSA3 00_0174 ABC transporter ATP- binding protein 8.87 33.74 2.15 0.58 SAUSA300 _RS05345 SAUSA3 00_0992 SAUSA300 _RS02635 SAUSA3 00_0491 YkyA family protein cysK cysteine synthase A 205.8 9 639.2 9 7140. 91 3976. 77 45.0 6 143. 08 13.18 389.9 1 not classifi ed SAUSA300 _RS09430 hypothetical protein 49.89 138.2 4 9.87 3.23 E P D SAUSA300 _RS02325 SAUSA3 00_0434 bifunctional cystathionine γ-lyase/homocysteine desulfhydrase 44.68 14.89 1.09 1.95 SAUSA300 _RS02335 SAUSA3 00_0436 methionine ABC transporter permease 18.51 76.09 2.72 2.91 SAUSA300 _RS09440 SAUSA3 00_1726 CrcB family protein 11.73 38.91 1.79 1.43 2.265 61963 5 2.193 65352 5 2.104 25690 8 2.053 18192 4 2.048 37860 8 2.035 04764 1 2.020 10295 2 0.0037 35 0.0069 66 0.0022 8 0.0126 6 0.0138 2 0.0119 9 0.0138 8 1.988 23317 1.976 59445 6 1.976 02321 2 0.0225 0.0203 6 0.0095 13 244 EP SAUSA300 _RS01060 SAUSA3 00_0201 ABC transporter permease Table A-2 (cont’d) S V P P I SAUSA300 _RS01875 SAUSA3 00_0354 SAUSA300 _RS13605 SAUSA3 00_2453 SAUSA300 _RS00920 SAUSA3 00_0175 SAUSA300 _RS02330 SAUSA3 00_0435 SAUSA300 _RS12685 SAUSA3 00_2296 S S E no homolo g found ET SAUSA300 _RS13940 SAUSA3 00_2511 SAUSA300 _RS04485 SAUSA300 _RS02855 SAUSA3 00_0831 SAUSA3 00_0534 SAUSA300 _RS07460 SAUSA3 00_1366 SAUSA300 _RS13025 SAUSA3 00_2359 low temperature requirement protein A 12.65 27.98 1.85 1.02 ATP-binding cassette domain-containing protein 67.04 179.7 3 13.0 6 5.48 ABC transporter substrate-binding protein methionine ABC transporter ATP-binding protein alpha/beta hydrolase DUF896 domain- containing protein DUF3055 domain- containing protein M20 family metallopeptidase 13.13 31.1 2.57 0.88 18.79 54.32 2.36 2.49 126.1 1 251.3 5 21.0 3 8.72 16.22 29.78 0.74 1.91 46.83 47.5 202.1 7 218.2 5 11.9 6.79 17.4 1 4.1 22.75 37.22 3.57 1.5 hypothetical protein 70.61 48.87 8.01 2.2 transporter substrate- binding domain-containing protein 70.83 138.4 5 11.7 2 6.67 245 1.933 38716 3 1.906 19927 1.904 44637 4 1.904 14439 5 1.878 24894 3 1.834 29987 2 1.824 78807 2 1.822 08257 1.784 98265 1.724 74927 6 1.693 26231 4 0.0140 4 0.0165 4 0.0224 9 0.0113 5 0.0321 1 0.0364 9 0.0462 3 0.0136 4 0.0225 0.0235 2 0.0266 3 Table A-2 (cont’d) S not classifi ed M K S P S M F SAUSA300 _RS13320 SAUSA3 00_2405 CPBP family lipoprotein N- acylation protein LnsB 52.3 92.92 9.76 3.9 SAUSA300 _RS10230 SAUSA3 00_1871 hypothetical protein 17.61 31.65 3.44 1.26 SAUSA300 _RS05540 SAUSA3 00_1029 isdA LPXTG-anchored heme- scavenging protein IsdA 148.9 229.4 9 28 9.65 SAUSA300 _RS12705 SAUSA3 00_2300 TetR/AcrR family transcriptional regulator 39.08 65.85 7.6 2.72 SAUSA300 _RS11450 SAUSA3 00_2080 DUF2529 domain- containing protein SAUSA300 _RS00605 SAUSA3 00_0117 sirA staphyloferrin B ABC transporter substrate- binding protein SirA 43.3 86.72 9.91 3.17 23.01 28.48 3.46 1.62 SAUSA300 _RS12440 SAUSA3 00_2253 CHAP domain-containing protein 332.0 1 177.4 1 35.7 1 14.09 SAUSA300 _RS03340 SAUSA3 00_0623 N- acetylglucosaminyldiphos phoundecaprenol N- acetyl-β-D- mannosaminyltransferase TarA 45.52 68.2 8.93 2.93 SAUSA300 _RS07465 SAUSA3 00_1367 cmk (d)CMP kinase 87.02 69.66 11.2 9 5.19 1.653 57398 7 1.646 39224 1 1.646 10995 7 1.620 36634 8 1.618 41672 3 1.608 09829 1 1.587 54982 5 1.531 81557 1 1.529 86373 1 0.0385 3 0.0300 8 0.0338 2 0.0453 2 0.0255 4 0.0278 2 0.0389 6 0.0300 8 0.0278 4 246 Table A-2 (cont’d) OU E not classifi ed E no homolo g found S SAUSA300 _RS04060 SAUSA3 00_0752 clpP SAUSA300 _RS00985 SAUSA3 00_0188 brnQ1 SAUSA300 _RS04205 SAUSA3 00_0780 SAUSA300 _RS07075 SAUSA3 00_1300 brnQ3 SAUSA300 _RS15260 SAUSA3 00_0937 SAUSA300 _RS09900 SAUSA3 00_1809 ATP-dependent Clp endopeptidase proteolytic subunit ClpP branched-chain amino acid transport system II carrier protein lipoprotein N-acylation protein LnsA branched-chain amino acid transport system II carrier protein hypothetical protein PTS transporter subunit IIC 1027. 25 715.2 4 81.89 139.0 1 109. 04 13.6 1 67.12 8.9 16.59 12.18 2.19 0.86 31.17 23.58 4.13 1.79 262.3 3 350.4 4 43.6 2 23.66 1.524 99860 2 1.502 77736 8 1.498 23469 3 1.468 59987 3 1.454 37516 2 40.1 47.08 6.38 3.33 -1.454 Genes downregulated in cymR::Tn CSSC when compared to WT CSSC COG Locus Old locus Gene Product TPM. 1 cymR TPM. 2 cymR TPM. 1 WT TPM. 2 WT Q H SAUSA300 _RS00950 SAUSA3 00_0181 non-ribosomal peptide synthetase 8.18 7.12 SAUSA300 _RS00955 SAUSA3 00_0182 4'-phosphopantetheinyl transferase superfamily protein 32.46 19.17 39.4 3 117. 58 35.55 96.52 DE Log2 FC - 3.459 24731 5 - 3.245 07261 2 0.0453 2 0.0498 3 0.0376 6 0.0453 2 0.0453 2 0.0453 2 DE Adj. P- value 1.966 E-09 3.242 E-08 247 Table A-2 (cont’d) C C F SAUSA300 _RS00800 SAUSA3 00_0151 adhE bifunctional acetaldehyde- CoA/alcohol dehydrogenase 3.63 2.35 SAUSA300 _RS03190 SAUSA3 00_0594 adhP alcohol dehydrogenase AdhP 80.52 51.88 SAUSA300 _RS14175 SAUSA3 00_2551 nrdD anaerobic ribonucleoside- triphosphate reductase 17.5 10.03 10.0 6 184. 05 28.9 3 12.51 250.8 55.94 no homolo g found no homolo g found no homolo g found no homolo g found SAUSA300 _RS15730 SAUSA300 _RS15735 SAUSA300 _RS15090 SAUSA300 _RS15740 phenol-soluble modulin PSM-alpha-4 9480. 7 29491 .47 6719 8.08 7335 0.18 phenol-soluble modulin PSM-alpha-2 15900 .9 49372 .47 1006 63.7 9 1280 39.36 phenol-soluble modulin PSM-alpha-3 15050 .63 48596 .27 9813 2.17 1258 06.27 phenol-soluble modulin PSM-alpha-1 12580 .61 37405 .9 8051 3.32 9083 5.34 - 3.132 78682 5 - 2.963 09354 1 - 2.862 05864 3 - 2.833 24622 2 - 2.804 71220 3 - 2.800 29420 6 - 2.785 89174 5 5.131 E-07 0.0000 02476 0.0000 3716 0.0000 9013 0.0001 313 0.0001 415 0.0001 135 248 Table A-2 (cont’d) no homolo g found no homolo g found SAUSA300 _RS11930 SAUSA3 00_2164 MAP domain-containing protein 112.6 7 54.61 158. 3 288.1 1 SAUSA300 _RS16065 hypothetical protein 5.51 2.81 F SAUSA300 _RS05225 SAUSA3 00_0972 purF amidophosphoribosyltrans ferase 28.41 4.72 10.9 1 38.0 2 10.1 34.89 no homolo g found SAUSA300 _RS15985 hypothetical protein 1916. 24 1268. 31 2477 .47 3277. 31 F G E M SAUSA300 _RS04085 SAUSA3 00_0757 phosphoglycerate kinase 35.37 43.12 SAUSA300 _RS04080 SAUSA3 00_0756 gap type I glyceraldehyde-3- phosphate dehydrogenase 1063. 41 916.2 5 SAUSA300 _RS06650 SAUSA3 00_1227 SAUSA300 _RS00400 SAUSA3 00_0079 thrC threonine synthase 14.77 13.7 YdhK family protein 531.3 3 177.0 6 53.9 7 881 26.5 5 555. 41 89.46 2226. 45 12.51 355.0 3 249 - 2.688 55628 5 - 2.507 91720 1 - 2.319 54928 1 - 2.224 68341 5 - 2.196 42910 9 - 2.032 92912 4 - 1.739 92651 - 1.710 13977 0.0001 321 0.0004 496 0.0041 3 0.0012 55 0.0030 47 0.0133 7 0.0108 3 0.0257 7 Table A-2 (cont’d) no homolo g found SAUSA300 _RS16085 SAUSA3 00_1988 delta-lysin family phenol- soluble modulin 13968 7.59 11566 8.38 1718 45.7 1 1415 12.48 KOT SAUSA300 _RS10935 SAUSA3 00_1989 agrB accessory gene regulator AgrB 1532. 17 771.5 1575 .92 1121. 46 O SAUSA300 _RS04245 SAUSA3 00_0786 organic hydroperoxide resistance protein 113.5 1 84.07 92.1 9 128.0 5 no homolo g found SAUSA300 _RS09675 gallidermin/nisin family lantibiotic 14.87 9.96 13.4 6 14.32 K SAUSA300 _RS10950 SAUSA3 00_1992 LytTR family DNA-binding domain-containing protein 886.0 4 496.3 1 978. 24 525.3 4 - 1.698 54326 3 - 1.628 80481 3 - 1.623 95907 3 - 1.580 81294 6 - 1.490 74049 7 0.0136 4 0.0238 9 0.0364 9 0.0453 2 0.0378 3 250 Table A-3. cymR::Tn sulfur starvation. Genes upregulated in cymR::Tn sulfur starvation when compared to cymR::Tn CSSC Share d with WT -S (Table 1) TPM. 1 Starv TPM. 2 Starv Gene Product COG Locus Old locus TPM. 1 CSSC TPM. 2 CSSC DE Log2 FC No K SAUSA300 _RS03085 SAUSA3 00_0577 Yes no homol og found SAUSA300 _RS16040 Yes H SAUSA300 _RS03730 SAUSA3 00_0695 Yes E SAUSA300 _RS11070 SAUSA3 00_2013 leuD Yes H SAUSA300 _RS03735 SAUSA3 00_0696 Yes no homol og found SAUSA300 _RS07125 redox-sensitive transcriptional regulator HypR imidazole glycerol phosphate synthase subunit HisF 7-carboxy-7- deazaguanine synthase QueE 3-isopropylmalate dehydratase small subunit 6- carboxytetrahydro pterin synthase QueD 129.2 2 208.2 9 2.85 2.38 62.91 69.67 1.25 1.08 158.3 8 170.6 1 5.52 3.19 46.54 87.07 2.11 1.25 123.2 2 128.8 7 4.38 2.46 4.455 32441 1 4.335 57811 2 3.790 84639 6 3.758 67167 7 3.734 84001 7 hypothetical protein 7557. 16 5803. 42 126.2 3 213.0 9 3.727 55005 1.15E -10 No E SAUSA300 _RS14510 SAUSA3 00_2610 hisC histidinol- phosphate transaminase 12.41 21.95 0.36 0.56 3.584 75176 5 3.14E -09 251 DE Adj. P- value 9.67E -19 1.43E -19 1.08E -13 1.6E- 11 8.85E -13 Table A-3 (cont’d) No J SAUSA300 _RS02765 SAUSA3 00_0516 Mini-ribonuclease 3 74.72 204.0 3 3.31 4.49 Yes E SAUSA300 _RS11055 SAUSA3 00_2010 leuA 2-isopropylmalate synthase 74.75 96.74 3.34 2.36 Yes E SAUSA300 _RS14090 SAUSA3 00_2539 Yes C SAUSA300 _RS02385 SAUSA3 00_0446 gltD Yes J SAUSA300 _RS02770 SAUSA3 00_0517 Yes E SAUSA300 _RS11075 SAUSA3 00_2014 ilvA 127.3 2 101.5 3 844.6 8 835.1 1 4.15 3.82 30.82 30.48 97.82 237.2 6 6.14 6.25 131.9 6 156.3 8 7.44 4.71 3.171 46442 1.47E -10 aspartate aminotransferase family protein glutamate synthase subunit β 23S rRNA (guanosine(2251) -2'-O)- methyltransferase RlmB threonine ammonia-lyase IlvA imidazoleglycerol- phosphate dehydratase HisB No Yes No Yes E S K H SAUSA300 _RS14505 SAUSA3 00_2609 hisB 10.94 17.9 0.28 0.71 SAUSA300 _RS02775 SAUSA3 00_0518 SAUSA300 _RS02780 SAUSA3 00_0519 NYN domain- containing protein 197.0 7 RNA polymerase sigma factor 52.53 407.7 5 171.8 8 13.49 12.5 4.57 4.36 SAUSA300 _RS11050 SAUSA3 00_2009 ilvC ketol-acid reductoisomerase 35.82 71.09 1.99 2.61 252 3.490 20884 9 3.486 00630 4 3.415 57416 2 3.362 78813 4 3.205 91305 6 2.98E -08 8.57E -13 1.6E- 11 2.28E -12 3.62E -08 3.161 53044 0.000 00581 3.054 29171 2 3.031 08725 3.006 18371 6 2.56E -08 0.000 00314 0.000 00020 8 Table A-3 (cont’d) No E SAUSA300 _RS11770 SAUSA3 00_2137 No EGP SAUSA300 _RS11780 SAUSA3 00_2139 staphyloferrin A biosynthesis protein SfaC staphyloferrin A export MFS transporter 161.8 9 189.0 7 10.54 6.29 19.37 36.64 0.73 1.54 No E SAUSA300 _RS11045 SAUSA3 00_2008 ilvN ACT domain- containing protein 40.75 71.61 1.7 3 Yes P SAUSA300 _RS10250 SAUSA3 00_1874 H-type ferritin FtnA 4155. 65 3220. 95 239.9 7 120.6 8 Yes P SAUSA300 _RS11525 SAUSA3 00_2092 dps Dps family protein 14143 .25 8173. 12 657.6 3 425.5 9 Yes O SAUSA300 _RS02020 SAUSA3 00_0379 ahpF Yes H SAUSA300 _RS11040 SAUSA3 00_2007 ilvB Yes K SAUSA300 _RS00650 SAUSA3 00_0126 No EGP SAUSA300 _RS00625 SAUSA3 00_0121 3971. 04 3699. 54 254.3 7 126.6 5 53.7 75.39 3.36 3.01 89.65 70.75 5.35 2.49 11.22 11.03 0.49 0.58 alkyl hydroperoxide reductase subunit F biosynthetic-type acetolactate synthase large subunit bifunctional transcriptional regulator/O- phospho-L-serine synthase SbnI staphyloferrin B export MFS transporter 253 2.993 80187 7 2.993 60462 9 2.987 88901 6 2.953 71107 3 2.948 65299 4 2.939 03260 6 2.936 86154 6 3.4E- 09 0.000 00405 0.000 00223 0.000 00016 4 0.000 00036 1 7.14E -08 2.48E -09 2.934 58969 8 0.000 00044 3 2.921 52969 1 0.000 00021 1 Table A-3 (cont’d) No E SAUSA300 _RS14495 SAUSA3 00_2607 hisA No P SAUSA300 _RS04800 SAUSA3 00_0890 oppF Yes S SAUSA300 _RS14550 SAUSA3 00_2618 Yes P SAUSA300 _RS12340 SAUSA3 00_2235 Yes O SAUSA300 _RS02025 SAUSA3 00_0380 ahpC No P SAUSA300 _RS05560 SAUSA3 00_1033 1-(5- phosphoribosyl)- 5-((5- phosphoribosyla mino)methylidene amino)imidazole- 4- carboxamide isomerase ATP-binding cassette domain- containing protein ECF-type riboflavin transporter substrate-binding protein ABC transporter substrate-binding protein alkyl hydroperoxide reductase subunit C hemin ABC transporter permease protein IsdF 12.66 18.8 0.61 0.82 103.2 5 184.9 3.44 8.95 59.48 61.13 1.84 3.77 539.0 1 417.5 4 22.65 26.15 7815. 91 7102. 56 433.1 5 381.5 8 35.02 25.1 1.07 1.91 Yes S SAUSA300 _RS04570 SAUSA3 00_0846 Na+/H+ antiporter family protein 178.1 4 170.7 7 7.69 10.87 2.914 91007 8 0.000 00093 7 2.887 78593 4 2.878 37950 6 2.876 61163 1 2.824 06232 8 2.816 18807 3 2.792 91763 1 0.000 0196 0.000 00367 7.62E -08 8.5E- 09 0.000 00694 0.000 00026 9 254 Table A-3 (cont’d) Yes E SAUSA300 _RS01070 SAUSA3 00_0203 Yes E SAUSA300 _RS11065 SAUSA3 00_2012 leuC No Q SAUSA300 _RS11775 SAUSA3 00_2138 No P SAUSA300 _RS14545 SAUSA3 00_2617 ABC transporter substrate-binding protein 3-isopropylmalate dehydratase large subunit staphyloferrin A synthetase SfaB ABC transporter ATP-binding protein 63.79 125.8 2 4.39 5.53 24.04 59.93 2.65 1.78 31.11 50.58 2.13 2.41 23.41 40.7 1.18 2.18 Yes F SAUSA300 _RS00725 SAUSA3 00_0138 deoD purine-nucleoside phosphorylase 45.01 45.75 3.84 1.42 No P SAUSA300 _RS03875 SAUSA3 00_0720 Yes E SAUSA300 _RS01075 SAUSA3 00_0204 ggt ABC transporter ATP-binding protein γ- glutamyltransfera se 15.44 26.79 1.18 1.2 281.5 7 322.8 7 20.97 15.89 No No Yes S F C SAUSA300 _RS05565 SAUSA3 00_1034 SAUSA300 _RS03740 SAUSA3 00_0697 SAUSA300 _RS07500 SAUSA3 00_1373 srtB class B sortase 40.4 24.82 1.21 2.41 7-cyano-7- deazaguanine synthase QueC ferredoxin 29.94 31.62 2.54 1.2 4511. 53 4046. 46 373.4 5 160.1 7 2.765 49278 9 2.747 84485 1 2.727 96356 7 2.710 45101 7 2.679 76217 1 2.678 60090 9 2.678 15357 4 2.638 14982 9 2.635 10957 2 2.619 42294 0.000 00108 0.000 00601 0.000 00029 7 0.000 0119 0.000 0144 0.000 00299 2.66E -08 0.000 0906 0.000 00581 0.000 00702 255 Table A-3 (cont’d) No J SAUSA300 _RS14535 SAUSA3 00_2615 GNAT family protein 20.23 28.74 1.13 1.78 Yes S SAUSA300 _RS10920 SAUSA3 00_1986 nitroreductase family protein 83.95 156.7 8 6.44 8.15 No G SAUSA300 _RS14530 SAUSA3 00_2614 polysaccharide deacetylase family protein 20.11 28.81 1.39 1.73 Yes no homol og found SAUSA300 _RS10765 SAUSA3 00_1963 DUF2482 family protein 20.32 109.1 6 0.63 4.63 Yes M SAUSA300 _RS14270 SAUSA3 00_2565 clfB No No E E SAUSA300 _RS14525 SAUSA3 00_2613 hisZ SAUSA300 _RS14515 SAUSA3 00_2611 hisD MSCRAMM family adhesin clumping factor ClfB ATP phosphoribosyltra nsferase regulatory subunit histidinol dehydrogenase 139.0 2 176 10.8 10.81 14.33 17.64 0.78 1.3 17.69 21.44 0.47 1.77 Yes NOU SAUSA300 _RS08765 SAUSA3 00_1609 A24 family peptidase 21.53 21.48 1.11 1.78 Yes H SAUSA300 _RS14190 SAUSA3 00_2553 NAD(P)-binding protein 581.7 8 450.4 1 46.1 28.47 2.576 86926 4 2.576 74117 1 2.537 72120 2 2.529 31563 6 0.000 0221 0.000 00606 0.000 00405 0.010 58 2.493 37701 7 0.000 00036 1 2.469 30811 7 2.467 9434 2.443 25680 5 2.441 96926 5 0.000 0603 0.001 37 0.000 0578 0.000 00746 256 Table A-3 (cont’d) Yes O SAUSA300 _RS10980 SAUSA3 00_1997 Yes C SAUSA300 _RS06395 SAUSA3 00_1183 Yes no homol og found SAUSA300 _RS14185 sulfurtransferase TusA family protein 2- oxoacid:ferredoxi n oxidoreductase subunit β 1885. 83 1726. 83 61.56 165.7 8 290.5 2 386.8 6 27.28 22.34 hypothetical protein 898.3 8 767.6 2 76.27 46.76 Yes C SAUSA300 _RS04255 SAUSA3 00_0788 nitroreductase 1218. 82 1274. 44 88.01 94.12 Yes EH SAUSA300 _RS11940 SAUSA3 00_2166 alsS acetolactate synthase AlsS 16.19 19.64 1.05 1.48 No M SAUSA300 _RS13525 SAUSA3 00_2440 fnbB fibronectin- binding protein FnbB 27.16 22.5 1.99 1.75 Yes S SAUSA300 _RS10760 SAUSA3 00_1962 DUF1108 family protein 17.32 116.3 2 1.16 5.34 Yes S SAUSA300 _RS14570 SAUSA3 00_2622 Yes P SAUSA300 _RS02335 SAUSA3 00_0436 rhodanese- related sulfurtransferase methionine ABC transporter permease 402.4 6 389.2 8 748.0 5 768.9 6 43.22 16.55 18.51 76.09 2.435 44524 7 2.413 91365 4 2.410 90450 4 2.410 06426 9 2.394 52469 7 2.376 0687 2.374 73191 1 2.353 50110 8 2.352 90244 7 0.000 4639 0.000 00080 2 0.000 00751 0.000 00141 0.000 0207 0.000 00603 0.015 38 0.000 1274 0.002 996 257 Table A-3 (cont’d) Yes HP SAUSA300 _RS03395 SAUSA3 00_0633 fhuA Yes L SAUSA300 _RS10740 SAUSA3 00_1958 Yes M SAUSA300 _RS13530 SAUSA3 00_2441 fnbA Yes E SAUSA300 _RS02380 SAUSA3 00_0445 gltB Yes I SAUSA300 _RS08990 SAUSA3 00_1647 accD No P SAUSA300 _RS14540 SAUSA3 00_2616 No P SAUSA300 _RS05555 SAUSA3 00_1032 ABC transporter ATP-binding protein single-stranded DNA-binding protein fibronectin- binding protein FnbA glutamate synthase large subunit acetyl-CoA carboxylase, carboxyltransfera se subunit β energy-coupling factor transporter transmembraneco mponent T heme ABC transporter substrate-binding protein IsdE 300.6 2 326.4 1 25.45 22.91 10.8 61.75 0.59 3.13 121.6 1 104.3 2 137.7 6 206.5 6 7.1 10.32 5.99 17.77 109.6 6 144.6 7 12.09 8.74 22.09 36.34 1.43 2.86 34.67 29.27 1.15 3.41 No CE SAUSA300 _RS11060 SAUSA3 00_2011 leuB 3-isopropylmalate dehydrogenase 22.09 55.34 3.91 2.03 No E SAUSA300 _RS04805 SAUSA3 00_0891 oppA 405.9 6 587.8 4 25.82 49.53 peptide ABC transporter substrate-binding protein 258 2.350 57439 3 2.322 23030 7 2.290 23314 5 2.272 72553 8 2.263 16651 7 2.251 06912 5 2.249 32260 1 2.245 74867 9 2.241 65553 1 0.000 00145 0.018 64 0.000 0635 0.001 912 0.000 00694 0.000 7266 0.002 651 0.000 6683 0.000 3603 Table A-3 (cont’d) No C SAUSA300 _RS03080 SAUSA3 00_0576 Yes L SAUSA300 _RS10735 SAUSA3 00_1957 Yes C SAUSA300 _RS05570 SAUSA3 00_1035 Yes S SAUSA300 _RS03690 SAUSA3 00_0687 Yes P SAUSA300 _RS07905 SAUSA3 00_1448 hypothiocyanous acid reductase MerA DnaD domain- containing protein 71.74 127.8 6 8.91 7.22 10.42 64.94 0.69 3.46 staphylobilin- forming heme oxygenase IsdG 233.8 9 hemolysin family protein 647.8 5 109 5.78 18.34 721.8 70.53 47.41 Fur family transcriptional regulator 1799. 9 1559. 7 187.2 1 105.6 6 Yes no homol og found SAUSA300 _RS10730 SAUSA3 00_1956 hypothetical protein 11.05 56.14 0.67 3.32 No J SAUSA300 _RS02760 SAUSA3 00_0515 cysS cysteine--tRNA ligase 100.5 1 218.8 17.01 9.95 Yes P SAUSA300 _RS01055 SAUSA3 00_0200 Yes D SAUSA300 _RS07900 SAUSA3 00_1447 xerD ABC transporter ATP-binding protein site-specific tyrosine recombinase XerD 560.5 9 480.7 6 28.76 56.72 350.6 8 327.5 5 38.84 23.94 2.241 52742 2 2.237 88533 2 2.231 62571 6 2.210 29214 5 2.191 56570 1 2.167 05141 5 2.156 79562 5 2.154 14874 6 2.113 07162 2 0.000 0298 0.024 57 0.006 752 0.000 0105 0.000 0697 0.031 23 0.000 384 0.000 8821 0.000 0709 259 Table A-3 (cont’d) Yes P SAUSA300 _RS02340 SAUSA3 00_0437 No C SAUSA300 _RS04425 SAUSA3 00_0821 Yes C SAUSA300 _RS06390 SAUSA3 00_1182 dipeptide ABC transporter glycylmethionine- binding lipoprotein SUF system NifU family Fe-S cluster assembly protein 2- oxoacid:acceptor oxidoreductase subunit α 2857. 81 3414. 82 123.3 4 372.0 9 73.21 154.2 8 12.48 7.44 50 64.63 4.66 5.58 Yes E SAUSA300 _RS02755 SAUSA3 00_0514 cysE serine O- acetyltransferase 103.8 9 143.9 4 10.54 11.66 No no homol og found SAUSA300 _RS05575 Yes S SAUSA300 _RS03075 SAUSA3 00_0575 hypothetical protein 29.79 15.46 0.76 2.46 DUF1450 domain- containing protein 287.3 5 198.4 1 26.21 18.61 No O SAUSA300 _RS08555 SAUSA3 00_1569 U32 family peptidase 78.89 Yes S SAUSA300 _RS10985 SAUSA3 00_1998 YeeE/YedE family protein 409.6 103.0 5 333.5 9 10.96 5.81 14.91 45.03 2.112 60531 1 2.109 19234 4 2.108 93281 4 2.107 87200 5 2.104 84400 3 2.104 12693 2 2.097 46648 8 2.094 93932 9 0.004 659 0.000 5562 0.000 0709 0.000 0635 0.027 46 0.000 2521 0.000 1548 0.005 985 260 Table A-3 (cont’d) No No P P SAUSA300 _RS03865 SAUSA3 00_0718 SAUSA300 _RS04795 SAUSA3 00_0889 oppD ABC transporter permease ABC transporter ATP-binding protein 10.81 14.08 1.2 1.04 26.5 64.84 2.4 5.34 Yes S SAUSA300 _RS10695 SAUSA3 00_1949 dut dUTP pyrophosphatase 12.62 67.03 0.87 4.58 No S SAUSA300 _RS04365 SAUSA3 00_0809 Yes F SAUSA300 _RS03850 SAUSA3 00_0717 nrdF Yes P SAUSA300 _RS03880 SAUSA3 00_0721 Yes K SAUSA300 _RS14555 SAUSA3 00_2619 No K SAUSA300 _RS11905 SAUSA3 00_2160 38.21 23.66 3.89 2.31 3385. 68 4233. 06 315.6 1 430.5 2 135.8 3 190.9 9 18.67 14.4 27.21 28.52 2.62 2.99 10.7 15.89 1.65 0.97 phage/plasmid primase, P4 family class 1b ribonucleoside- diphosphate reductase subunit β siderophore ABC transporter substrate-binding protein S-adenosyl-L- methionine hydroxide adenosyltransfera se family protein MerR family transcriptional regulator 261 2.089 81840 8 2.001 73570 2 2.000 99240 4 1.988 16571 1 1.971 42060 5 1.970 96601 5 1.958 07828 3 1.934 52935 4 0.000 1951 0.008 266 0.049 4 0.001 511 0.000 4094 0.000 1214 0.000 4355 0.003 95 Table A-3 (cont’d) Yes P SAUSA300 _RS02330 SAUSA3 00_0435 No T SAUSA300 _RS06020 SAUSA3 00_1112 methionine ABC transporter ATP- binding protein protein- serine/threonine phosphatase Stp1 381.5 2 394.4 8 18.79 54.32 52.4 86.83 7.62 6.89 Yes E SAUSA300 _RS11035 SAUSA3 00_2006 ilvD dihydroxy-acid dehydratase 117.8 7 89.78 11.72 10.72 Yes K SAUSA300 _RS14655 SAUSA3 00_2639 cold-shock protein 31165 .95 38909 .15 2344 4676. 49 No F SAUSA300 _RS14520 SAUSA3 00_2612 hisG No no homol og found SAUSA300 _RS16065 Yes C SAUSA300 _RS14195 SAUSA3 00_2554 ATP phosphoribosyltra nsferase hypothetical protein assimilatory sulfite reductase (NADPH) flavoprotein subunit 12.44 16.58 0.44 2.09 32.67 47.74 5.51 2.81 78.46 71.39 7.45 8.83 Yes EP SAUSA300 _RS01065 SAUSA3 00_0202 ABC transporter permease 100.2 4 118.0 3 10.39 13.38 Yes S SAUSA300 _RS00890 SAUSA3 00_0169 YbaN family protein 132.1 7 114.6 5 9.43 16.76 1.901 02864 2 1.900 20096 5 1.888 74799 2 1.878 81243 2 1.878 07789 2 0.012 46 0.000 5763 0.000 7114 0.004 656 0.038 23 1.859 0956 0.008 444 1.858 93782 5 1.838 53411 3 1.829 43090 1 0.000 838 0.001 002 0.005 769 262 Table A-3 (cont’d) No no homol og found SAUSA300 _RS04375 SAUSA3 00_0811 hypothetical protein 34.03 20.2 4.18 1.66 Yes F SAUSA300 _RS03840 SAUSA3 00_0715 nrdI class Ib ribonucleoside- diphosphate reductase assembly flavoprotein NrdI 430.2 7 617.2 17 82.06 Yes CH SAUSA300 _RS13660 SAUSA3 00_2463 ddh D-lactate dehydrogenase 392.3 3 291.1 2 59.74 18.72 Yes C SAUSA300 _RS12320 SAUSA3 00_2231 fdhD Yes S SAUSA300 _RS07495 SAUSA3 00_1372 formate dehydrogenase accessory sulfurtransferase FdhD helix-turn-helix domain- containing protein 144.1 3 150.3 5 20.34 14.5 150.7 3 178.2 1 26.69 11.79 No No Yes S E no homol og found SAUSA300 _RS05285 SAUSA3 00_0982 hypothetical protein 420.3 4 696.2 4 62.01 65.54 SAUSA300 _RS12580 SAUSA3 00_2276 SAUSA300 _RS10255 M20 peptidase aminoacylase family protein hypothetical protein 263 22.11 25.47 1.99 3.31 86.18 69.13 13.79 3.7 1.819 72603 6 0.021 93 1.789 11683 7 1.786 97592 2 1.778 02569 9 1.776 21052 2 1.768 09358 1 0.047 7 0.015 8 0.000 7485 0.004 28 0.001 622 1.767 26895 0.006 011 1.754 66383 9 0.032 1 Table A-3 (cont’d) No No P F SAUSA300 _RS13240 SAUSA3 00_2392 opuC b ABC transporter permease 32.79 44.43 5.52 3.65 SAUSA300 _RS06655 SAUSA3 00_1228 thrB homoserine kinase 40.14 69.39 6.45 6.29 No M SAUSA300 _RS13235 SAUSA3 00_2391 opuC c No - SAUSA300 _RS04355 SAUSA3 00_0807 Yes L SAUSA300 _RS08900 SAUSA3 00_1631 No No S V SAUSA300 _RS04360 SAUSA3 00_0808 SAUSA300 _RS05030 SAUSA3 00_0936 Yes U SAUSA300 _RS11755 SAUSA3 00_2135 osmoprotectant ABC transporter substrate- bindingprotein hypothetical protein replication initiation and membrane attachment family protein DUF1474 family protein ABC transporter ATP-binding protein iron ABC transporter permease 77.03 99.33 12.26 9.11 32.42 19.37 3.7 2.51 85.06 154.1 2 9.57 17.22 44 26.88 4.5 4.02 21.87 15.19 1.6 2.71 108.5 124.4 8 15.58 13.37 Yes E SAUSA300 _RS14085 SAUSA3 00_2538 amino acid permease 731.1 6 576.5 62.48 93.99 1.749 40789 2 1.741 86855 1 1.732 26185 3 1.724 92731 3 1.715 86395 5 1.706 82078 1 1.700 45988 2 1.700 18686 2 1.699 46733 6 0.002 075 0.002 579 0.001 069 0.013 87 0.012 69 0.014 23 0.019 88 0.001 069 0.007 759 264 Table A-3 (cont’d) Yes S SAUSA300 _RS12540 SAUSA3 00_2268 No not classif ied SAUSA300 _RS15215 No J SAUSA300 _RS06015 SAUSA3 00_1111 No O SAUSA300 _RS04410 SAUSA3 00_0818 sufC bile acid:sodium symporter family protein hypothetical protein 23S rRNA (adenine(2503)- C(2))- methyltransferase RlmN Fe-S cluster assembly ATPase SufC 79.33 62.56 4.15 11.71 63.8 39.7 9.06 3.56 40.29 74.21 8.63 5.48 210.9 8 289.5 6 30.02 32.6 Yes E SAUSA300 _RS02320 SAUSA3 00_0433 cysM cysteine synthase family protein 50.07 39.27 6.04 5.48 No not classif ied SAUSA300 _RS04345 SAUSA3 00_0805 Yes I SAUSA300 _RS08985 SAUSA3 00_1646 accA Yes U SAUSA300 _RS04915 SAUSA3 00_0914 Yes C SAUSA300 _RS01085 SAUSA3 00_0206 helix-turn-helix domain- containing protein acetyl-CoA carboxylase carboxyltransfera se subunit α alanine/glycine:ca tion symporter family protein FMN-dependent NADH- azoreductase 81.86 44.92 11.97 4.21 288.9 6 304.7 51.18 27.08 84.57 75.06 11.04 10.02 169.9 8 178.9 7 25.6 21.21 1.691 04558 5 1.681 07054 8 1.673 79614 8 1.660 95247 3 1.648 56741 6 1.633 90507 6 1.629 91413 1 1.621 16381 4 1.601 82215 5 0.035 54 0.030 59 0.006 054 0.002 513 0.005 019 0.042 07 0.005 878 0.003 35 0.002 651 265 Table A-3 (cont’d) Yes E SAUSA300 _RS13265 SAUSA3 00_2395 APC family permease 39.16 45.52 3.75 6.86 No No No E E no homol og found SAUSA300 _RS06650 SAUSA3 00_1227 thrC threonine synthase SAUSA300 _RS04420 SAUSA3 00_0820 sufS cysteine desulfurase 82.97 85.92 133.3 6 166.6 5 14.77 13.7 19.61 13.63 SAUSA300 _RS13715 hypothetical protein 180.0 2 305.2 5 36.61 27.93 Yes C SAUSA300 _RS00885 SAUSA3 00_0168 Yes Yes I I SAUSA300 _RS00930 SAUSA3 00_0177 SAUSA300 _RS02535 SAUSA3 00_0472 ipk Yes S SAUSA300 _RS13755 SAUSA3 00_2479 cidA Yes H SAUSA300 _RS01160 SAUSA3 00_0221 pflA staphylobilin- forming heme oxygenase IsdI acyl-CoA/acyl- ACP dehydrogenase 4-(cytidine 5'- diphospho)-2-C- methyl-D-erythritol kinase holin-like murein hydrolase modulator CidA pyruvate formate- lyase-activating protein 526.1 2 366.6 1 456.2 8 496.2 1 37.96 75.76 50.29 74.56 108 182.7 6 15.31 22.22 35.32 33.97 4.33 5.01 23.14 67.26 7.33 4.26 1.599 91745 3 1.589 69601 2 1.581 47456 2 1.577 36895 1 1.574 73217 1 1.574 63279 3 1.564 84735 3 0.018 33 0.005 027 0.010 14 0.007 251 0.034 66 0.010 62 0.015 49 1.558 70114 0.012 09 1.557 73392 7 0.039 8 266 Table A-3 (cont’d) No O SAUSA300 _RS04415 SAUSA3 00_0819 sufD No C SAUSA300 _RS12475 SAUSA3 00_2258 Yes P SAUSA300 _RS11750 SAUSA3 00_2134 Fe-S cluster assembly protein SufD formate dehydrogenase subunit α iron chelate uptake ABC transporter family permease subunit 84.79 163.6 6 16.42 16.94 30.3 67.91 7.26 5.91 1.533 59869 5 0.013 25 1.530 48046 0.018 84 178.1 6 175.2 9 30.27 20.31 Yes L SAUSA300 _RS08280 SAUSA3 00_1517 deoxyribonucleas e IV 198.6 8 214.1 4 38.88 21 Yes E SAUSA300 _RS06640 SAUSA3 00_1225 Yes L SAUSA300 _RS07130 SAUSA3 00_1309 aspartate kinase 45.43 52.12 4.69 8.7 IS200/IS605 family transposase 289.4 3 227.2 2 50.14 27.04 Yes no homol og found SAUSA300 _RS15665 SAUSA3 00_2604 hypothetical protein 203.0 6 247.7 1 34.59 31.87 No L SAUSA300 _RS06155 SAUSA3 00_1137 Yes C SAUSA300 _RS00940 SAUSA3 00_0179 rnhB ribonuclease HII 13.27 21.65 2.93 2.14 NAD-dependent formate dehydrogenase 36.3 41.91 5.91 5.82 267 1.522 59177 4 1.500 51121 3 1.477 24340 2 1.465 62803 3 1.456 72541 8 1.449 17036 8 1.431 73794 1 0.006 752 0.012 54 0.034 52 0.023 68 0.009 251 0.023 42 0.011 68 Table A-3 (cont’d) Yes F SAUSA300 _RS05655 SAUSA3 00_1050 XTP/dITP diphosphatase 291.5 295.1 7 33.89 51.69 No not classif ied SAUSA300 _RS04370 SAUSA3 00_0810 hypothetical protein 49.06 33.44 7.63 4.9 No M SAUSA300 _RS05650 SAUSA3 00_1049 murI glutamate racemase 137.0 5 140.3 2 14.61 25.67 Yes H SAUSA300 _RS00040 SAUSA3 00_0007 NAD(P)H-hydrate dehydratase 152.0 4 111.4 8 16.95 23.56 No P SAUSA300 _RS05255 SAUSA3 00_0978 Yes P SAUSA300 _RS06680 SAUSA3 00_1232 ABC transporter ATP-binding protein catalase 25.48 24.9 4.49 3.51 606.8 2 615.4 9 93.79 98.44 No no homol og found SAUSA300 _RS01180 SAUSA3 00_0224 coa staphylocoagulas e 12.09 14.67 1.96 2.21 No BQ SAUSA300 _RS09180 SAUSA3 00_1681 acuC acetoin utilization protein AcuC 25.27 25.74 4.67 3.48 Yes J SAUSA300 _RS06190 SAUSA3 00_1144 gid 260.2 365.2 2 61.61 38.45 methylenetetrahy drofolate--tRNA- (uracil(54)- C(5))- methyltransferase (FADH(2)- oxidizing) TrmFO 268 1.429 82128 7 1.428 56537 8 1.416 22281 6 1.374 45170 1 1.371 74870 6 1.371 29191 7 1.363 08916 7 1.360 43943 1 1.354 26774 6 0.027 99 0.038 95 0.040 6 0.044 15 0.018 77 0.016 09 0.024 99 0.020 85 0.023 2 Table A-3 (cont’d) Yes L SAUSA300 _RS03790 SAUSA3 00_0705 recQ DNA helicase RecQ 153.9 7 141.1 4 17.71 28.07 Yes JKL SAUSA300 _RS08285 SAUSA3 00_1518 Yes S SAUSA300 _RS00020 SAUSA3 00_0003 No Yes L L SAUSA300 _RS00025 SAUSA3 00_0004 recF SAUSA300 _RS02485 SAUSA3 00_0463 Yes EGP SAUSA300 _RS11720 SAUSA3 00_2128 Yes C SAUSA300 _RS00280 SAUSA3 00_0055 No F SAUSA300 _RS03815 SAUSA3 00_0711 Yes V SAUSA300 _RS09580 SAUSA3 00_1751 hsdS Yes G SAUSA300 _RS11545 SAUSA3 00_2096 manA 64.94 83.11 12.31 11.62 763.5 7 867.3 102.0 4 155.9 8 284.3 9 343.6 6 49.13 53.42 174.1 2 216.6 38.02 27.16 78.96 93.2 18.13 10.78 95.33 87.76 19.62 11.34 163.7 260.5 5 33.34 36.19 42.79 41.67 7.33 7.15 107 94.53 20.09 15.44 DEAD/DEAH box helicase S4 domain- containing protein YaaA DNA replication/repair protein RecF DNA replication initiation control protein YabA multidrug efflux MFS transporter SdrM zinc-dependent alcohol dehydrogenase family protein diacylglycerol kinase family lipid kinase restriction endonuclease subunit S class I mannose- 6-phosphate isomerase 269 1.345 65723 3 1.329 44090 3 1.316 66309 7 1.308 92491 7 1.301 56573 8 1.299 00995 3 1.298 75954 7 1.282 61428 3 1.257 22753 2 1.237 01318 1 0.047 75 0.019 19 0.047 02 0.024 35 0.024 99 0.032 3 0.039 33 0.037 62 0.035 04 0.040 6 Table A-3 (cont’d) No V SAUSA300 _RS01645 SAUSA3 00_0308 No E SAUSA300 _RS07385 SAUSA3 00_1355 aroA Yes J SAUSA300 _RS08600 SAUSA3 00_1578 mnm A FtsX-like permease family protein 3- phosphoshikimate 1- carboxyvinyltransf erase tRNA 2- thiouridine(34) synthase MnmA 27.83 39.43 5.35 6.07 32.35 50.24 7.72 6.76 197.3 1 212.3 2 40.31 34.26 1.236 01849 9 1.207 27057 8 1.184 78652 6 0.049 4 0.049 36 0.040 32 Genes downregulated in cymR::Tn sulfur starvation when compared to cymR::Tn CSSC Share d with WT -S (Table 1) TPM. 1 CSSC TPM. 1 Starv TPM. 2 Starv Gene Product COG Locus Old locus TPM. 2 CSSC DE Log2 FC Yes F SAUSA300 _RS05210 SAUSA3 00_0969 purS Yes O SAUSA300 _RS09140 SAUSA3 00_1674 Yes F SAUSA300 _RS05245 SAUSA3 00_0976 purD 2.47 3.94 71.99 13.69 85.06 85.76 1357. 31 388.2 8 19.9 25.17 550.4 6 48.93 - 4.149 57726 7 - 4.029 53461 8 - 3.957 56855 2 phosphoribosylfor mylglycinamidine synthase subunit PurS trypsin-like peptidase domain- containing protein phosphoribosyla mine--glycine ligase 270 DE Adj. P- value 1.81E -08 1.47E -10 0.000 00131 Table A-3 (cont’d) No S SAUSA300 _RS01635 SAUSA3 00_0307 5'-nucleotidase, lipoprotein e(P4) family 18.71 13.8 108.6 9 134.9 No Yes not classif ied not classif ied SAUSA300 _RS15375 SAUSA300 _RS03000 Yes S SAUSA300 _RS03005 Yes Yes Yes no homol og found no homol og found no homol og found SAUSA300 _RS15740 SAUSA300 _RS15735 SAUSA300 _RS15090 hypothetical protein 121.8 164.6 571 1344. 17 hypothetical protein 791.7 7 650.7 8 5797. 02 3465. 16 38489 .16 31696 .59 22391 1.13 20544 5.74 4289. 85 3326. 41 12580 .61 37405 .9 5730. 32 4555. 63 15900 .9 49372 .47 6345. 06 5161. 13 15050 .63 48596 .27 C1q-binding complement inhibitor VraX phenol-soluble modulin PSM- alpha-1 phenol-soluble modulin PSM- alpha-2 phenol-soluble modulin PSM- alpha-3 271 - 3.807 58815 5 - 3.614 37204 2 - 3.589 74782 4 - 3.571 43228 1 - 3.495 37831 6 - 3.446 53104 7 - 3.267 40945 4 1.36E -12 0.000 00000 8 3.42E -12 7.47E -13 0.000 00031 2 0.000 00058 9 0.000 00328 Table A-3 (cont’d) No M SAUSA300 _RS09800 SAUSA3 00_1790 prsA peptidylprolyl isomerase 421.5 3 472.9 5 2316. 62 1527. 7 Yes F SAUSA300 _RS05195 SAUSA3 00_0966 purE 5- (carboxyamino)im idazole ribonucleotide mutase 3.99 5.78 34.58 12.45 Yes E SAUSA300 _RS04565 SAUSA3 00_0845 ampA M17 family metallopeptidase 36.03 30.19 174.0 9 110.9 7 Yes no homol og found SAUSA300 _RS15730 phenol-soluble modulin PSM- alpha-4 4781. 15 3722. 92 9480. 7 29491 .47 Yes S SAUSA300 _RS04760 SAUSA3 00_0883 MAP domain- containing protein 124.6 7 114.6 6 461.4 6 414.0 3 Yes no homol og found SAUSA300 _RS08745 hypothetical protein 197.3 4 199.9 7 814 614.8 6 No E SAUSA300 _RS06940 SAUSA3 00_1278 pepF oligoendopeptida se F 75.95 58.57 300.9 4 192.9 4 - 3.102 76006 3 - 3.096 27570 1 - 3.064 39436 - 3.031 54555 8 - 2.890 12139 1 - 2.865 49480 9 - 2.837 84688 4 1.47E -10 0.000 00084 1 3.2E- 09 0.000 0163 3.65E -09 3.65E -09 9.86E -08 272 Table A-3 (cont’d) No GH SAUSA300 _RS00995 SAUSA3 00_0190 ipdC No J SAUSA300 _RS10275 SAUSA3 00_1878 rumA alpha-keto acid decarboxylase family protein 23S rRNA (uracil(1939)- C(5))- methyltransferase RlmD 10.1 6.08 45.98 18.64 32.74 30.2 110.1 3 106.3 3 Yes Yes Yes no homol og found no homol og found no homol og found SAUSA300 _RS09670 SAUSA3 00_1767 epiA gallidermin/nisin family lantibiotic 14.79 14.8 62.42 43.05 SAUSA300 _RS13850 SAUSA3 00_2493 SAUSA300 _RS04395 SAUSA3 00_0815 ear cell wall inhibition responsive protein CwrA DUF4888 domain- containing protein 494.4 1 405.7 6 1253. 62 1809. 15 285.7 8 247.5 7 514.5 4 1337. 86 No S SAUSA300 _RS04990 SAUSA3 00_0929 IDEAL domain- containing protein 546.2 2 445.0 1 1192. 94 2058. 93 Yes H SAUSA300 _RS04995 SAUSA3 00_0930 lipoate--protein ligase 21.94 21.55 83.77 52.52 - 2.821 40982 1 - 2.807 67328 - 2.802 09471 4 - 2.761 38450 9 - 2.721 08987 5 - 2.694 53938 8 - 2.651 77849 5 0.000 0101 1.45E -08 0.000 00019 2 0.000 00082 5 0.000 056 0.000 00617 0.000 00022 4 273 Table A-3 (cont’d) Yes J SAUSA300 _RS02850 SAUSA3 00_0533 tuf elongation factor Tu 2098. 2 1576. 56 9598. 42 2950. 33 No M SAUSA300 _RS10340 SAUSA3 00_1890 cysteine protease staphopain A 23.86 17.48 90.7 41.01 No S SAUSA300 _RS08750 SAUSA3 00_1606 DUF4930 family protein 115.4 5 102.3 7 381.6 4 263.3 5 No C SAUSA300 _RS00705 SAUSA3 00_0135 superoxide dismutase 327.1 4 159.5 9 813.0 5 687.4 1 No no homol og found SAUSA300 _RS10505 SAUSA3 00_1918 phospholipase 231.8 8 358.2 4 247.8 6 1370. 52 No S SAUSA300 _RS03335 SAUSA3 00_0622 M50 family metallopeptidase 33.18 27.28 90.42 59.45 No no homol og found SAUSA300 _RS12715 hypothetical protein 29.21 27.54 62.52 74.48 No K SAUSA300 _RS12480 SAUSA3 00_2259 LCP family protein 71.48 67.66 173.8 2 151.6 2 - 2.599 00773 - 2.580 73020 5 - 2.577 56366 - 2.538 40489 9 - 2.331 74297 4 - 2.319 37854 2 - 2.308 03231 2 - 2.284 43859 2 0.000 0999 0.000 0165 0.000 00041 6 0.000 0398 0.006 75249 0.000 0144 0.000 0341 0.000 00482 274 Table A-3 (cont’d) No H SAUSA300 _RS09355 SAUSA3 00_1712 ribH No not classif ied SAUSA300 _RS10455 SAUSA3 00_1910 6,7-dimethyl-8- ribityllumazine synthase phenol-soluble modulin export ABC transporter permease subunit PmtD 125.9 4 114.0 1 276.5 1 281.8 4 95.33 102.7 7 117.4 3 363.0 8 Yes J SAUSA300 _RS06310 SAUSA3 00_1166 rpsO 30S ribosomal protein S15 1299. 07 1092. 22 5369. 18 1160 Yes G SAUSA300 _RS04670 SAUSA3 00_0865 pgi No I SAUSA300 _RS12685 SAUSA3 00_2296 No V SAUSA300 _RS03590 SAUSA3 00_0669 glucose-6- phosphate isomerase alpha/beta hydrolase undecaprenyl- diphosphate phosphatase 173.2 1 188.5 7 531.0 8 307.5 4 74.21 90.01 126.1 1 251.3 5 95.13 92.82 380.4 100.7 7 No S SAUSA300 _RS10345 staphostatin A 59.61 55.49 168.8 2 96.37 - 2.276 22248 1 - 2.253 53176 6 - 2.251 18427 4 - 2.244 10416 2 - 2.243 17413 7 - 2.222 61993 - 2.216 19622 1 0.000 00889 0.002 32582 1 0.003 27264 7 0.000 0186 0.000 38308 8 0.001 84399 7 0.000 0495 275 Table A-3 (cont’d) No K SAUSA300 _RS10185 SAUSA3 00_1865 vraR No S SAUSA300 _RS12500 SAUSA3 00_2262 two-component system response regulator VraR CPBP family intramembrane glutamic endopeptidaseSd pB 124.0 8 133.1 3 359.4 5 217.2 4 63.68 66.42 223.4 7 84.83 Yes S SAUSA300 _RS13155 SAUSA3 00_2378 membrane protein 114.1 4 105.7 4 368.7 143.9 No S SAUSA300 _RS06705 SAUSA3 00_1236 CAP domain- containing protein 46.6 56.85 133.6 5 88.75 No not classif ied SAUSA300 _RS11565 SAUSA3 00_2100 lytic regulatory protein 211.9 5 349.9 9 728.4 9 464.1 1 Yes J SAUSA300 _RS02575 SAUSA3 00_0479 50S ribosomal protein L25/general stress proteinCtc 1009. 33 776.5 9 3437. 61 870.1 4 No O SAUSA300 _RS04625 SAUSA3 00_0857 peptidylprolyl isomerase 143.4 9 218.3 5 532.0 7 240.1 4 - 2.203 43570 3 0.000 0221 - 2.196 61617 0.000 43873 6 - 2.170 79242 5 - 2.165 30558 6 - 2.145 36723 7 - 2.116 03300 3 - 2.103 18800 8 0.000 52119 0.000 0196 0.000 0709 0.004 84533 4 0.000 43873 6 276 Table A-3 (cont’d) No S SAUSA300 _RS12865 SAUSA3 00_2328 DUF4889 domain- containing protein 146.9 5 178.1 3 295.0 8 348.6 9 No not classif ied SAUSA300 _RS10200 SAUSA3 00_1868 hypothetical protein 33.4 42.96 69.49 79.98 Yes M SAUSA300 _RS02965 SAUSA3 00_0556 6-phospho-3- hexuloisomerase 63.36 91.24 204.5 5 108.4 9 No C SAUSA300 _RS00155 SAUSA3 00_0030 glycerophosphory l diester phosphodiesteras e 27.56 42.18 64.24 68.28 No S SAUSA300 _RS04765 SAUSA3 00_0884 YjzD family protein 396.4 4 436.4 4 787.7 775.4 2 No EJ SAUSA300 _RS07470 SAUSA3 00_1368 ansA asparaginase 27.49 29.51 68.76 41.19 No OU SAUSA300 _RS04060 SAUSA3 00_0752 clpP ATP-dependent Clp endopeptidase proteolytic subunit ClpP 440.6 4 477.3 6 1027. 25 715.2 4 - 2.079 52448 9 - 2.062 92856 8 - 2.057 30151 4 - 2.025 99487 9 - 2.002 29876 5 - 1.997 35650 3 - 1.995 96247 1 0.000 0709 0.000 099 0.000 26039 1 0.000 14352 8 0.000 0697 0.000 19452 9 0.000 082 277 Table A-3 (cont’d) No S SAUSA300 _RS05185 SAUSA3 00_0964 No K SAUSA300 _RS12390 SAUSA3 00_2245 DUF5011 domain- containing protein HTH-type transcriptional regulator SarR 1334. 4 993.7 7 1790. 96 2632. 43 1604. 75 1808. 51 2792. 06 3279. 78 No no homol og found SAUSA300 _RS07815 SAUSA3 00_1432 hypothetical protein 230.1 9 344.1 6 486.1 6 523.1 Yes S SAUSA300 _RS09825 SAUSA3 00_1795 YlbF/YmcA family competence regulator 1580. 86 1536. 39 3185. 12 2401. 33 No No no homol og found no homol og found SAUSA300 _RS07460 SAUSA3 00_1366 hypothetical protein 36.42 28.53 70.61 48.87 SAUSA300 _RS06620 SAUSA3 00_1221 hypothetical protein 45.16 89.89 130.3 1 106.4 5 Yes C SAUSA300 _RS14075 SAUSA3 00_2537 L-lactate dehydrogenase 95.89 87.56 172.6 2 150.2 6 No no homol og found SAUSA300 _RS16085 SAUSA3 00_1988 delta-lysin family phenol-soluble modulin 77389 .75 67469 .88 13968 7.59 11566 8.38 - 1.971 72832 8 - 1.931 71892 3 - 1.922 80399 4 - 1.916 19496 - 1.903 30707 8 - 1.896 75278 8 - 1.894 15454 8 - 1.889 06454 7 0.001 36213 4 0.000 26039 1 0.000 32817 8 0.000 18578 7 0.001 40393 1 0.001 23968 9 0.000 26964 8 0.000 32817 8 278 Table A-3 (cont’d) No not classif ied SAUSA300 _RS08150 SAUSA3 00_1493 SA1362 family protein 73.65 114.3 3 171.7 5 149.8 2 No K SAUSA300 _RS07035 SAUSA3 00_1295 cold shock protein CspA 6674. 63 7960. 83 8195. 49 17190 .01 Yes G SAUSA300 _RS02960 SAUSA3 00_0555 No S SAUSA300 _RS10195 SAUSA3 00_1867 3-hexulose-6- phosphate synthase cell wall-active antibiotics response protein LiaF 31.5 53.55 80.68 63.24 51.05 51.63 83.33 82.76 No O SAUSA300 _RS09055 SAUSA3 00_1659 tpx thiol peroxidase 541.7 543.1 7 1042. 42 725.7 No E SAUSA300 _RS02635 SAUSA3 00_0491 cysK cysteine synthase A 3633. 25 2980. 47 7140. 91 3976. 77 Yes not classif ied SAUSA300 _RS13040 SAUSA3 00_2361 putative metal homeostasis protein 1679. 47 1321. 49 3210. 33 1826. 03 - 1.877 27516 3 - 1.868 59809 8 - 1.855 73885 9 - 1.792 03336 9 - 1.782 58335 6 - 1.778 97610 4 - 1.774 18544 6 0.000 43873 6 0.005 78183 8 0.000 76234 2 0.000 66746 9 0.000 72659 4 0.002 60445 3 0.002 87907 279 Table A-3 (cont’d) Yes C SAUSA300 _RS03045 SAUSA3 00_0569 heme-dependent peroxidase 363.7 2 351.6 698.1 3 442.7 7 Yes S SAUSA300 _RS13645 SAUSA3 00_2460 GNAT family N- acetyltransferase 65.19 95.56 123.2 3 120.2 2 No T SAUSA300 _RS10190 SAUSA3 00_1866 vraS sensor histidine kinase 48.93 50.75 86.62 63.01 Yes S SAUSA300 _RS07160 SAUSA3 00_1314 YozE family protein 436.4 1 557.2 8 824.2 7 630.1 6 No S SAUSA300 _RS10910 SAUSA3 00_1984 CPBP family intramembrane glutamic endopeptidaseMr oQ 111.7 1 104.0 9 126.2 1 189.5 2 No K SAUSA300 _RS05805 SAUSA3 00_1070 N- acetyltransferase 267.3 4 256.5 9 400.9 353.6 7 Yes J SAUSA300 _RS03960 SAUSA3 00_0736 yfiA 5021. 99 4039. 9 5701. 51 6755. 99 ribosome- associated translation inhibitor RaiA 280 - 1.740 31010 8 - 1.717 48581 2 - 1.676 41484 4 - 1.658 36285 2 - 1.645 90080 7 - 1.627 09675 1 - 1.553 14604 1 0.001 50938 5 0.001 59011 4 0.001 61703 9 0.001 58791 1 0.008 32515 7 0.002 38303 3 0.009 95202 1 Table A-3 (cont’d) No S SAUSA300 _RS12720 SAUSA3 00_2302 tcaA Yes G SAUSA300 _RS07165 SAUSA3 00_1315 crr glycopeptide resistance protein TcaA PTS glucose transporter subunit IIA 72.33 66.7 100.7 3 88.6 503.1 567.8 752.0 4 659.1 3 No H SAUSA300 _RS09425 SAUSA3 00_1725 transaldolase 380.7 4 299.7 509.0 3 398.7 4 Yes no homol og found SAUSA300 _RS15985 No F SAUSA300 _RS02705 SAUSA3 00_0505 hypothetical protein 1270. 74 1137. 97 1916. 24 1268. 31 pyridoxal 5'- phosphate synthase glutaminase subunit PdxT 152.5 5 146.5 9 154.7 6 231.1 7 No not classif ied SAUSA300 _RS06630 SAUSA3 00_1223 hypothetical protein 98.31 107.2 2 156.9 3 107.3 1 Yes S SAUSA300 _RS13765 SAUSA3 00_2481 sterile α motif-like domain- containing protein 5481. 61 5785. 93 6156. 04 8057. 18 - 1.547 15648 - 1.518 42218 5 - 1.495 84226 9 - 1.482 95495 6 - 1.480 55009 1 - 1.464 07273 7 - 1.460 91964 1 0.004 90233 6 0.004 31367 4 0.010 58877 8 0.010 58877 8 0.021 00904 7 0.008 44839 6 0.014 81135 7 281 Table A-3 (cont’d) No no homol og found SAUSA300 _RS15910 hypothetical protein 467.2 9 484.7 8 547.1 1 613.5 7 No J SAUSA300 _RS08135 SAUSA3 00_1490 efp elongation factor P 536.8 7 635.1 4 581.3 7 712.1 6 No no homol og found SAUSA300 _RS15830 hypothetical protein 95.61 104.4 4 114.8 5 103.4 2 - 1.412 59882 3 - 1.287 34146 7 - 1.248 38044 2 0.014 59714 5 0.033 7843 0.040 10672 282 Table B-1. Cysteine (Cys) differentially expressed genes. APPENDIX B: Chapter 2 sulfur source Tables Genes upregulated in Cys when compared to cystine (CSSC) TPM.1 Cys Old locus Gene Locus SAUSA300_ RS15735 SAUSA300_ RS15740 SAUSA300_ RS15090 SAUSA300_ RS15730 Product phenol-soluble modulin PSM-alpha- 2 phenol-soluble modulin PSM-alpha- 1 phenol-soluble modulin PSM-alpha- 3 phenol-soluble modulin PSM-alpha- 4 TPM.2 Cys TPM.1 CSSC TPM.2 CSSC DE Log2 FC DE Adj. P-value 6414.0 7 22311. 29 5005.5 7 17730. 55 6602.5 9 24391. 96 4712.0 8 16717. 7 1675.5 4447.18 1.198345 086 7.64841E -05 1294.02 3576.07 1.193139 353 8.3552E- 05 1880.69 4471.81 1.190219 679 8.90957E -05 1439.61 3355.4 1.130860 907 0.000192 115 Genes downregulated in Cys when compared to CSSC Locus Old locus Gene SAUSA300_ RS04580 SAUSA30 0_0848 SAUSA300_ RS06690 SAUSA30 0_1234 Product FAD/NAD(P)- binding protein TPM.1 Cys TPM.2 Cys TPM.1 CSSC TPM.2 CSSC 9.93 10.43 29.4 171.15 rpsN 30S ribosomal 554.57 552.48 991.64 2246.49 protein S14 283 DE Log2 FC - 1.769049 937 - 1.060341 408 DE Adj. P-value 4.77602E -09 0.000256 525 Table B-2. Oxidized glutathione (GSSG) differentially expressed genes. Genes upregulated in GSSG when compared to CSSC Locus Old locus Gene Product SAUSA300 _RS00930 SAUSA300 _RS02185 SAUSA300 _RS00925 SAUSA300 _RS00915 SAUSA300 _0177 SAUSA300 _0407 SAUSA300 _0176 SAUSA300 _0174 SAUSA300 _RS02340 SAUSA300 _0437 gmpC SAUSA300 _RS03085 SAUSA300 _0577 SAUSA300 _RS13745 SAUSA300 _2477 SAUSA300 _RS02325 SAUSA300 _0434 SAUSA300 _RS00920 SAUSA300 _RS00910 SAUSA300 _RS13525 SAUSA300 _0175 SAUSA300 _0173 SAUSA300 _2440 TPM.1 GSSG TPM.2 GSSG TPM. 1 CSSC TPM. 2 CSSC 115.41 240.12 2.69 9.11 60.15 76.27 2.18 5.91 78.28 96.38 4.22 2.74 14.09 28.88 1.31 1.85 667.4 817.5 30.16 144.4 4 79.95 217.72 12.24 7.56 acyl-CoA/acyl-ACP dehydrogenase superantigen-like protein SSL11 ABC transporter permease ABC transporter ATP- binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein redox-sensitive transcriptional regulator HypR pyruvate oxidase 176.31 224.87 16.24 30.98 bifunctional cystathionine γ-lyase/homocysteine desulfhydrase ABC transporter substrate-binding protein DUF4242 domain- containing protein fibronectin-binding protein FnbB 66.24 118.57 6.96 15.5 20.65 35.29 2.69 2.58 174.59 290.64 14.98 49.04 60.43 59.28 5.83 8.01 fnbB 284 DE Log2 FC 4.1575 43995 3.4390 76082 3.3033 13167 2.8307 62445 2.6543 14417 2.5501 01035 2.4597 29837 2.4241 27828 2.4089 38291 2.3711 31373 2.3573 41809 DE Adj. P-value 3.04382E -22 5.42187E -17 9.01551E -10 9.05749E -09 9.05749E -09 1.54999E -05 9.05749E -09 2.57027E -08 4.67518E -06 6.13964E -08 7.51319E -07 Table B-2 (cont’d) SAUSA300 _RS02035 SAUSA300 _RS02190 SAUSA300 _RS10920 SAUSA300 _RS13910 SAUSA300 _RS07500 SAUSA300 _RS10985 SAUSA300 _RS04170 SAUSA300 _RS01055 SAUSA300 _RS02315 SAUSA300 _RS01185 SAUSA300 _RS01180 SAUSA300 _RS07495 SAUSA300 _RS02335 SAUSA300 _RS14570 SAUSA300 _0382 SAUSA300 _0408 SAUSA300 _1986 SAUSA300 _2505 SAUSA300 _1373 SAUSA300 _1998 SAUSA300 _0773 SAUSA300 _0200 SAUSA300 _0432 SAUSA300 _0224 SAUSA300 _1372 SAUSA300 _0436 SAUSA300 _2622 SAUSA300 _RS02330 SAUSA300 _0435 L-cystine transporter FKLRK protein nitroreductase family protein GNAT family N- acetyltransferase ferredoxin 1284.5 9 132.34 1070. 76 186.8 1 127.0 4 125.6 2 14.95 26.75 74.65 74.1 5.47 16.95 18.59 30.85 0.55 7 2280.4 2 2592. 1 274.4 6 309.7 9 YeeE/YedE family protein 45.26 77.02 4.7 14.19 vwb von Willebrand factor binding protein Vwb ABC transporter ATP- binding protein sodium-dependent transporter 22.43 39.39 2.02 8.27 82.97 88.98 9.74 14.41 160.87 163.7 3 16.69 35.07 hypothetical protein 61.49 77.54 6.01 16.16 coa staphylocoagulase 37.03 72.45 4.59 14 2.3063 20567 2.2892 37921 2.2764 37635 2.2243 12445 2.2193 60193 2.1964 73104 2.1398 49726 2.1368 24537 2.1081 73714 2.1021 02385 2.0561 29945 2.0390 23845 1.9860 24342 1.9830 40622 1.28398E -05 1.87629E -07 4.49694E -07 0.000261 289 8.76536E -06 7.51319E -07 7.0435E- 06 6.18049E -06 2.0706E- 06 1.90088E -05 7.69607E -06 1.48728E -05 5.44287E -05 9.43471E -05 153.75 146.5 7 17.77 29.53 58.66 85.25 4.85 23.05 169.47 471.8 6 25.72 85.02 45.39 68.57 6.07 13.82 1.9821 14069 7.69607E -06 helix-turn-helix domain- containing protein methionine ABC transporter permease rhodanese-related sulfurtransferase methionine ABC transporter ATP-binding protein 285 Table B-2 (cont’d) SAUSA300 _RS10980 SAUSA300 _1997 SAUSA300 _RS13025 SAUSA300 _2359 SAUSA300 _0204 SAUSA300 _1026 SAUSA300 _0201 SAUSA300 _0433 SAUSA300 _2165 SAUSA300 _1203 SAUSA300 _0178 SAUSA300 _0268 SAUSA300 _1222 SAUSA300 _2196 SAUSA300 _RS01075 SAUSA300 _RS05520 SAUSA300 _RS01060 SAUSA300 _RS02320 SAUSA300 _RS16005 SAUSA300 _RS11935 SAUSA300 _RS06225 SAUSA300 _RS06500 SAUSA300 _RS00935 SAUSA300 _RS01440 SAUSA300 _RS06625 SAUSA300 _RS12110 SAUSA300 _RS10510 sulfurtransferase TusA family protein transporter substrate- binding domain-containing protein 270.7 8 167.4 1 321.0 8 263.0 8 39.77 54.14 1.9391 89828 7.77652E -05 19.34 69.69 ggt γ-glutamyltransferase 28.66 33.84 4.18 7.39 DUF177 domain-containing protein 1413. 86 2722. 12 153.0 2 729.1 7 ABC transporter permease 20.73 22.11 3.3 3.88 cysteine synthase family protein 15.84 16.98 2.64 2.54 hypothetical protein 61.05 73.6 3.59 26.52 budA acetolactate decarboxylase 22.63 52.06 4.02 10.73 hypothetical protein 466.0 1 690.1 7 71.03 167.1 7 hypothetical protein 23.75 20.62 1.84 7.85 DUF2294 domain- containing protein 598.6 1 688.1 8 92.52 169.3 3 MFS transporter 19.46 21.64 2.57 6.44 thermonuclease family protein 47.77 64.22 4.64 22.12 rpmC 50S ribosomal protein L29 96.44 hypothetical protein 474.9 9 188.7 3 538.8 1 14.13 49.61 47.79 197.9 3 286 1.8684 32696 1.8423 47726 1.8393 26382 1.8153 53512 1.8061 0258 1.7889 17197 1.7862 63268 1.7779 37733 1.7649 8125 1.7371 66884 1.7328 19023 1.7221 56579 1.7170 32349 1.7123 90344 5.3321E- 05 7.74037E -05 0.000295 32 0.000587 577 0.001534 208 0.002760 437 0.000363 127 7.77652E -05 0.002486 839 0.000187 161 0.000196 649 0.000769 904 0.000561 759 0.000472 33 Table B-2 (cont’d) SAUSA300 _RS05670 SAUSA300 _RS13755 SAUSA300 _RS10255 SAUSA300 _RS12415 SAUSA300 _RS06695 SAUSA300 _RS09830 SAUSA300 _RS02805 SAUSA300 _RS08875 SAUSA300 _RS12105 SAUSA300 _RS06705 SAUSA300 _RS13915 SAUSA300 _RS10250 SAUSA300 _RS02845 SAUSA300 _RS12090 SAUSA300 _RS03735 SAUSA300 _1052 SAUSA300 _2479 ecb cidA complement convertase inhibitor Ecb holin-like murein hydrolase modulator CidA 10430 .33 8507. 61 1011. 3 3581. 72 83.68 81.85 10.97 26.05 SAUSA300 _2249 SAUSA300 _1235 SAUSA300 _1796 SAUSA300 _0524 SAUSA300 _1627 SAUSA300 _2195 SAUSA300 _1236 SAUSA300 _2506 SAUSA300 _1874 SAUSA300 _0532 SAUSA300 _2192 SAUSA300 _0696 hypothetical protein 22.66 36.96 3.61 8.17 CHAP domain-containing protein 1972. 19 2369. 74 375.4 6 388.1 3 guaC GMP reductase 17.25 31.11 2.02 9.81 DUF445 domain-containing protein rplJ 50S ribosomal protein L10 infC translation initiation factor IF-3 rpsQ 30S ribosomal protein S17 CAP domain-containing protein isaA lytic transglycosylase IsaA ftnA H-type ferritin FtnA fusA elongation factor G rplE 50S ribosomal protein L5 queD 6-carboxytetrahydropterin synthase QueD 115.1 4 11616 .08 430.3 8 113.0 3 132.1 9 7884. 88 492.8 519.9 4 154.4 5 89.24 7.76 47.79 13656 .03 791.6 2 200.2 9 145.2 8 8642. 19 949.3 6 1145. 37 1888. 15 69.57 3950. 63 229.9 3 20.36 53.63 12.84 63.9 1471. 51 122.5 9 121.7 7 2548. 49 214.3 4 295.8 6 292 35.53 75.61 15.48 26.72 3.84 6.07 1.7004 4758 1.6981 77302 1.6954 15345 1.6951 27202 1.6588 68724 1.6197 63721 1.6172 89669 1.6112 53418 1.6042 31927 1.5688 3287 1.4884 56096 1.4836 56263 1.4768 87914 1.4690 91457 1.4440 43835 0.000630 005 0.000352 912 0.005250 193 0.001996 199 0.002041 038 0.005623 452 0.000415 338 0.000796 613 0.000769 904 0.003192 373 0.002608 647 0.003553 971 0.003553 971 0.002999 274 0.010359 352 287 Table B-2 (cont’d) SAUSA300 _RS12085 SAUSA300 _RS12075 SAUSA300 _2191 SAUSA300 _2189 SAUSA300 _RS03880 SAUSA300 _0721 SAUSA300 _RS04410 SAUSA300 _RS13020 SAUSA300 _RS02840 SAUSA300 _RS08430 SAUSA300 _RS01435 SAUSA300 _RS12080 SAUSA300 _RS03445 SAUSA300 _RS02795 SAUSA300 _RS11455 SAUSA300 _RS06220 SAUSA300 _RS09505 SAUSA300 _RS01470 SAUSA300 _0818 SAUSA300 _2358 SAUSA300 _0531 SAUSA300 _1546 SAUSA300 _0267 SAUSA300 _2190 SAUSA300 _0642 SAUSA300 _0522 SAUSA300 _2081 SAUSA300 _1149 SAUSA300 _1738 SAUSA300 _0274 type Z 30S ribosomal protein S14 rplF 50S ribosomal protein L6 sufC siderophore ABC transporter substrate- binding protein Fe-S cluster assembly ATPase SufC amino acid ABC transporter permease rpsG 30S ribosomal protein S7 holA DNA polymerase III subunit delta 249.5 7 193.0 4 222.0 8 71.9 119.5 3 642.5 6 376.9 9 286.3 3 58.41 92.01 43.19 76.27 263.1 18 134.3 125.9 1 376.5 8 1202. 65 9.8 48.93 19.62 162.6 3 127.3 1 325.5 34.2 35.18 4.79 16.68 IS1182 family transposase 12.61 16 2.04 6.4 rpsH 30S ribosomal protein S8 hypothetical protein rplK 50S ribosomal protein L11 CTP synthase rpsB 30S ribosomal protein S2 DUF4909 domain- containing protein 240.1 5 175.7 3 6887. 07 112.3 1 2338. 33 345.3 5 162.0 3 8204. 74 246.9 7 3407. 46 62.37 82.95 16.71 97.23 1597. 14 2381. 7 25.14 82.44 547.4 9 1071. 67 16.8 14.12 3.24 5.12 hypothetical protein 76.11 61.94 8.81 37.54 1.4414 64039 1.4369 41322 1.4354 17673 1.4314 76197 1.3771 26022 1.3732 34814 1.3663 20996 1.3459 79451 1.3411 18802 1.3403 10845 1.3234 76865 1.3228 00643 1.3155 80016 1.3139 33411 1.3122 30047 0.005140 203 0.003727 587 0.021157 034 0.010359 352 0.040614 674 0.006761 354 0.008896 376 0.021157 034 0.015622 243 0.027666 854 0.013419 835 0.014780 092 0.008059 084 0.031711 545 0.022468 518 288 Table B-2 (cont’d) SAUSA300 _RS01175 SAUSA300 _RS03655 SAUSA300 _RS12020 SAUSA300 _RS05300 SAUSA300 _RS13015 SAUSA300 _RS02800 SAUSA300 _RS05600 SAUSA300 _RS05880 SAUSA300 _RS08870 SAUSA300 _RS02345 SAUSA300 _RS09325 SAUSA300 _RS02835 SAUSA300 _RS06750 SAUSA300 _RS09395 SAUSA300 _RS12150 SAUSA300 _0223 SAUSA300 _0681 SAUSA300 _2178 SAUSA300 _0985 SAUSA300 _2357 SAUSA300 _0523 SAUSA300 _1040 SAUSA300 _1085 SAUSA300 _1626 SAUSA300 _0438 SAUSA300 _1708 SAUSA300 _0530 SAUSA300 _1243 SAUSA300 _1720 SAUSA300 _2204 complement inhibitor SCIN family protein hypothetical protein DNA-directed RNA polymerase subunit α glutaredoxin-like protein NrdH amino acid ABC transporter ATP-binding protein nrdH rplA 50S ribosomal protein L1 zapA cell division protein ZapA RNA-binding protein rpmI 50S ribosomal protein L35 aaa autolysin/adhesin Aaa MarR family transcriptional regulator rpsL 30S ribosomal protein S12 1078. 39 194 111.6 6 152.3 5 587.8 7 2815. 97 150.5 8 134.0 9 1426. 77 136.6 1856. 63 449.4 6 749.0 6 164.0 9 241.2 4 101.2 5 1126. 44 4907. 22 163.4 8 132.9 6 2117. 93 235.5 6 1352. 39 921.6 4 218.6 7 246.2 35.4 71.4 27.47 79.22 23.84 53.83 153.5 5 649.3 9 347.3 1722. 04 20.25 87.95 16.44 77.46 348.3 6 707.8 9 32.67 82.52 372.3 8 114.3 5 532.5 7 311.3 4 exonuclease subunit SbcC 24.78 25.9 4.61 11.82 N-acetylglucosaminidase 348.4 4 rplC 50S ribosomal protein L3 61.97 399.6 3 135.6 3 56.98 199.5 18.58 39.74 1.2866 69411 1.2839 8078 1.2826 24568 1.2822 92065 1.2793 74623 1.2737 65546 1.2659 09775 1.2612 47318 1.2526 20214 1.2501 36779 1.2426 53826 1.2421 40915 1.2385 71117 1.2371 68972 1.2270 8297 0.045188 836 0.017977 586 0.017455 078 0.028431 659 0.013517 702 0.011596 632 0.024410 672 0.028738 26 0.013387 071 0.014232 282 0.040537 541 0.020375 529 0.015981 333 0.018201 307 0.027466 148 289 Table B-2 (cont’d) SAUSA300 _RS14655 SAUSA300 _RS07285 SAUSA300 _RS04180 SAUSA300 _RS05430 SAUSA300 _RS12115 SAUSA300 _RS09090 SAUSA300 _RS11210 SAUSA300 _2639 SAUSA300 _1336 SAUSA300 _0775 SAUSA300 _1009 SAUSA300 _2197 SAUSA300 _1666 SAUSA300 _2037 cold-shock protein class I SAM-dependent RNA methyltransferase 85383. 85 73729. 38 15403 .29 36858 .2 23.54 38.19 6.02 13.94 hypothetical protein 107.1 137.98 18.31 70.73 typA translational GTPase TypA 43.52 66.53 10.76 25.73 rplP 50S ribosomal protein L16 100.6 225.21 34.63 62.4 rpsD 30S ribosomal protein S4 8545.8 10848. 68 2335. 75 3461. 35 DEAD/DEAH box helicase 461.01 801.74 96.45 354 SAUSA300 _RS11405 SAUSA300 _2071 prmC peptide chain release factor N(5)-glutamine methyltransferase 74.85 150.33 20.07 54.97 SAUSA300 _RS12070 SAUSA300 _RS11990 SAUSA300 _2188 SAUSA300 _2172 rplR 50S ribosomal protein L18 224.25 336.27 67.79 97.07 rplM 50S ribosomal protein L13 2485.6 4 3347.5 710.5 7 1079. 1 1.2128 49751 1.1717 57401 1.1674 47496 1.1584 696 1.1572 64551 1.1539 68675 1.1539 09403 1.1535 0969 1.1456 81757 1.1237 28432 0.023895 526 0.027320 561 0.034250 049 0.025045 599 0.049442 479 0.040708 41 0.037063 284 0.036318 346 0.048416 068 0.048067 748 Genes downregulated in GSSG when compared to CSSC Locus Old locus Gene Product SAUSA300 _RS05795 SAUSA300 _1068 TPM.1 GSSG TPM.2 GSSG 157.97 172.34 TPM. 1 CSSC TPM. 2 CSSC 3464. 54 3534. 23 DE Log2 FC - 4.3047 27876 DE Adj. P-value 1.5934E- 20 290 Table B-2 (cont’d) SAUSA300 _RS05790 SAUSA300 _1067 beta-class phenol-soluble modulin 231.7 6 231.6 4 4820. 71 SAUSA300 _RS04580 SAUSA300 _0848 beta-class phenol-soluble modulin 3.18 4.13 29.4 4917 171.1 5 SAUSA300 _RS13360 SAUSA300 _2413 cntL FAD/NAD(P)-binding protein 3.35 2.17 42.35 67.31 SAUSA300 _RS15090 SAUSA300 _RS15740 SAUSA300 _RS15735 SAUSA300 _RS15730 SAUSA300 _RS03005 D-histidine (S)-2- aminobutanoyltransferase CntL 152.5 9 251.8 2 1880. 69 4471. 81 phenol-soluble modulin PSM-alpha-3 119.2 1 201.3 9 1294. 02 3576. 07 phenol-soluble modulin PSM-alpha-1 153.8 8 267.8 1 1675. 5 4447. 18 phenol-soluble modulin PSM-alpha-2 128.5 5 212.5 7 1439. 61 3355. 4 vraX phenol-soluble modulin PSM-alpha-4 14226 .21 7060. 01 13009 8.29 60616 .24 SAUSA300 _RS13040 SAUSA300 _2361 C1q-binding complement inhibitor VraX 7009. 33 4703. 8 51134 .91 41439 .61 SAUSA300 _RS03000 putative metal homeostasis protein 350 157.3 4 2775. 63 1589. 92 - 4.2788 90597 - 4.2079 16659 - 4.0635 14424 - 3.8975 8922 - 3.8075 26925 - 3.7626 71691 - 3.7540 98002 - 3.0293 80781 - 2.9819 50513 - 2.9719 6365 3.97915E -20 3.97915E -20 1.42843E -18 3.04382E -22 4.6765E- 21 1.00147E -20 1.00147E -20 7.51319E -07 3.35575E -08 7.51319E -07 291 Table B-2 (cont’d) SAUSA300 _RS13355 SAUSA300 _2412 cntM hypothetical protein 4.26 2.14 16.89 38.39 SAUSA300 _RS13330 SAUSA300 _2407 staphylopine dehydrogenase CntM 3.36 4.52 11.71 53.39 SAUSA300 _RS13340 SAUSA300 _2409 ABC transporter ATP- binding protein 5.79 4.06 20.31 54.35 SAUSA300 _RS06690 SAUSA300 _1234 SAUSA300 _RS10275 SAUSA300 _1878 SAUSA300 _RS08880 SAUSA300 _1628 SAUSA300 _RS07010 SAUSA300 _1290 SAUSA300 _RS13325 SAUSA300 _2406 SAUSA300 _RS07015 SAUSA300 _1291 SAUSA300 _RS16085 SAUSA300 _1988 rpsN ABC transporter permease 295.6 1 181.4 991.6 4 2246. 49 rlmD 30S ribosomal protein S14 12.68 16.55 86.77 64.72 23S rRNA (uracil(1939)- C(5))-methyltransferase RlmD dapD amino acid permease 2,3,4,5-tetrahydropyridine- 2,6-dicarboxylate N- acetyltransferase 32.89 39.93 113.0 6 312.0 7 108.4 6 111.7 455.4 9 710.3 2 93.2 55.58 310.4 9 534.5 3 MFS transporter 15.12 19.69 63.58 90.07 amidohydrolase 7032. 08 5470. 08 18503 .83 35902 .11 - 2.8752 51245 - 2.7921 07182 - 2.7631 2879 - 2.6425 68704 - 2.5752 87956 - 2.5423 14586 - 2.5206 51649 - 2.4704 29805 - 2.3269 63795 - 2.1718 67176 1.07614E -08 2.8376E- 09 3.44703E -09 3.35575E -08 2.0706E- 06 1.2333E- 09 2.56794E -08 7.51319E -07 6.21097E -07 4.12707E -06 292 Table B-2 (cont’d) SAUSA300 _RS14175 SAUSA300 _2551 nrdD delta-lysin family phenol- soluble modulin 6.42 13.29 34.12 39.52 SAUSA300 _RS14170 SAUSA300 _2550 nrdG anaerobic ribonucleoside- triphosphate reductase 15.87 30.72 89.47 81 SAUSA300 _RS09670 SAUSA300 _1767 anaerobic ribonucleoside- triphosphate reductase activating protein 10.86 17.86 68.29 38.64 SAUSA300 _RS12890 SAUSA300 _2333 gallidermin/nisin family lantibiotic 39.45 38.95 112.3 9 200.4 2 SAUSA300 _RS01625 SAUSA300 _0305 nitrate/nitrite transporter 115.3 5 157.0 4 429.3 9 544.9 9 SAUSA300 _RS05195 SAUSA300 _0966 purE formate/nitrite transporter family protein 7.16 7.74 17.97 42.28 SAUSA300 _RS07025 SAUSA300 _1293 lysA 5-(carboxyamino)imidazole ribonucleotide mutase 67.99 119.4 9 195.4 7 534.7 SAUSA300 _RS01635 SAUSA300 _0307 diaminopimelate decarboxylase 49.76 43.86 160.9 7 150.1 3 SAUSA300 _RS07000 SAUSA300 _1288 dapA 5'-nucleotidase, lipoprotein e(P4) family 10.77 19.16 33.36 64.28 SAUSA300 _RS10485 4-hydroxy- tetrahydrodipicolinate synthase 83.85 97.64 375.5 9 160.5 7 - 2.1593 93763 - 2.1556 09012 - 2.1500 37555 - 2.1224 75632 - 2.0810 32901 - 2.0663 62184 - 2.0392 70385 - 1.9686 53763 - 1.8928 34032 - 1.8925 03751 2.99305E -05 7.81136E -05 0.000540 373 3.60293E -06 1.90088E -05 7.22394E -06 4.12707E -06 0.000368 55 4.93298E -05 0.004619 96 293 Table B-2 (cont’d) SAUSA300 _RS06990 SAUSA300 _1286 SAUSA300 _RS06995 SAUSA300 _1287 hypothetical protein 12.16 22.17 29.3 94.79 aspartate kinase 12.53 22.2 36.57 76.45 SAUSA300 _RS07020 SAUSA300 _1292 aspartate-semialdehyde dehydrogenase 7.21 12.84 19 45.97 SAUSA300 _RS12480 SAUSA300 _2259 SAUSA300 _RS13350 SAUSA300 _2411 SAUSA300 _RS13345 SAUSA300 _2410 SAUSA300 _RS13860 SAUSA300 _2495 SAUSA300 _RS05185 SAUSA300 _0964 alanine racemase 40.79 50.98 113.1 8 157.6 3 cntA LCP family protein 32.03 26.97 38.2 staphylopine-dependent metal ABC transporter substrate-binding protein CntA 40.11 30.46 59.12 205.1 1 204.0 3 copZ ABC transporter permease 3092. 61 2654. 74 10217 .43 5935. 91 copper chaperone CopZ 1478. 3 715.6 4004. 37 2605. 7 SAUSA300 _RS07005 SAUSA300 _1289 dapB DUF5011 domain- containing protein 23.2 44.89 54.26 SAUSA300 _RS10195 SAUSA300 _1867 liaF 4-hydroxy- tetrahydrodipicolinate reductase 26.5 29.03 56.96 165.9 8 110.1 4 - 1.8873 25956 - 1.8728 70427 - 1.8256 69371 - 1.8103 49562 - 1.8093 68607 - 1.8060 48803 - 1.7996 06649 - 1.7803 12978 - 1.7617 7941 - 1.7508 26039 4.93298E -05 4.85665E -05 7.74037E -05 0.000223 37 0.000740 24 0.000295 32 0.004998 035 0.008012 672 0.000196 649 0.000169 321 294 Table B-2 (cont’d) SAUSA300 _RS10185 SAUSA300 _1865 vraR cell wall-active antibiotics response protein LiaF 96.99 137.4 7 277.5 9 350.5 SAUSA300 _RS04855 SAUSA300 _0902 pepF two-component system response regulator VraR 38.22 65.6 87.93 218.1 4 SAUSA300 _RS13045 SAUSA300 _2362 SAUSA300 _RS12545 SAUSA300 _2269 SAUSA300 _RS15795 oligoendopeptidase F 2,3-diphosphoglycerate- dependent phosphoglycerate mutase pepA1 hypothetical protein 872.1 5 1164. 6 1514. 2 4828. 17 252.3 8 174.5 6 573.5 1 583.6 9 786.7 3 766.4 7 2456. 95 1290. 29 SAUSA300 _RS04985 SAUSA300 _0928 type I toxin-antitoxin system Fst family toxin PepA1 9.63 12.1 19.18 40.77 SAUSA300 _RS06940 SAUSA300 _1278 pepF competence protein ComK 95.77 95.44 207.7 2 271.2 2 SAUSA300 _RS10555 oligoendopeptidase F 657.5 4 372.4 3 1740. 21 716.3 3 SAUSA300 _RS09355 SAUSA300 _1712 ribE hypothetical protein 111.6 2 124.0 6 240.5 9 334.7 3 SAUSA300 _RS00995 SAUSA300 _0190 6,7-dimethyl-8- ribityllumazine synthase 13.28 10.78 25.77 34.11 - 1.7153 2027 - 1.7024 72242 - 1.6841 02273 - 1.6668 65506 - 1.6393 9493 - 1.6206 72046 - 1.5854 20486 - 1.5619 43065 - 1.5538 46145 - 1.5399 30896 0.000792 279 0.000195 422 0.000212 094 0.004942 313 0.015438 398 0.000667 423 0.002808 289 0.038624 179 0.002773 409 0.005623 452 295 Table B-2 (cont’d) SAUSA300 _RS01265 SAUSA300 _0237 SAUSA300 _RS12970 alpha-keto acid decarboxylase family protein 21.17 25.76 50.02 59.68 nucleoside hydrolase 35.76 27.36 81.4 67.56 SAUSA300 _RS03335 SAUSA300 _0622 hypothetical protein 65.9 56.11 140.3 3 140.7 3 SAUSA300 _RS13725 SAUSA300 _2473 M50 family metallopeptidase 102.7 3 95.64 212.7 7 237.7 8 SAUSA300 _RS04185 SAUSA300 _0776 alpha/beta hydrolase 37.61 25.45 55.04 109.1 5 SAUSA300 _RS12780 SAUSA300 _2313 thermonuclease family protein 330.1 5 656.8 3 887.0 6 1282. 53 SAUSA300 _RS14075 SAUSA300 _2537 SAUSA300 _RS05245 SAUSA300 _0976 L-lactate permease 130.8 1 164.8 8 241.4 9 410.5 7 purD L-lactate dehydrogenase 37.61 46.72 68 118.9 4 SAUSA300 _RS00400 SAUSA300 _0079 phosphoribosylamine-- glycine ligase 198.6 1 283.2 2 361.2 6 716.4 3 SAUSA300 _RS00215 SAUSA300 _0040 YdhK family protein 7.35 7.29 16.36 14.43 - 1.5336 85135 - 1.5264 47487 - 1.4995 98783 - 1.4786 66015 - 1.4777 80793 - 1.4553 96328 - 1.3961 83733 - 1.3935 1826 - 1.3893 5943 - 1.3849 91387 0.005005 037 0.018992 461 0.012737 296 0.010359 352 0.006955 209 0.007016 909 0.005316 458 0.005316 458 0.004227 754 0.040708 41 296 Table B-2 (cont’d) SAUSA300 _RS09140 SAUSA300 _1674 hypothetical protein 148.6 5 184.7 5 242.4 5 500.7 5 SAUSA300 _RS12865 SAUSA300 _2328 trypsin-like peptidase domain-containing protein 511.7 1 418.1 1025. 36 853.3 6 SAUSA300 _RS09800 SAUSA300 _1790 DUF4889 domain- containing protein 381.9 6 370.9 8 476.9 7 1357. 86 SAUSA300 _RS01235 SAUSA300 _0233 SAUSA300 _RS01990 SAUSA300 _0374 SAUSA300 _RS04565 SAUSA300 _0845 peptidylprolyl isomerase 19.82 38.78 48.66 69.56 hypothetical protein GlsB/YeaQ/YmgE family stress response membrane protein 3030. 82 4042. 18 6286. 49 7095. 9 54.52 70.8 93.39 150.8 5 SAUSA300 _RS10935 SAUSA300 _1989 agrB M17 family metallopeptidase 561.5 4 883.9 9 1150. 16 1544. 07 SAUSA300 _RS00395 SAUSA300 _0078 accessory gene regulator AgrB 39.14 77.35 66.5 171.5 5 SAUSA300 _RS10950 SAUSA300 _1992 heavy metal translocating P-type ATPase 173.3 6 289.9 8 304.6 4 592.3 2 SAUSA300 _RS07160 SAUSA300 _1314 LytTR family DNA-binding domain-containing protein 237.9 6 273.3 5 397.0 4 556.2 1 - 1.3621 42313 - 1.3607 50229 - 1.3584 39536 - 1.3275 72511 - 1.2881 02453 - 1.2497 80323 - 1.2485 30597 - 1.2175 30251 - 1.2162 70388 - 1.2101 66496 0.004998 035 0.039399 129 0.006449 607 0.024225 047 0.028749 116 0.019408 834 0.028033 267 0.022101 684 0.020463 346 0.033546 539 297 Table B-2 (cont’d) SAUSA300 _RS13150 SAUSA300 _2377 DUF4064 domain- containing protein 33.59 34.15 42.16 91.11 - 1.1608 98774 0.028749 116 298 Table B-3. Reduced glutathione (GSH) differentially expressed genes. Genes upregulated in GSH when compared to CSSC Gene Product acyl-CoA/acyl-ACP dehydrogenase TPM. 1 GSH 121.5 3 ABC transporter permease 66.92 SAUSA300 _RS02340 SAUSA3 00_0437 gmpC Locus SAUSA300 _RS00930 SAUSA300 _RS00925 SAUSA300 _RS02185 SAUSA300 _RS00915 Old locus SAUSA3 00_0177 SAUSA3 00_0176 SAUSA3 00_0407 SAUSA3 00_0174 SAUSA300 _RS02330 SAUSA300 _RS00920 SAUSA300 _RS11740 SAUSA300 _RS01185 SAUSA300 _RS06580 SAUSA300 _RS06505 SAUSA300 _RS06500 SAUSA300 _RS12110 SAUSA3 00_0435 SAUSA3 00_0175 SAUSA3 00_2132 SAUSA3 00_1213 SAUSA3 00_1203 SAUSA3 00_2196 superantigen-like protein SSL11 ABC transporter ATP-binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein methionine ABC transporter ATP-binding protein ABC transporter substrate- binding protein hypothetical protein TPM. 2 GSH 319.7 6 130.6 7 122.9 5 TPM. 1 CSSC TPM. 2 CSSC 2.69 9.11 4.22 2.74 2.18 5.91 DE Log2 FC 4.04672 1405 3.12583 3599 3.08354 8435 2.98901 4416 DE Adj. P-value 8.67351 E-15 5.35039 E-07 1.13366 E-07 3.54407 E-07 30.16 144.4 4 2.31567 9543 4.10697 E-05 17.3 47.95 1.31 1.85 34.84 579.8 4 68.43 889.8 9 117.6 8 6.07 13.82 18.04 50.12 2.69 2.58 757.2 5 1059. 95 76.55 120.0 9 2.29040 1543 2.28240 9423 2.17337 2354 2.15008 3564 2.11181 0188 2.10878 522 2.10687 5855 2.10265 0383 1.35302 E-05 0.00054 7181 8.53142 E-05 0.00029 9038 0.00684 5352 0.00447 5634 0.00074 0514 0.00267 7665 hypothetical protein 90.86 88.68 6.01 16.16 hypothetical protein 44.88 53.03 0.47 12.46 hypothetical protein 18.79 21.99 0.61 4.2 hypothetical protein 32.66 40.54 1.84 7.85 rpmC 50S ribosomal protein L29 87.92 495.6 3 14.13 49.61 299 Table B-3 (cont’d) SAUSA300 _RS01180 SAUSA300 _RS02190 SAUSA300 _RS00910 SAUSA300 _RS12105 SAUSA300 _RS06575 SAUSA300 _RS02845 SAUSA300 _RS02335 SAUSA300 _RS04425 SAUSA300 _RS10920 SAUSA300 _RS02315 SAUSA300 _RS03730 SAUSA300 _RS10510 SAUSA300 _RS02035 SAUSA300 _RS08430 SAUSA300 _RS16005 SAUSA3 00_0224 SAUSA3 00_0408 SAUSA3 00_0173 SAUSA3 00_2195 SAUSA3 00_1212 SAUSA3 00_0532 SAUSA3 00_0436 SAUSA3 00_0821 SAUSA3 00_1986 SAUSA3 00_0432 SAUSA3 00_0695 SAUSA3 00_0382 SAUSA3 00_1546 coa staphylocoagulase FKLRK protein DUF4242 domain-containing protein rpsQ 30S ribosomal protein S17 polymorphic toxin type 50 domain-containing protein fusA elongation factor G methionine ABC transporter permease SUF system NifU family Fe-S cluster assembly protein 38.28 141.8 217.4 7 104.2 3 115.6 7 206.0 4 240.9 8 549.3 6 4.59 14 14.95 26.75 14.98 49.04 20.36 53.63 92.16 106.2 2.68 27.94 1772. 98 52.72 1197. 1 145.6 6 121.7 7 295.8 6 4.85 23.05 16.24 72.76 2.17 9.09 nitroreductase family protein 93.24 55.54 5.47 16.95 queE sodium-dependent transporter 7-carboxy-7-deazaguanine synthase QueE hypothetical protein L-cystine transporter holA DNA polymerase III subunit delta hypothetical protein 221.1 4 143.4 2 16.69 35.07 23.67 32.52 1.9 7.02 801.2 8 1328. 76 621.0 5 696.7 4 47.79 127.0 4 197.9 3 125.6 2 48.34 81.67 4.79 16.68 56.24 127.0 8 3.59 26.52 2.10046 3489 2.08750 2758 2.08181 6612 2.01809 6815 2.01468 4719 2.00858 7337 1.99519 5436 1.98977 3533 1.96734 647 1.95067 1187 1.89704 6743 1.86021 0621 1.83826 2363 1.83115 6883 1.82585 9258 0.00054 9967 0.00012 3001 0.00014 0982 0.00335 1341 0.00386 1321 0.00077 3725 0.00176 9395 0.00414 9892 0.00176 9395 0.00141 8977 0.00119 1171 0.00253 0158 0.00898 218 0.00141 8977 0.00925 3583 300 Table B-3 (cont’d) SAUSA300 _RS06695 SAUSA300 _RS04170 SAUSA300 _RS13910 SAUSA300 _RS12090 SAUSA300 _RS13525 SAUSA300 _RS05520 SAUSA300 _RS13025 SAUSA300 _RS08875 SAUSA300 _RS12150 SAUSA300 _RS12045 SAUSA300 _RS05375 SAUSA300 _RS06705 SAUSA300 _RS01440 SAUSA300 _RS01075 SAUSA300 _RS01435 SAUSA3 00_1235 SAUSA3 00_0773 SAUSA3 00_2505 SAUSA3 00_2192 SAUSA3 00_2440 SAUSA3 00_1026 SAUSA3 00_2359 SAUSA3 00_1627 SAUSA3 00_2204 SAUSA3 00_2183 SAUSA3 00_0998 SAUSA3 00_1236 SAUSA3 00_0268 SAUSA3 00_0204 SAUSA3 00_0267 guaC GMP reductase 21.38 49.87 2.02 9.81 vwb von Willebrand factor binding protein Vwb GNAT family N- acetyltransferase rplE 50S ribosomal protein L5 fnbB infC fibronectin-binding protein FnbB DUF177 domain-containing protein transporter substrate-binding domain-containing protein translation initiation factor IF- 3 rplC 50S ribosomal protein L3 19.7 41.67 2.02 8.27 12.17 33.32 0.55 7 165.4 6 642.5 3 35.53 75.61 41.09 55.47 5.83 8.01 1746. 21 174.4 7 614.0 8 120.9 4 3104. 06 335.8 9 1101. 33 250.5 1 153.0 2 729.1 7 19.34 69.69 69.57 229.9 3 18.58 39.74 adenylate kinase 64.45 331.5 17.28 36.57 XRE family transcriptional regulator CAP domain-containing protein 29.92 66.4 3.57 12.93 176.9 8 225.9 4 12.84 63.9 MFS transporter 37.36 14.92 2.57 6.44 ggt γ-glutamyltransferase 35.54 35.95 4.18 7.39 IS1182 family transposase 34.68 12.85 2.04 6.4 1.81753 7523 1.80091 5624 1.79700 4954 1.79182 7537 1.76549 1147 1.75597 9103 1.75369 4235 1.74829 5789 1.74718 4815 1.74604 1028 1.74023 0711 1.73788 6173 1.72865 6723 1.72170 7092 1.70786 7216 0.00414 9892 0.00306 1771 0.01941 728 0.00682 9885 0.00312 0213 0.00378 1697 0.00267 7665 0.00224 7546 0.00262 9918 0.01342 3043 0.00436 8127 0.00421 5597 0.01348 4163 0.00329 95 0.02103 1039 301 Table B-3 (cont’d) SAUSA300 _RS05535 SAUSA300 _RS01055 SAUSA300 _RS04145 SAUSA300 _RS06570 SAUSA300 _RS15905 SAUSA300 _RS12025 SAUSA300 _RS04410 SAUSA300 _RS09470 SAUSA300 _RS09830 SAUSA300 _RS12085 SAUSA300 _RS00935 SAUSA300 _RS12115 SAUSA300 _RS16000 SAUSA300 _RS12075 SAUSA300 _RS04390 SAUSA3 00_1028 SAUSA3 00_0200 SAUSA3 00_0769 SAUSA3 00_1211 SAUSA3 00_2179 SAUSA3 00_0818 SAUSA3 00_1731 SAUSA3 00_1796 SAUSA3 00_2191 SAUSA3 00_0178 SAUSA3 00_2197 SAUSA3 00_2189 SAUSA3 00_0814 isdB heme uptake protein IsdB 17.83 21.34 1.43 6.1 ABC transporter ATP-binding protein DUF5067 domain-containing protein 93.69 55.55 9.74 14.41 19.39 24.04 0.43 8.37 hypothetical protein hypothetical protein 229.5 5 277.4 2 rpsK 30S ribosomal protein S11 62.73 sufC pckA Fe-S cluster assembly ATPase SufC phosphoenolpyruvate carboxykinase (ATP) DUF445 domain-containing protein type Z 30S ribosomal protein S14 DUF2294 domain-containing protein rplP 50S ribosomal protein L16 207.0 1 219.2 8 394.5 5 206.3 1 16.71 72.57 17.01 86.2 20.99 45.01 9.8 48.93 94.03 31.12 45.89 4.33 9.46 107.5 5 236.7 6 649.7 4 115.7 9 157.8 4 744.2 4 819.4 1 522.5 5 7.76 47.79 58.41 92.01 92.52 169.3 3 34.63 62.4 minor capsid protein 58.13 70.81 5.38 21.52 rplF 50S ribosomal protein L6 173.3 7 567.8 1 43.19 76.27 Abi family protein 582.4 670 29.73 254.5 302 1.70209 7085 1.66739 4083 1.65665 9585 1.64334 9984 1.64334 9642 1.63820 6467 1.63445 5126 1.63095 1725 1.62334 6219 1.60149 5869 1.59783 0273 1.58883 3404 1.58826 9635 1.57031 1496 1.56365 6708 0.00447 5634 0.01218 702 0.03955 7101 0.00876 62 0.01231 2678 0.02809 6832 0.01102 0586 0.00394 9272 0.01253 3987 0.01563 3984 0.00544 0727 0.02481 9405 0.00873 4929 0.01764 915 0.02763 3932 Table B-3 (cont’d) SAUSA300 _RS02840 SAUSA300 _RS07030 SAUSA3 00_0531 SAUSA3 00_1294 SAUSA300 _RS02325 SAUSA3 00_0434 SAUSA300 _RS12020 SAUSA300 _RS05670 SAUSA300 _RS05600 SAUSA300 _RS01060 SAUSA300 _RS03445 SAUSA300 _RS12095 SAUSA300 _RS08565 SAUSA300 _RS09690 SAUSA300 _RS12080 SAUSA300 _RS06625 SAUSA300 _RS07500 SAUSA300 _RS06635 SAUSA3 00_2178 SAUSA3 00_1052 SAUSA3 00_1040 SAUSA3 00_0201 SAUSA3 00_0642 SAUSA3 00_2193 SAUSA3 00_1571 SAUSA3 00_1770 SAUSA3 00_2190 SAUSA3 00_1222 SAUSA3 00_1373 SAUSA3 00_1224 rpsG 30S ribosomal protein S7 msaC ecb sarA expression modulator MsaC bifunctional cystathionine γ- lyase/homocysteine desulfhydrase DNA-directed RNA polymerase subunit α complement convertase inhibitor Ecb zapA cell division protein ZapA 1277. 12 1284. 06 162.6 3 325.5 17.75 18.1 2.12 4.81 46.92 69.42 6.96 15.5 132.5 6 8660. 72 247.9 8 445.8 6 12958 .51 234.5 1 27.47 79.22 1011. 3 3581. 72 20.25 87.95 ABC transporter permease 21.52 21.05 3.3 3.88 hypothetical protein rplX 50S ribosomal protein L24 methyltransferase domain- containing protein 207.4 124.7 4 295.4 8 558.7 4 16.71 97.23 41.81 65.15 12.83 25.92 1.63 6.46 hypothetical protein 13.9 16.32 1.08 5.73 rpsH 30S ribosomal protein S8 230.6 7 663.4 4 62.37 82.95 thermonuclease family protein 65.51 47.86 4.64 22.12 ferredoxin hypothetical protein 1852. 46 121.8 8 1504. 25 147.1 6 274.4 6 309.7 9 18.86 34.11 1.55718 4282 1.54271 4621 0.00797 5117 0.01545 6886 1.53930 7173 0.00741 5367 1.53762 0634 1.53370 34 1.52931 0202 1.52400 8315 1.52212 8826 1.50850 5601 1.50193 637 1.49500 6061 1.48702 4905 1.47938 6077 1.47847 8639 1.46792 3086 0.01944 3584 0.00876 62 0.01493 5747 0.02015 7954 0.01943 2255 0.03675 2967 0.01969 4909 0.02763 3932 0.02806 6756 0.02809 6832 0.02809 6832 0.01330 2658 303 Table B-3 (cont’d) SAUSA300 _RS09695 SAUSA300 _RS02815 SAUSA300 _RS06390 SAUSA300 _RS02835 SAUSA300 _RS08295 SAUSA300 _RS12145 SAUSA300 _RS03735 SAUSA300 _RS08400 SAUSA300 _RS04255 SAUSA300 _RS13775 SAUSA300 _RS08015 SAUSA300 _RS00010 SAUSA300 _RS04180 SAUSA300 _RS08555 SAUSA300 _RS06750 SAUSA3 00_1771 SAUSA3 00_0526 SAUSA3 00_1182 SAUSA3 00_0530 SAUSA3 00_1520 SAUSA3 00_2203 SAUSA3 00_0696 SAUSA3 00_1541 SAUSA3 00_0788 SAUSA3 00_1468 SAUSA3 00_0001 SAUSA3 00_0775 SAUSA3 00_1569 SAUSA3 00_1243 DUF1828 domain-containing protein class I SAM-dependent methyltransferase 2-oxoacid:acceptor oxidoreductase subunit α rpsL 30S ribosomal protein S12 tRNA (adenine(22)-N(1))- methyltransferase TrmK rplD 50S ribosomal protein L4 queD grpE 6-carboxytetrahydropterin synthase QueD nucleotide exchange factor GrpE nitroreductase hypothetical protein 129.9 9 57.03 153.4 3 117.7 6 9.95 58.37 6.44 33.14 12.95 25.82 1.98 6.27 850.5 4 1081. 74 114.3 5 311.3 4 13.6 43.2 1.83 10.5 274.4 6 210.0 1 36.9 61.43 24.28 26.15 3.84 6.07 34.77 97.47 4.95 24.8 499.0 8 436.5 5 521.3 5 370.8 2 53.82 34.13 191.2 3 169.6 6 recN DNA repair protein RecN 27.49 87.77 4.41 21.76 dnaA chromosomal replication initiator protein DnaA hypothetical protein 49.1 192.8 7 100.9 2 163.2 6 6.12 29.82 18.31 70.73 U32 family peptidase 19.76 55.56 3.9 12.58 exonuclease subunit SbcC 26.76 46.2 4.61 11.82 1.46283 2805 1.46021 2935 1.45878 6422 1.44297 068 1.44243 5919 1.42442 5489 1.42353 6204 1.41518 437 1.40664 8838 1.40162 398 1.38679 0721 1.38666 2694 1.37887 6713 1.37597 343 1.36188 1461 0.02721 8672 0.02721 8672 0.01696 0367 0.01218 702 0.04698 6359 0.02721 8672 0.02800 4122 0.04081 6815 0.01969 4909 0.03657 7794 0.04909 9554 0.03551 508 0.03109 4167 0.03675 2967 0.02073 2042 304 Table B-3 (cont’d) SAUSA300 _RS06225 SAUSA300 _RS01470 SAUSA300 _RS07495 SAUSA300 _RS10985 SAUSA300 _RS05880 SAUSA300 _RS14570 SAUSA300 _RS03505 SAUSA300 _RS09570 SAUSA300 _RS12140 SAUSA3 00_0274 SAUSA3 00_1372 SAUSA3 00_1998 SAUSA3 00_1085 SAUSA3 00_2622 SAUSA3 00_0654 SAUSA3 00_1749 SAUSA3 00_2202 hypothetical protein hypothetical protein helix-turn-helix domain- containing protein 359.1 4 76.55 114.1 7 729.7 9 106.0 4 105.2 8 71.03 167.1 7 8.81 37.54 17.77 29.53 YeeE/YedE family protein 33.44 42.54 4.7 14.19 RNA-binding protein rhodanese-related sulfurtransferase HTH-type transcriptional regulator SarX DUF1433 domain-containing protein sarX rplW 50S ribosomal protein L23 150.4 5 209.2 5 200.0 8 216.0 6 16.44 77.46 25.72 85.02 25.49 38.97 4.17 12.09 48.44 59.34 7.05 21.44 300.7 4 391.0 1 55.07 115.7 5 Genes downregulated in GSH when compared to CSSC Locus Old locus Gene Product SAUSA300 _RS03005 vraX C1q-binding complement inhibitor VraX TPM. 1 GSH 7355. 24 TPM. 2 GSH 5156. 82 TPM. 1 CSSC TPM. 2 CSSC 13009 8.29 60616 .24 SAUSA300 _RS13040 SAUSA3 00_2361 putative metal homeostasis protein 4606. 12 5956. 22 51134 .91 41439 .61 1.35017 3181 1.34280 6873 1.34050 4493 1.30498 8845 1.29383 7497 1.28821 0782 1.24392 7166 1.22571 3833 1.21705 224 DE Log2 FC - 3.96794 5975 - 3.48392 362 0.02721 8672 0.03120 5807 0.03430 1114 0.03024 6065 0.04397 0121 0.03605 4453 0.04914 1271 0.04829 7087 0.04362 6347 DE Adj. P-value 1.11797 E-09 1.7629E -09 305 Table B-3 (cont’d) SAUSA300 _RS03000 hypothetical protein 278.9 2 126 2775. 63 1589. 92 SAUSA300 _RS04580 SAUSA3 00_0848 SAUSA300 _RS12890 SAUSA3 00_2333 SAUSA300 _RS13360 SAUSA3 00_2413 cntL SAUSA300 _RS15375 SAUSA300 _RS13860 SAUSA3 00_2495 SAUSA300 _RS06690 SAUSA3 00_1234 SAUSA300 _RS10485 FAD/NAD(P)-binding protein 8.92 9.34 29.4 171.1 5 nitrate/nitrite transporter 20.71 16.94 112.3 9 200.4 2 D-histidine (S)-2- aminobutanoyltransferase CntL hypothetical protein copZ copper chaperone CopZ rpsN 30S ribosomal protein S14 5.63 10.01 42.35 67.31 127.4 5 127.3 8 1755. 08 300.1 4 1014. 44 1716. 65 10217 .43 5935. 91 254.1 6 210.7 6 991.6 4 2246. 49 hypothetical protein 37.25 52.36 SAUSA300 _RS01625 SAUSA3 00_0305 formate/nitrite transporter family protein 96.03 78.13 SAUSA300 _RS13325 SAUSA3 00_2406 MFS transporter 91.76 56.32 306 375.5 9 160.5 7 429.3 9 544.9 9 310.4 9 534.5 3 - 3.47821 0201 - 3.29698 9547 - 3.27583 3554 - 3.17898 958 - 3.15352 3215 - 3.01854 4811 - 3.00510 6974 - 2.98346 4068 - 2.82685 1365 - 2.73389 307 2.09417 E-07 2.82912 E-09 7.74351 E-10 1.11797 E-09 2.80009 E-05 1.92626 E-06 5.57662 E-09 1.22502 E-05 5.35039 E-07 1.70962 E-06 Table B-3 (cont’d) SAUSA300 _RS13340 SAUSA3 00_2409 ABC transporter permease 6.94 6.37 20.31 54.35 SAUSA300 _RS10275 SAUSA3 00_1878 rlmD 23S rRNA (uracil(1939)-C(5))- methyltransferase RlmD 10.55 25.98 86.77 64.72 SAUSA300 _RS07015 SAUSA3 00_1291 amidohydrolase 14.52 20.48 63.58 90.07 SAUSA300 _RS07010 SAUSA3 00_1290 dapD 2,3,4,5-tetrahydropyridine- 2,6-dicarboxylate N- acetyltransferase 147.9 1 82.44 455.4 9 710.3 2 SAUSA300 _RS13355 SAUSA3 00_2412 cntM staphylopine dehydrogenase CntM 4.91 7.1 16.89 38.39 SAUSA300 _RS08880 SAUSA3 00_1628 amino acid permease 44.79 44.97 113.0 6 312.0 7 SAUSA300 _RS14175 SAUSA3 00_2551 nrdD anaerobic ribonucleoside- triphosphate reductase 6.63 12.59 34.12 39.52 SAUSA300 _RS01635 SAUSA3 00_0307 5'-nucleotidase, lipoprotein e(P4) family 43.27 35.25 160.9 7 150.1 3 SAUSA300 _RS13850 SAUSA3 00_2493 cwrA cell wall inhibition responsive protein CwrA 620.2 8 482.2 3 3279. 08 941.3 SAUSA300 _RS13765 SAUSA3 00_2481 sterile α motif-like domain- containing protein 5849. 07 9772. 13 34671 .29 16151 .83 - 2.69541 6261 - 2.59701 828 - 2.59251 0948 - 2.57781 0751 - 2.55612 3075 - 2.49740 6008 - 2.46304 0159 - 2.43008 7067 - 2.38590 1587 - 2.30896 2369 3.1823E -07 6.42746 E-05 1.48713 E-06 1.80121 E-05 5.35039 E-07 1.33354 E-06 2.44652 E-05 0.00010 6264 0.00196 2663 0.00122 9708 307 Table B-3 (cont’d) SAUSA300 _RS14170 SAUSA3 00_2550 nrdG anaerobic ribonucleoside- triphosphate reductase activating protein SAUSA300 _RS09670 SAUSA3 00_1767 gallidermin/nisin family lantibiotic 20.93 31.36 89.47 81 16.77 15.01 68.29 38.64 SAUSA300 _RS12480 SAUSA3 00_2259 SAUSA300 _RS06590 SAUSA3 00_1215 SAUSA300 _RS11550 SAUSA3 00_2097 LCP family protein 35.78 48.58 hypothetical protein 48.95 74.96 113.1 8 157.6 3 241.9 3 125.4 SDR family oxidoreductase 15.49 14.45 31.72 75.47 SAUSA300 _RS15795 pepA 1 type I toxin-antitoxin system Fst family toxin PepA1 715.0 7 553.1 8 2456. 95 1290. 29 SAUSA300 _RS07025 SAUSA3 00_1293 lysA diaminopimelate decarboxylase 74.55 150.8 9 195.4 7 534.7 SAUSA300 _RS13345 SAUSA3 00_2410 SAUSA300 _RS00400 SAUSA3 00_0079 SAUSA300 _RS01990 SAUSA3 00_0374 ABC transporter permease 34.71 39.89 59.12 204.0 3 YdhK family protein GlsB/YeaQ/YmgE family stress response membrane protein 94.18 273.6 7 361.2 6 716.4 3 2038. 13 2912. 41 6286. 49 7095. 9 - 2.27217 4034 - 2.24727 971 - 2.19433 8867 - 2.18573 6423 - 2.15818 5258 - 2.10272 1683 - 2.07578 2801 - 2.06866 9945 - 2.02505 2602 - 2.01535 6648 0.00029 5853 0.00176 9395 0.00010 572 0.00210 4691 9.08117 E-05 0.00366 6205 0.00015 0994 0.00015 5603 0.00084 7597 0.00092 0149 308 Table B-3 (cont’d) SAUSA300 _RS01265 SAUSA3 00_0237 nucleoside hydrolase 21.77 15.4 50.02 59.68 SAUSA300 _RS13280 SAUSA3 00_2398 fetB iron export ABC transporter permease subunit FetB 38.38 40.36 106.8 2 105.3 3 SAUSA300 _RS09355 SAUSA3 00_1712 ribE 6,7-dimethyl-8-ribityllumazine synthase 105.4 7 96.68 240.5 9 334.7 3 SAUSA300 _RS12545 SAUSA3 00_2269 hypothetical protein 243.5 7 181.8 9 573.5 1 583.6 9 SAUSA300 _RS05185 SAUSA3 00_0964 DUF5011 domain-containing protein 1499. 07 1054. 32 4004. 37 2605. 7 SAUSA300 _RS13330 SAUSA3 00_2407 ABC transporter ATP-binding protein 9.03 10.4 11.71 53.39 SAUSA300 _RS13725 SAUSA3 00_2473 SAUSA300 _RS06940 SAUSA3 00_1278 alpha/beta hydrolase 99.78 68.21 pepF oligoendopeptidase F 97.95 85.16 212.7 7 237.7 8 207.7 2 271.2 2 SAUSA300 _RS13855 SAUSA3 00_2494 heavy metal translocating P- type ATPase 152.5 5 175.0 7 318.0 7 537.5 5 SAUSA300 _RS04855 SAUSA3 00_0902 pepF oligoendopeptidase F 35.3 79.69 87.93 218.1 4 - 2.00685 8994 - 1.98968 3824 - 1.98586 7081 - 1.94285 3336 - 1.92071 4136 - 1.90339 4709 - 1.89490 8447 - 1.88249 2346 - 1.87471 9351 - 1.85138 4637 0.00176 9395 0.00176 9395 0.00092 3237 0.00306 1771 0.00764 851 0.00159 4128 0.00378 1697 0.00226 7767 0.00095 0944 0.00138 7792 309 Table B-3 (cont’d) SAUSA300 _RS14010 SAUSA3 00_2525 fructosamine kinase family protein 27.65 23 62.37 62.14 SAUSA300 _RS05795 SAUSA3 00_1068 beta-class phenol-soluble modulin 1868. 35 569.9 5 3464. 54 3534. 23 SAUSA300 _RS15490 type I toxin-antitoxin system toxin PepG1 775.3 6 570.2 4 2420. 73 678.5 4 SAUSA300 _RS13645 SAUSA3 00_2460 GNAT family N- acetyltransferase 101.2 9 90.05 252.1 3 188.1 2 SAUSA300 _RS12865 SAUSA3 00_2328 DUF4889 domain-containing protein 434.6 9 380.4 1025. 36 853.3 6 SAUSA300 _RS05995 SAUSA3 00_1107 TM2 domain-containing protein 1924. 53 2043. 23 5682. 01 2971. 77 SAUSA300 _RS04765 SAUSA3 00_0884 SAUSA300 _RS14670 SAUSA3 00_2642 YjzD family protein 684.4 3 791.2 3 1986. 21 1210. 13 DUF3147 family protein 8.84 5.38 16.61 19.99 SAUSA300 _RS02195 SAUSA3 00_0409 spn myeloperoxidase inhibitor SPIN 1850. 15 1455. 3 4360. 22 3178. 19 SAUSA300 _RS04565 SAUSA3 00_0845 M17 family metallopeptidase 51.4 48.49 93.39 150.8 5 - 1.83725 5588 - 1.82868 7326 - 1.81450 2823 - 1.80240 4738 - 1.79248 9741 - 1.79230 2309 - 1.78446 5382 - 1.78435 4321 - 1.77767 8341 - 1.76895 9834 0.00521 7474 0.01604 228 0.02721 8672 0.01003 4607 0.00876 62 0.01493 5747 0.01253 3987 0.01013 7322 0.01218 702 0.00286 2569 310 Table B-3 (cont’d) SAUSA300 _RS10185 SAUSA3 00_1865 vraR two-component system response regulator VraR 108.1 8 172.7 9 277.5 9 350.5 SAUSA300 _RS01620 SAUSA3 00_0304 DUF4064 domain-containing protein 23.16 30.35 41.54 89.51 SAUSA300 _RS15740 phenol-soluble modulin PSM- alpha-1 1137. 55 579.1 5 1294. 02 3576. 07 SAUSA300 _RS09140 SAUSA3 00_1674 trypsin-like peptidase domain- containing protein 146.6 9 172.0 7 242.4 5 500.7 5 SAUSA300 _RS00995 SAUSA3 00_0190 alpha-keto acid decarboxylase family protein 15.79 9.96 25.77 34.11 SAUSA300 _RS01235 SAUSA3 00_0233 SAUSA300 _RS10555 SAUSA300 _RS13045 SAUSA3 00_2362 SAUSA300 _RS06715 SAUSA300 _RS12715 hypothetical protein 18.92 37.34 48.66 69.56 hypothetical protein 2,3-diphosphoglycerate- dependent phosphoglycerate mutase DNA damage-induced cell division inhibitor SosA 778.6 1 395.6 1 1740. 21 716.3 3 1347. 72 1092. 58 1514. 2 4828. 17 30.21 39.84 75.14 62.69 hypothetical protein 38.89 63.88 113.6 5 82.31 - 1.76615 5442 - 1.74364 7507 - 1.70151 4808 - 1.68447 348 - 1.67176 0935 - 1.65763 1234 - 1.64639 5888 - 1.64403 2898 - 1.64139 3003 - 1.63477 4724 0.00368 2729 0.00203 0812 0.01013 7322 0.00275 7425 0.01220 1753 0.00891 7687 0.04397 0121 0.00631 0057 0.01709 1893 0.02175 1487 311 Table B-3 (cont’d) SAUSA300 _RS14565 SAUSA3 00_2621 SAUSA300 _RS01090 SAUSA3 00_0207 SAUSA300 _RS03335 SAUSA3 00_0622 SAUSA300 _RS14075 SAUSA3 00_2537 SMP- 30/gluconolactonase/LRE family protein 11.83 13.89 27.91 22.36 M23 family metallopeptidase 48.53 29.99 80.85 88.84 M50 family metallopeptidase 81.56 50.23 140.3 3 140.7 3 L-lactate dehydrogenase 166.3 2 128.2 1 241.4 9 410.5 7 SAUSA300 _RS13385 SAUSA3 00_2418 carboxymuconolactone decarboxylase family protein 130.5 5 127.8 235.1 2 291.7 1 SAUSA300 _RS00960 SAUSA3 00_0183 SAUSA300 _RS09425 SAUSA3 00_1725 SAUSA300 _RS12970 YagU family protein transaldolase 655.2 9 562.2 6 1441. 83 854.0 1 260.8 8 205.7 6 368.4 6 647.2 6 hypothetical protein 33.77 46.81 81.4 67.56 SAUSA300 _RS06685 SAUSA3 00_1233 rpmG 50S ribosomal protein L33 6062. 78 8757. 21 16477 .46 9467. 32 SAUSA300 _RS03960 SAUSA3 00_0736 raiA ribosome-associated translation inhibitor RaiA 7751. 35 7263. 3 17578 .62 9706. 41 - 1.62850 8384 - 1.61163 7437 - 1.61162 9485 - 1.59655 9574 - 1.59625 6095 - 1.58270 9357 - 1.57163 5626 - 1.56715 7838 - 1.55780 0202 - 1.55702 8276 0.01904 8836 0.02002 8183 0.02175 1487 0.01052 5476 0.01218 702 0.03551 508 0.01135 6589 0.02481 9405 0.03657 7794 0.03980 0629 312 Table B-3 (cont’d) SAUSA300 _RS04760 SAUSA3 00_0883 MAP domain-containing protein 85.49 70.38 121.1 1 211.7 1 SAUSA300 _RS10805 transcriptional regulator 28.08 25.44 43.65 67.39 SAUSA300 _RS07035 SAUSA3 00_1295 cspA cold shock protein CspA 2573 9.07 32953 .44 65893 .82 36585 .83 SAUSA300 _RS04990 SAUSA3 00_0929 IDEAL domain-containing protein 1430. 09 1320. 39 2511. 05 2756. 31 SAUSA300 _RS11520 SAUSA3 00_2091 deoD purine-nucleoside phosphorylase 605.7 1 750.3 9 887.3 1889. 62 SAUSA300 _RS06835 SAUSA3 00_1258 2-hydroxymuconate tautomerase 1261. 11 1709. 56 3231. 43 1800. 33 SAUSA300 _RS05195 SAUSA3 00_0966 purE 5-(carboxyamino)imidazole ribonucleotide mutase 11.98 16.99 17.97 42.28 SAUSA300 _RS12780 SAUSA3 00_2313 SAUSA300 _RS01560 SAUSA3 00_0292 L-lactate permease hypothetical protein 386.3 8 788.4 5 887.0 6 1282. 53 109.6 1 156.4 6 274.7 3 179.2 5 SAUSA300 _RS15735 phenol-soluble modulin PSM- alpha-2 1729. 12 776.9 1 1675. 5 4447. 18 - 1.55429 9896 - 1.54639 5517 - 1.54589 9549 - 1.53178 4305 - 1.51571 1571 - 1.51485 9276 - 1.51294 4011 - 1.51253 439 - 1.51070 4485 - 1.50259 2842 0.01218 702 0.01539 9326 0.03934 5525 0.02015 7954 0.00821 5915 0.04397 0121 0.01003 4607 0.01545 6886 0.03930 6475 0.03109 4167 313 Table B-3 (cont’d) SAUSA300 _RS04210 hypothetical protein 130.4 4 109.1 6 247.6 4 182.7 9 SAUSA300 _RS12940 SAUSA3 00_2344 cobA uroporphyrinogen-III C- methyltransferase 10.7 10.23 17.6 21.02 SAUSA300 _RS07160 SAUSA3 00_1314 SAUSA300 _RS02365 SAUSA3 00_0442 YozE family protein YibE/F family protein 237.9 7 282.1 9 397.0 4 556.2 1 153.1 5 96.62 216.8 4 251.2 5 SAUSA300 _RS04030 SAUSA3 00_0747 trxB thioredoxin-disulfide reductase 108.6 128.6 3 142.2 5 317.5 9 SAUSA300 _RS09030 SAUSA300 _RS12420 hypothetical protein 53.77 91.22 107.4 7 144.2 5 hypothetical protein 397.7 7 556.7 5 836.4 8 685.1 9 SAUSA300 _RS14620 SAUSA3 00_2632 HdeD family acid-resistance protein 96.03 76.17 130.2 9 182.1 6 SAUSA300 _RS09800 SAUSA3 00_1790 peptidylprolyl isomerase 409.0 1 527.8 6 476.9 7 1357. 86 SAUSA300 _RS02965 SAUSA3 00_0556 hxlB 6-phospho-3- hexuloisomerase 23.35 63 52.33 96.07 - 1.49465 1806 - 1.46714 2223 - 1.46312 0185 - 1.43273 7656 - 1.42582 9929 - 1.42464 5271 - 1.40067 8637 - 1.39224 8821 - 1.38254 4145 - 1.36930 5342 0.04081 6815 0.02721 8672 0.01850 5881 0.04081 6815 0.01366 2837 0.02790 4275 0.04753 3674 0.03560 0642 0.01703 8754 0.03661 048 314 Table B-3 (cont’d) SAUSA300 _RS04985 SAUSA3 00_0928 competence protein ComK 17.45 13.42 19.18 40.77 SAUSA300 _RS04080 SAUSA3 00_0756 gap type I glyceraldehyde-3- phosphate dehydrogenase 1232. 21 2113. 44 2006. 84 3819. 2 SAUSA300 _RS05095 SAUSA3 00_0948 menB 1,4-dihydroxy-2-naphthoyl- CoA synthase 293.4 7 269.2 9 423.4 7 509.8 8 SAUSA300 _RS11560 SAUSA3 00_2099 czrB CDF family zinc efflux transporter CzrB 533.5 4 691.0 2 759.5 6 1273. 14 SAUSA300 _RS07000 SAUSA3 00_1288 dapA SAUSA300 _RS04245 SAUSA3 00_0786 SAUSA300 _RS08745 4-hydroxy- tetrahydrodipicolinate synthase organic hydroperoxide resistance protein hypothetical protein 16.22 43.3 33.36 64.28 64.77 62.98 75.88 346.8 5 345.6 8 424.3 8 146.1 9 756.5 SAUSA300 _RS07165 SAUSA3 00_1315 PTS glucose transporter subunit IIA 235.0 2 286.3 7 315.1 4 535.9 4 SAUSA300 _RS01250 SAUSA3 00_0235 L-lactate dehydrogenase 2378. 38 2353. 91 2610. 86 5659. 02 SAUSA300 _RS03665 SAUSA3 00_0683 DeoR/GlpR family DNA- binding transcription regulator 104.0 8 141.3 3 151.4 7 239.3 9 - 1.36845 6829 - 1.36505 4426 - 1.33361 3795 - 1.30853 1176 - 1.29896 3468 - 1.29385 6257 - 1.29280 0049 - 1.27587 8242 - 1.27542 8749 - 1.27328 3026 0.03354 9663 0.02177 0348 0.04642 7827 0.03170 1317 0.04768 4211 0.03675 2967 0.03733 0.03733 0.03643 0885 0.04023 8276 315 Table B-3 (cont’d) SAUSA300 _RS05540 SAUSA3 00_1029 isdA LPXTG-anchored heme- scavenging protein IsdA 247.9 6 244.8 4 297.7 9 505.1 4 SAUSA300 _RS11300 SAUSA3 00_2052 single-stranded DNA-binding protein 60.13 62.2 73.21 SAUSA300 _RS08320 SAUSA3 00_1525 glycine--tRNA ligase 405.3 9 572.4 4 460.2 125.8 5 1101. 39 - 1.24662 1213 - 1.24535 7734 - 1.17621 1964 0.04753 3674 0.04768 4211 0.04843 413 316 Table B-4. Sodium thiosulfate (sTS) differentially expressed genes. Genes upregulated in sTS when compared to CSSC Locus SAUSA300 _RS00930 SAUSA300 _RS10250 SAUSA300 _RS10920 SAUSA300 _RS04665 SAUSA300 _RS00915 SAUSA300 _RS11935 SAUSA300 _RS00925 SAUSA300 _RS02330 Old locus SAUSA3 00_0177 SAUSA3 00_1874 SAUSA3 00_1986 SAUSA3 00_0864 SAUSA3 00_0174 SAUSA3 00_2165 SAUSA3 00_0176 SAUSA3 00_0435 Gene Product acyl-CoA/acyl-ACP dehydrogenase ftnA H-type ferritin FtnA nitroreductase family protein argininosuccinate synthase ABC transporter ATP-binding protein budA acetolactate decarboxylase ABC transporter permease methionine ABC transporter ATP-binding protein SAUSA300_RS10255 hypothetical protein SAUSA300 _RS13755 SAUSA300 _RS13745 SAUSA300 _RS11605 SAUSA3 00_2479 SAUSA3 00_2477 SAUSA3 00_2105 SAUSA300 _RS03085 SAUSA3 00_0577 cidA holin-like murein hydrolase modulator CidA pyruvate oxidase PTS mannitol transporter subunit IICB redox-sensitive transcriptional regulator HypR TPM. 1 sTS 166. 22 4777 .36 251. 11 26.2 2 34.9 6 95.5 4 80.1 8 182. 15 59.6 9 282. 2 334. 37 42.3 3 137. 39 TPM. 2 sTS 182. 5 5582 .06 259. 56 TPM. 1 CSSC TPM. 2 CSSC 2.69 9.11 122.5 9 214.3 4 5.47 16.95 42.8 0.59 2.39 37.4 8 152. 69 90.4 7 117. 61 130. 21 243. 42 317. 48 265. 36 205. 18 1.31 1.85 4.02 10.73 4.22 2.74 6.07 13.82 3.61 8.17 10.97 26.05 16.24 30.98 3.52 19.81 12.24 7.56 DE Log2 FC DE Adj. P-value 3.975153 529 3.844318 382 3.610523 144 3.596379 992 3.342311 59 3.126458 411 3.098636 848 2.952964 98 2.905815 163 2.901618 74 2.797331 264 2.697969 241 2.612761 904 5.97E-23 8.99E-22 1.63E-19 1.94E-15 1.77E-13 9.50E-14 1.70E-08 1.52E-11 4.00E-08 1.35E-12 7.44E-12 2.47E-05 5.59E-06 317 SAUSA300 _RS02340 SAUSA3 00_0437 gmpC Table B-4 (Cont’d) SAUSA300 _RS02335 SAUSA300 _RS00910 SAUSA300 _RS13025 SAUSA300 _RS01060 SAUSA300 _RS11940 SAUSA300 _RS00920 SAUSA3 00_0436 SAUSA3 00_0173 SAUSA3 00_2359 SAUSA3 00_0201 SAUSA3 00_2166 SAUSA3 00_0175 SAUSA300 _RS09895 SAUSA300 _RS01215 SAUSA300 _RS02345 SAUSA300 _RS04425 SAUSA300 _RS11740 SAUSA300 _RS13910 SAUSA300 _RS07500 SAUSA300 _RS06580 SAUSA3 00_1808 SAUSA3 00_0229 SAUSA3 00_0438 SAUSA3 00_0821 SAUSA3 00_2132 SAUSA3 00_2505 SAUSA3 00_1373 SAUSA3 00_1213 methionine ABC transporter permease DUF4242 domain-containing protein transporter substrate-binding domain-containing protein ABC transporter permease alsS acetolactate synthase AlsS ABC transporter substrate- binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein ABC transporter permease subunit acyl CoA:acetate/3-ketoacid CoA transferase aaa autolysin/adhesin Aaa SUF system NifU family Fe- S cluster assembly protein hypothetical protein GNAT family N- acetyltransferase ferredoxin hypothetical protein 318 167. 18 398. 39 402 58.8 7 63.7 4 30.7 7 818. 78 30.4 15.2 5 472. 91 38.6 5 560. 61 28 2767 .83 56.8 6 115. 53 264. 86 472. 45 34.0 5 64.6 8 32.1 4 605. 64 59.3 6 34.6 3 464. 3 4.85 23.05 14.98 49.04 19.34 69.69 3.3 3.88 2.66 11.08 2.69 2.58 2.573132 474 2.546576 593 2.502235 403 2.500861 509 2.456850 704 2.331970 676 1.07E-07 1.70E-08 3.10E-09 1.50E-06 3.47E-08 7.16E-06 30.16 144.4 4 2.298614 243 2.06E-06 1.67 9.24 1.58 3.76 32.67 82.52 47.9 2.17 9.09 1318 .04 26.0 6 2819 .97 76.55 120.0 9 0.55 7 274.4 6 309.7 9 35.1 0.47 12.46 2.268943 961 2.258843 535 2.170454 206 2.160861 821 2.152383 371 2.131333 234 2.096101 62 2.088354 863 1.29E-05 3.99E-06 1.29E-07 5.46E-06 2.11E-05 0.000552 202 2.11E-05 0.003000 985 Table B-4 (Cont’d) SAUSA300 _RS05865 SAUSA3 00_1082 SAUSA300 _RS04410 SAUSA300 _RS08560 SAUSA300 _RS03730 SAUSA3 00_0818 SAUSA3 00_1570 SAUSA3 00_0695 YggS family pyridoxal phosphate-dependent enzyme Fe-S cluster assembly ATPase SufC sufC peptidase U32 family protein queE 7-carboxy-7-deazaguanine synthase QueE SAUSA300_RS06505 hypothetical protein SAUSA300 _RS01075 SAUSA300 _RS02320 SAUSA300 _RS08565 SAUSA300 _RS02535 SAUSA300 _RS08015 SAUSA300 _RS05880 SAUSA300 _RS05300 SAUSA300 _RS06500 SAUSA300 _RS01065 SAUSA300 _RS07495 SAUSA3 00_0204 SAUSA3 00_0433 SAUSA3 00_1571 SAUSA3 00_0472 SAUSA3 00_1468 SAUSA3 00_1085 SAUSA3 00_0985 SAUSA3 00_1203 SAUSA3 00_0202 SAUSA3 00_1372 ggt g-glutamyltransferase cysteine synthase family protein methyltransferase domain- containing protein 4-(cytidine 5'-diphospho)-2- C-methyl-D-erythritol kinase ispE recN DNA repair protein RecN RNA-binding protein nrdH glutaredoxin-like protein NrdH hypothetical protein ABC transporter permease helix-turn-helix domain- containing protein 86.4 209. 2 20.0 5 26.9 8 22.5 49.0 1 23.8 4 23.2 2 115. 46 87.2 7 257. 23 257. 89 31.7 3 21.0 4 167. 96 143. 38 187. 03 28.3 1 35.9 6 14.9 7 42.4 5 7.63 23.43 9.8 48.93 1.26 5.73 1.9 7.02 0.61 4.2 4.18 7.39 24.3 2.64 2.54 32.3 8 162. 67 75.2 2 327. 74 278. 21 30.9 5 14.2 2 175. 45 1.63 6.46 8.1 34.39 4.41 21.76 16.44 77.46 23.84 53.83 1.84 7.85 1.75 3.11 17.77 29.53 2.053900 828 2.049308 555 2.028225 132 2.025380 044 2.016536 706 2.012821 806 2.001501 731 1.991107 69 1.968938 636 1.937444 192 1.926835 28 1.907622 231 1.894343 714 1.888041 935 1.875386 447 5.29E-06 2.44E-05 3.36E-05 1.29E-05 0.003526 285 6.77E-06 0.000177 93 3.27E-05 2.51E-05 7.31E-05 5.07E-05 7.77E-06 0.000357 239 0.000125 814 2.63E-05 319 Table B-4 (Cont’d) SAUSA300 _RS03080 SAUSA300 _RS12110 SAUSA300 _RS07420 SAUSA300 _RS09470 SAUSA300 _RS07030 SAUSA300 _RS06575 SAUSA300 _RS09400 SAUSA3 00_0576 SAUSA3 00_2196 SAUSA3 00_1361 SAUSA3 00_1731 SAUSA3 00_1294 SAUSA3 00_1212 SAUSA3 00_1721 SAUSA300 _RS06390 SAUSA3 00_1182 SAUSA300 _RS05610 SAUSA300 _RS06610 SAUSA300 _RS12105 SAUSA300 _RS02845 SAUSA3 00_1042 SAUSA3 00_1219 SAUSA3 00_2195 SAUSA3 00_0532 hypothiocyanous acid reductase MerA rpmC 50S ribosomal protein L29 pckA msaC polX heptaprenyl diphosphate synthase component 1 phosphoenolpyruvate carboxykinase (ATP) sarA expression modulator MsaC polymorphic toxin type 50 domain-containing protein hypothetical protein 2-oxoacid:acceptor oxidoreductase subunit alpha DNA polymerase/3'-5' exonuclease PolX sensor histidine kinase rpsQ 30S ribosomal protein S17 fusA elongation factor G SAUSA300_RS11585 hypothetical protein SAUSA300 _RS08285 SAUSA300 _RS06535 SAUSA3 00_1518 SAUSA3 00_1208 DEAD/DEAH box helicase hypothetical protein 274. 24 202 152. 81 24.6 22.6 1 94.6 394. 34 21.8 4 12.7 1 18.4 7 244. 44 1115 .28 23.1 3 70.3 5 39.8 5 263. 92 193. 75 157. 99 74.7 2 23.8 6 73.2 7 368. 21 28.3 6 19.2 7 19.4 1 209. 29 1446 .07 23.9 4 99.9 1 28.5 8 32.03 32.87 14.13 49.61 6.16 48.55 4.33 9.46 2.12 4.81 2.68 27.94 17.92 120.1 1.98 6.27 0.96 4.52 0.93 5.8 20.36 53.63 121.7 7 295.8 6 1.45 6.13 3.81 28.93 3.69 7.04 1.870936 99 1.870234 088 1.857742 232 1.852519 043 1.845486 059 1.815437 068 1.812727 87 1.812403 244 1.807853 608 1.803045 824 1.792468 104 1.764528 575 1.734975 149 1.728418 368 1.727369 268 0.000299 457 3.36E-05 0.000902 686 0.000616 728 0.000122 678 0.003210 931 0.000758 264 4.40E-05 0.000310 34 0.000704 809 4.66E-05 2.94E-05 0.007131 029 0.002203 13 0.000893 544 320 Table B-4 (Cont’d) SAUSA300 _RS12045 SAUSA300 _RS09395 SAUSA300 _RS08400 SAUSA300 _RS04415 SAUSA300 _RS01055 SAUSA300 _RS08430 SAUSA300 _RS11580 SAUSA300 _RS05615 SAUSA300 _RS06000 SAUSA300 _RS07300 SAUSA300 _RS04145 SAUSA300 _RS12020 SAUSA300 _RS04800 SAUSA300 _RS06470 SAUSA300 _RS08765 SAUSA3 00_2183 SAUSA3 00_1720 SAUSA3 00_1541 SAUSA3 00_0819 SAUSA3 00_0200 SAUSA3 00_1546 SAUSA3 00_2101 SAUSA3 00_1043 SAUSA3 00_1108 SAUSA3 00_1339 SAUSA3 00_0769 SAUSA3 00_2178 SAUSA3 00_0890 SAUSA3 00_1198 SAUSA3 00_1609 grpE sufD holA adenylate kinase N-acetylglucosaminidase nucleotide exchange factor GrpE Fe-S cluster assembly protein SufD ABC transporter ATP-binding protein DNA polymerase III subunit delta SAP domain-containing protein endonuclease MutS2 peptide deformylase YppE family protein DUF5067 domain-containing protein DNA-directed RNA polymerase subunit alpha ATP-binding cassette domain-containing protein hflX GTPase HflX A24 family peptidase 159. 09 713. 02 66.9 4 62.9 8 83.0 3 47.2 9 35.6 1 16.8 7 17.6 7 103. 26 23.6 7 241. 15 33.2 13.9 6 15 169. 86 690. 23 91.3 9 67.7 6 73.9 2 70.2 4 32.5 2 19.6 5 21.9 8 123. 55 17.7 1 327. 11 26.4 5 20.1 1 13.3 9 17.28 36.57 56.98 199.5 4.95 24.8 5.79 17.93 9.74 14.41 4.79 16.68 1.47 12.29 1.13 6.14 1.1 6.63 6.7 38.94 0.43 8.37 27.47 79.22 3.17 7.76 1.23 5.57 1.72 3.11 1.723116 664 1.712393 268 1.712372 773 1.688682 79 1.685882 24 1.683984 12 1.678149 92 1.652461 587 1.652173 429 1.645638 316 1.617434 315 1.616044 804 1.606820 984 1.602081 278 1.601724 494 6.26E-05 0.000113 45 0.000634 316 0.000108 67 0.000393 937 0.000268 858 0.004011 335 0.001302 251 0.002996 801 0.001749 293 0.021566 263 0.000215 03 0.000538 775 0.001606 474 0.001404 406 321 Table B-4 (Cont’d) SAUSA300 _RS09505 SAUSA300 _RS10970 SAUSA300 _RS11965 SAUSA300 _RS06635 SAUSA300 _RS13020 SAUSA300 _RS03740 SAUSA300 _RS03655 SAUSA3 00_1738 SAUSA3 00_1995 SAUSA3 00_2169 SAUSA3 00_1224 SAUSA3 00_2358 SAUSA3 00_0697 SAUSA3 00_0681 SAUSA300 _RS02325 SAUSA3 00_0434 SAUSA300 _RS14245 SAUSA300 _RS07325 SAUSA300 _RS06615 SAUSA300 _RS08875 SAUSA300 _RS05040 SAUSA300 _RS12025 SAUSA300 _RS02035 SAUSA3 00_2560 SAUSA3 00_1343 SAUSA3 00_1220 SAUSA3 00_1627 SAUSA3 00_0938 SAUSA3 00_2179 SAUSA3 00_0382 DUF4909 domain-containing protein LacI family DNA-binding transcriptional regulator 29.1 7 24.2 queC DUF6414 family protein hypothetical protein amino acid ABC transporter permease 7-cyano-7-deazaguanine synthase QueC hypothetical protein bifunctional cystathionine g- lyase/homocysteine desulfhydrase hypothetical protein nth endonuclease III infC response regulator transcription factor translation initiation factor IF- 3 hypothetical protein rpsK 30S ribosomal protein S11 L-cystine transporter 23.3 5 126. 41 282. 36 11.3 4 379. 98 55.4 7 38.0 3 64.8 32.0 9 553. 3 43.3 3 151. 2 862. 92 21.1 1 54.5 7 23.9 6 182. 58 411. 6 14.6 3 213. 74 65.4 1 46.9 5 87.8 4 37.3 8 949. 95 39.1 3 203. 18 768. 97 3.24 5.12 2.24 13.74 0.86 9.3 18.86 34.11 19.62 127.3 1 1.12 3.89 35.4 71.4 1.600583 121 1.596151 079 1.593421 868 1.593057 072 1.590410 837 1.574831 082 1.573087 702 0.002224 654 0.005972 472 0.010578 232 0.000529 061 0.003709 448 0.001831 038 0.001783 967 6.96 15.5 1.562598 853 0.000357 239 2.45 15.39 3.97 29.49 2.75 11.65 69.57 229.9 3 1.59 16.71 20.99 45.01 127.0 4 125.6 2 1.558169 728 1.553667 971 1.552786 993 1.550780 996 1.541104 259 1.539937 665 1.528200 303 0.006174 18 0.006569 744 0.001657 565 0.001005 053 0.013906 827 0.000550 683 0.004872 848 322 Table B-4 (Cont’d) SAUSA300 _RS02815 SAUSA3 00_0526 SAUSA300 _RS14655 SAUSA3 00_2639 SAUSA300 _RS10985 SAUSA300 _RS03445 SAUSA300 _RS05600 SAUSA300 _RS09835 SAUSA300 _RS05135 SAUSA300 _RS07205 SAUSA300 _RS08305 SAUSA3 00_1998 SAUSA3 00_0642 SAUSA3 00_1040 SAUSA3 00_1797 SAUSA3 00_0955 SAUSA3 00_1323 SAUSA3 00_1522 SAUSA300 _RS05360 SAUSA3 00_0995 SAUSA300 _RS14570 SAUSA300 _RS04660 SAUSA300 _RS08955 SAUSA300 _RS11415 SAUSA3 00_2622 SAUSA3 00_0863 SAUSA3 00_1641 SAUSA3 00_2073 class I SAM-dependent methyltransferase cold-shock protein YeeE/YedE family protein hypothetical protein zapA cell division protein ZapA helix-turn-helix transcriptional regulator bifunctional autolysin NifU N-terminal domain- containing protein dnaG DNA primase dihydrolipoamide acetyltransferase family protein rhodanese-related sulfurtransferase argH argininosuccinate lyase citrate synthase thymidine kinase 78.7 3 1279 92.8 6 50.9 9 257. 61 237. 88 449. 17 147. 89 46.8 6 64.8 5 85.7 7 245. 63 31.8 5 43.3 2 49.5 6 103. 41 1391 10.1 5 41.3 9 251. 32 262. 88 597. 96 156. 65 50.7 4 99.7 3 122. 32 271. 2 95.9 5 77.9 3 97.1 1 6.44 33.14 1.528094 211 0.002980 786 15403 .29 36858 .2 1.520163 913 4.7 14.19 16.71 97.23 20.25 87.95 63.39 137.0 4 21.05 33.47 3.82 17.28 6.34 30.02 14.37 23.34 25.72 85.02 7.81 15.19 6.68 17.62 5.39 26.81 1.519930 81 1.519712 443 1.517895 145 1.508302 565 1.505201 421 1.498358 274 1.488297 936 1.481352 945 1.480012 125 1.478895 537 1.478202 694 1.477917 301 0.000388 284 0.001033 925 0.004102 487 0.001723 98 0.000605 046 0.001197 262 0.003835 402 0.003419 209 0.001759 275 0.000909 205 0.008440 785 0.001906 952 0.006561 5 323 Table B-4 (Cont’d) SAUSA300 _RS10440 SAUSA300 _RS04640 SAUSA300 _RS09830 SAUSA300 _RS12845 SAUSA300 _RS07330 SAUSA300 _RS04390 SAUSA300 _RS07905 SAUSA300 _RS06110 SAUSA300 _RS08030 SAUSA300 _RS02530 SAUSA300 _RS03690 SAUSA300 _RS08830 SAUSA300 _RS00935 SAUSA300 _RS01475 SAUSA300 _RS06030 SAUSA3 00_1907 SAUSA3 00_0860 SAUSA3 00_1796 SAUSA3 00_2324 SAUSA3 00_1344 SAUSA3 00_0814 SAUSA3 00_1448 SAUSA3 00_1129 SAUSA3 00_1471 SAUSA3 00_0471 SAUSA3 00_0687 SAUSA3 00_1620 SAUSA3 00_0178 SAUSA3 00_0275 SAUSA3 00_1114 DUF1700 domain-containing protein ornithine--oxo-acid transaminase DUF445 domain-containing protein sucrose-specific PTS transporter subunit IIBC DnaD domain-containing protein Abi family protein Fur family transcriptional regulator putative DNA-binding protein exodeoxyribonuclease VII small subunit Veg family protein hemolysin family protein ribosome biogenesis GTP- binding protein YihA/YsxC DUF2294 domain-containing protein DUF5079 family protein yihA rsgA ribosome small subunit- dependent GTPase A 324 58.4 9 17.2 2 117. 02 46.8 8 47.9 1 549. 34 1004 .45 22.8 5 45.7 6 6854 .46 518. 55 25.8 7 699. 17 16.0 9 26.9 1 66.1 8 21.3 7 120. 44 101. 45 80.8 6 623. 37 1049 .92 29.8 1 47.1 6 5742 .38 571. 66 31.5 9 635. 15 13.7 1 41.8 9 3.85 25.08 2.03 6.05 7.76 47.79 9.24 19.02 5.1 23.78 29.73 254.5 138.1 8 264.8 9 1.68 10.38 2.18 19.9 861.0 2 39.62 1623. 4 218.9 2 2.65 10.14 92.52 169.3 3 0.96 6.08 2.17 14.22 1.471677 067 1.469146 272 1.467489 302 1.456119 968 1.451306 077 1.450299 962 1.446574 906 1.443984 846 1.442471 384 1.437194 902 1.436842 442 1.433920 608 1.433256 821 1.424540 089 1.420277 101 0.007713 829 0.001533 869 0.006595 66 0.004064 768 0.005335 616 0.014178 774 0.001194 478 0.011462 055 0.018971 358 0.001770 2 0.005868 545 0.003650 79 0.001759 275 0.013343 771 0.012219 224 Table B-4 (Cont’d) SAUSA300 _RS02630 SAUSA300 _RS08405 SAUSA3 00_0490 SAUSA3 00_1542 hslO hrcA Hsp33 family molecular chaperone HslO heat-inducible transcriptional repressor HrcA SAUSA300_RS16000 minor capsid protein SAUSA300 _RS00010 SAUSA300 _RS09815 SAUSA3 00_0001 SAUSA3 00_1793 dnaA SAUSA300 _RS08175 SAUSA3 00_1498 gcvT SAUSA300 _RS05640 SAUSA300 _RS03170 SAUSA300 _RS06055 SAUSA300 _RS10245 SAUSA3 00_1047 SAUSA3 00_0590 SAUSA3 00_1119 SAUSA3 00_1873 sdhA fakA murT SAUSA300 _RS06015 SAUSA3 00_1111 rlmN SAUSA300 _RS07885 SAUSA3 00_1444 scpB chromosomal replication initiator protein DnaA exonuclease SbcCD subunit D glycine cleavage system aminomethyltransferase GcvT succinate dehydrogenase flavoprotein subunit flavodoxin family protein fatty acid kinase catalytic subunit FakA lipid II isoglutaminyl synthase subunit MurT 23S rRNA (adenine(2503)- C(2))-methyltransferase RlmN SMC-Scp complex subunit ScpB SAUSA300_RS10510 hypothetical protein SAUSA300 _RS12075 SAUSA3 00_2189 rplF 50S ribosomal protein L6 26.3 6 64.4 8 64.7 7 67.8 3 149. 25 20.7 5 80.3 1 168. 71 300. 4 47.4 7 30.9 9 53.9 1 570. 15 278. 74 51.2 1 78.3 1 51.6 7 84.1 4 133. 02 28.4 4 159. 32 353. 81 414. 08 63.4 1 24.5 8 96.8 7 437. 9 302. 35 3.45 13.85 7.29 24.75 5.38 21.52 6.12 29.82 10.9 57.23 2.29 8.93 14.37 35.52 32.37 74.97 34.03 132 5.4 20.43 2.2 11.44 6.52 29.18 47.79 197.9 3 43.19 76.27 1.419876 334 1.414974 279 1.414265 218 1.413894 401 1.409258 628 1.405429 661 1.402206 756 1.395715 06 1.394456 625 1.383188 175 1.374626 49 1.369529 428 1.355984 544 1.353699 98 0.006705 135 0.002020 778 0.004559 983 0.005219 82 0.006866 043 0.004182 027 0.004148 534 0.005254 432 0.003388 922 0.003741 699 0.010331 72 0.009255 663 0.006946 407 0.003358 653 325 Table B-4 (Cont’d) SAUSA300 _RS07055 SAUSA3 00_1298 SAUSA300 _RS08555 SAUSA300 _RS08475 SAUSA300 _RS13525 SAUSA300 _RS03525 SAUSA3 00_1569 SAUSA3 00_1555 SAUSA3 00_2440 SAUSA3 00_0657 5-bromo-4-chloroindolyl phosphate hydrolysis family protein U32 family peptidase aroE shikimate dehydrogenase fnbB fibronectin-binding protein FnbB DUF402 domain-containing protein SAUSA300_RS10880 hypothetical protein SAUSA300 _RS13770 SAUSA300 _RS03075 SAUSA300 _RS12090 SAUSA300 _RS09175 SAUSA300 _RS06160 SAUSA300 _RS14005 SAUSA300 _RS08020 SAUSA3 00_2482 SAUSA3 00_0575 SAUSA3 00_2192 SAUSA3 00_1680 SAUSA3 00_1138 SAUSA3 00_2524 SAUSA3 00_1469 CHAP domain-containing protein DUF1450 domain-containing protein rplE 50S ribosomal protein L5 hypothetical protein ADP-forming succinate--CoA ligase subunit b TIGR04197 family type VII secretion effector transcriptional regulator ArgR sucC argR SAUSA300_RS12465 hypothetical protein SAUSA300 _RS04920 SAUSA3 00_0915 esterase family protein 90.7 2 30.9 4 20.0 1 33.4 2 131. 95 28.1 6 327. 51 136. 04 241. 32 28.8 5 39.7 9 10.9 4 254. 79 79.8 2 28.3 3 120. 45 38.7 1 19.3 1 36.9 9 119. 81 23.5 9 475. 05 155. 43 261. 54 44.3 7 75.4 5 13.6 9 197. 21 68.5 8 35.5 6 9.78 41.59 3.9 12.58 2.41 6.61 5.83 8.01 10.61 53.72 2.18 10.71 69.74 79.07 16.77 52.54 35.53 75.61 3.77 13.81 5.34 22.88 1.2 4.49 33.1 66.06 6.51 31.23 2.92 13.15 1.347639 201 1.328773 323 1.328408 177 1.325807 162 1.324846 698 1.323400 914 1.321758 295 1.320392 738 1.316847 299 1.316413 745 1.316067 708 1.311195 86 1.310396 701 1.307818 655 1.306127 155 0.006393 396 0.004179 148 0.005343 835 0.007685 236 0.010740 75 0.015076 794 0.013171 009 0.004002 855 0.003248 204 0.008367 857 0.012297 821 0.026708 744 0.005868 545 0.013808 772 0.010544 027 326 Table B-4 (Cont’d) SAUSA300 _RS06570 SAUSA300 _RS06655 SAUSA3 00_1211 SAUSA3 00_1228 hypothetical protein thrB homoserine kinase SAUSA300 _RS07355 SAUSA3 00_1349 bshA N-acetyl-alpha-D- glucosaminyl L-malate synthase BshA SAUSA300_RS00405 hypothetical protein SAUSA300 _RS02780 SAUSA3 00_0519 RNA polymerase sigma factor SAUSA300_RS07450 hypothetical protein SAUSA300 _RS08900 SAUSA3 00_1631 SAUSA300 _RS06625 SAUSA300 _RS02840 SAUSA300 _RS10445 SAUSA300 _RS08295 SAUSA300 _RS06425 SAUSA300 _RS07310 SAUSA300 _RS12115 SAUSA3 00_1222 SAUSA3 00_0531 SAUSA3 00_1908 SAUSA3 00_1520 SAUSA3 00_1189 SAUSA3 00_1340 SAUSA3 00_2197 replication initiation and membrane attachment family protein thermonuclease family protein rpsG 30S ribosomal protein S7 membrane protein tRNA (adenine(22)-N(1))- methyltransferase TrmK DNA mismatch repair endonuclease MutL Holliday junction resolvase RecU mutL recU rplP 50S ribosomal protein L16 198. 09 39 21.7 5 42.0 6 12.8 2 38.7 9 44.2 5 47.4 9 974. 86 67.4 3 21.4 8 25.0 6 96.4 9 219. 55 152. 7 34.0 8 29.7 4 41.6 8 11.1 8 36.9 3 49.6 4 55.8 1 1209 .2 75.8 7 25.0 9 21.6 3 110. 36 216. 18 16.71 72.57 4.04 14.12 2.94 9.58 2.57 19.13 1.2 4.68 5.6 11.16 4.2 20.37 4.64 22.12 162.6 3 325.5 5.72 32.43 1.83 10.5 1.63 11.25 12.52 38.73 34.63 62.4 1.299124 566 1.288882 425 1.286338 38 1.285264 513 1.284738 694 1.284484 71 1.280018 335 1.279999 427 1.277221 364 1.276273 141 1.267260 338 1.262983 714 1.261666 313 1.254890 186 0.011886 151 0.007685 236 0.007026 76 0.037556 934 0.017956 927 0.013171 009 0.012297 821 0.013151 395 0.004752 685 0.018329 865 0.021818 12 0.029577 139 0.005882 961 0.007228 347 327 Table B-4 (Cont’d) SAUSA300 _RS07415 SAUSA300 _RS03560 SAUSA300 _RS12085 SAUSA3 00_1360 SAUSA3 00_0663 SAUSA3 00_2191 demethylmenaquinone methyltransferase hypothetical protein type Z 30S ribosomal protein S14 SAUSA300_RS06225 hypothetical protein SAUSA300 _RS06205 SAUSA300 _RS02720 SAUSA300 _RS14280 SAUSA300 _RS03505 SAUSA3 00_1147 SAUSA3 00_0508 SAUSA3 00_2567 SAUSA3 00_0654 sarX SAUSA300 _RS08920 SAUSA3 00_1635 mutM SAUSA300 _RS04420 SAUSA300 _RS08265 SAUSA300 _RS12095 SAUSA300 _RS14320 SAUSA300 _RS10145 SAUSA3 00_0820 SAUSA3 00_1514 SAUSA3 00_2193 SAUSA3 00_2574 SAUSA3 00_1857 hslU ATP-dependent protease ATPase subunit HslU UvrB/UvrC motif-containing protein arcC carbamate kinase HTH-type transcriptional regulator SarX bifunctional DNA- formamidopyrimidine glycosylase/DNA-(apurinic or apyrimidinic site) lyase cysteine desulfurase Fur family transcriptional regulator rplX 50S ribosomal protein L24 hypothetical protein SE1561 family protein 328 100. 37 149. 88 354. 72 364. 19 61.9 8 20.1 7 28.3 5 32.6 1 31.4 2 50.9 1 52.0 6 260. 5 15.1 7 318. 39 106. 38 205. 1 342. 18 664. 67 88.1 5 22.8 7 41.9 7 33.3 3 7.9 48.88 10.32 88.81 58.41 92.01 71.03 167.1 7 8.16 30.34 1.86 9.58 3.63 14.53 4.17 12.09 1.254021 602 1.253841 21 1.252383 913 1.250373 186 1.247877 063 1.244763 755 1.244717 224 1.241607 397 0.023354 301 0.040617 602 0.009994 056 0.010677 443 0.009638 217 0.022624 056 0.013177 658 0.011218 307 39.7 3.16 15.97 1.239891 761 0.019075 819 53.6 7 34.2 3 225. 81 23.4 3 292. 23 7.1 18.45 5.53 16.08 41.81 65.15 1.76 8.39 35.05 125.0 5 1.238878 63 1.225087 199 1.224551 786 1.224351 82 1.222208 253 0.006064 628 0.017956 927 0.013171 009 0.029445 073 0.010896 691 Table B-4 (Cont’d) SAUSA300 _RS14680 SAUSA3 00_2644 rsmG SAUSA300 _RS02315 SAUSA300 _RS12670 SAUSA300 _RS02835 SAUSA3 00_0432 SAUSA3 00_2293 SAUSA3 00_0530 corA 16S rRNA (guanine(527)- N(7))-methyltransferase RsmG sodium-dependent transporter magnesium/cobalt transporter CorA rpsL 30S ribosomal protein S12 SAUSA300 _RS10470 SAUSA3 00_1913 pmtA SAUSA300 _RS14240 SAUSA3 00_2559 nsaR phenol-soluble modulin export ABC transporter ATP- binding protein PmtA nisin susceptibility- associated two-component system response regulator NsaR SAUSA300 _RS06195 SAUSA300 _RS00220 SAUSA300 _RS03735 SAUSA3 00_1145 SAUSA3 00_0041 SAUSA3 00_0696 SAUSA300 _RS02830 SAUSA3 00_0529 SAUSA300 _RS12475 SAUSA300 _RS07410 SAUSA300 _RS12040 SAUSA3 00_2258 SAUSA3 00_1359 SAUSA3 00_2182 xerC tyrosine recombinase XerC hypothetical protein 6-carboxytetrahydropterin synthase QueD ribosomal L7Ae/L30e/S12e/Gadd45 family protein formate dehydrogenase subunit a polyprenyl synthetase family protein translation initiation factor IF- 1 queD fdhF infA 329 18.0 2 87.0 6 29.3 6 735. 55 41.8 8 22.8 9 84.9 9 19.3 6 19.9 6 511. 03 20.8 1 80.3 6 135. 03 22.1 5 133. 5 32 965. 43 54.3 1 34.3 7 86.9 7 24.3 7 693. 13 29.0 8 99.8 8 118. 68 2.46 7.7 16.69 35.07 2.08 15.47 114.3 5 311.3 4 5.71 19.38 1.221177 741 1.213364 032 1.213160 134 1.212672 535 1.206438 904 0.012297 821 0.009638 217 0.040617 602 0.007131 029 0.011021 615 3.63 10.86 1.202127 945 0.014199 959 7.95 40.15 14.3 1.17 8.53 3.84 6.07 1.201073 232 1.199774 158 1.198927 896 0.021708 573 0.048851 336 0.022523 689 97.37 182.6 4 1.196781 397 0.010544 027 2.52 11.14 13.46 30.66 19.89 41.42 1.193655 17 1.193036 326 1.189494 372 0.019075 819 0.008587 112 0.012784 97 Table B-4 (Cont’d) SAUSA300 _RS03500 SAUSA300 _RS02185 SAUSA300 _RS12150 SAUSA300 _RS02615 SAUSA3 00_0653 SAUSA3 00_0407 SAUSA3 00_2204 SAUSA3 00_0487 SAUSA300 _RS03785 SAUSA3 00_0704 SAUSA300 _RS12080 SAUSA3 00_2190 AraC family transcriptional regulator superantigen-like protein SSL11 rplC 50S ribosomal protein L3 tilS tRNA lysidine(34) synthetase TilS ABC-F family ATP-binding cassette domain-containing protein rpsH 30S ribosomal protein S8 SAUSA300_RS15905 hypothetical protein typA translational GTPase TypA SAUSA300 _RS05430 SAUSA300 _RS09695 SAUSA3 00_1009 SAUSA3 00_1771 SAUSA300 _RS12320 SAUSA3 00_2231 fdhD SAUSA300 _RS11025 SAUSA3 00_2005 tsaE SAUSA300 _RS05375 SAUSA300 _RS10475 SAUSA3 00_0998 SAUSA3 00_1914 DUF1828 domain-containing protein formate dehydrogenase accessory sulfurtransferase FdhD tRNA (adenosine(37)-N6)- threonylcarbamoyltransferas e complex ATPase subunit type 1 TsaE XRE family transcriptional regulator GntR family transcriptional regulator 330 27.4 1 12.7 1 100. 3 30.3 5 44.0 3 341. 79 194. 38 71.3 3 119. 66 148. 55 24.9 8 31.7 4 50.9 6 29.3 6 19.4 9 140. 34 3.45 11.73 2.18 5.91 18.58 39.74 41 3.81 15.72 56.4 6 321. 9 167. 85 72.4 4 111. 16 252. 62 4.2 24.78 62.37 82.95 17.01 86.2 10.76 25.73 9.95 58.37 28.69 73.94 1.184023 304 1.181097 954 1.180865 562 1.180486 675 1.176623 601 1.174216 686 1.170149 598 1.166293 484 1.152749 612 1.149986 618 0.011714 796 0.020454 61 0.010918 646 0.018507 41 0.033205 504 0.022233 967 0.031065 572 0.009638 217 0.037717 454 0.016791 141 35.4 2.57 14.83 1.148885 744 0.045211 561 28.0 4 54.3 9 3.57 12.93 8.23 18.34 1.147416 438 1.147386 94 0.023733 68 0.015563 226 Table B-4 (Cont’d) SAUSA300 _RS00135 SAUSA3 00_0026 rlmH SAUSA300 _RS05520 SAUSA300 _RS06185 SAUSA3 00_1026 SAUSA3 00_1143 SAUSA300 _RS11135 SAUSA3 00_2025 SAUSA300 _RS04070 SAUSA3 00_0754 SAUSA300 _RS08465 SAUSA3 00_1553 nadD recG rimP SAUSA300 _RS06060 SAUSA300 _RS06750 SAUSA300 _RS10980 SAUSA300 _RS06270 SAUSA300 _RS05255 SAUSA3 00_1120 SAUSA3 00_1243 SAUSA3 00_1997 SAUSA3 00_1158 SAUSA3 00_0978 SAUSA300 _RS00415 SAUSA3 00_0081 SAUSA300 _RS04805 SAUSA3 00_0891 23S rRNA (pseudouridine(1915)-N(3))- methyltransferase RlmH DUF177 domain-containing protein topA type I DNA topoisomerase PP2C family protein- serine/threonine phosphatase DUF4887 domain-containing protein nicotinate (nicotinamide) nucleotide adenylyltransferase ATP-dependent DNA helicase RecG exonuclease subunit SbcC sulfurtransferase TusA family protein ribosome maturation factor RimP ABC transporter ATP-binding protein TIGR04141 family sporadically distributed protein peptide ABC transporter substrate-binding protein 331 81.3 9 1408 .85 22.3 8 90.6 6 31.5 7 82.5 3 1628 .89 28.6 9 96.4 3 46.6 5 10.08 35.28 153.0 2 729.1 7 2.93 11.36 7.51 48.79 3.56 19.17 34.8 40.1 3.4 18.51 22.9 4 35.4 7 238. 49 44.5 5 25.4 8 26.2 7 164. 14 27.3 9 26.6 8 174. 32 2.75 11.61 4.61 11.82 39.77 54.14 52.1 4.81 23.17 34.9 3.08 14.38 28.9 2.61 13.76 162. 96 17.14 79.74 1.143342 514 1.142321 273 1.142306 307 1.140625 951 1.137056 414 1.136999 294 1.136698 83 1.133667 703 1.133582 231 1.131858 283 1.128877 854 1.125988 829 1.115766 728 0.017545 434 0.026375 528 0.020254 051 0.045722 533 0.040902 383 0.038664 002 0.023385 793 0.018329 227 0.035077 68 0.033082 679 0.031545 527 0.036477 48 0.030017 983 Table B-4 (Cont’d) SAUSA300 _RS12850 SAUSA300 _RS12070 SAUSA300 _RS03360 SAUSA3 00_2325 SAUSA3 00_2188 SAUSA3 00_0627 SAUSA300 _RS06405 SAUSA3 00_1185 miaB SAUSA300 _RS02095 SAUSA300 _RS06065 SAUSA300 _RS01160 SAUSA3 00_0392 SAUSA3 00_1121 SAUSA3 00_0221 pflA SAUSA300 _RS05810 SAUSA3 00_1071 bshC SAUSA300 _RS09810 SAUSA300 _RS02020 SAUSA300 _RS05515 SAUSA300 _RS07350 SAUSA300 _RS09220 SAUSA300 _RS09260 SAUSA3 00_1792 SAUSA3 00_0379 SAUSA3 00_1025 SAUSA3 00_1348 SAUSA3 00_1687 SAUSA3 00_1695 YbgA family protein rplR 50S ribosomal protein L18 glycosyltransferase family A protein tRNA (N6-isopentenyl adenosine(37)-C2)- methylthiotransferase MiaB hypothetical protein fapR transcription factor FapR pyruvate formate-lyase- activating protein bacillithiol biosynthesis cysteine-adding enzymeBshC AAA family ATPase ahpF alkyl hydroperoxide reductase subunit F nucleotidyltransferase CCA tRNA nucleotidyltransferase DNA translocase FtsK phosphotransferase family protein 332 1266 .06 337. 86 75.4 1 1099 .47 370. 31 99.2 5 186.4 3 436.8 6 67.79 97.07 8.99 42.82 29.5 9 57.4 5 30.2 3 122. 11 44.6 8 74.6 9 642. 39 22.6 1 18.1 4 21.2 3 383. 86 31.5 2 71.7 6 53.7 9 183. 25 78.3 8 76.4 8 954. 36 35.1 8 20.5 7 23.8 4 419. 38 2.91 15.56 12.74 17.62 6.74 14.99 25.7 52.94 9.52 23.21 7.93 38.54 147.5 3 247.9 6 3.63 13.25 3.12 7.55 3.07 10.24 42.82 209.4 9 1.113251 503 1.113241 203 1.105268 315 1.103402 63 1.089216 778 1.083891 117 1.082166 05 1.078932 507 1.072779 291 1.069844 011 1.057905 641 1.047992 951 1.045253 893 1.043455 34 0.015836 168 0.027131 822 0.034605 756 0.041811 058 0.046819 375 0.033671 936 0.023291 555 0.028569 35 0.040617 602 0.030156 128 0.037891 511 0.027808 317 0.027886 142 0.047336 317 Table B-4 (Cont’d) SAUSA300 _RS08000 SAUSA300 _RS09760 SAUSA300 _RS08445 SAUSA300 _RS09570 SAUSA3 00_1465 SAUSA3 00_1783 SAUSA3 00_1549 SAUSA3 00_1749 hemE alpha-ketoacid dehydrogenase subunit beta uroporphyrinogen decarboxylase ComEA family DNA-binding protein DUF1433 domain-containing protein 35.7 4 27.9 3 11.2 6 54.3 4 46.7 2 34.7 7 13.0 4 42.1 8 6.11 17.44 3.77 15.39 1.76 5.24 7.05 21.44 1.041265 361 1.032689 086 1.027530 569 1.021661 185 0.028413 27 0.043331 707 0.047629 476 0.045862 518 Genes downregulated in sTS when compared to CSSC Locus Old locus Gene Product SAUSA300 _RS13360 SAUSA3 00_2413 cntL D-histidine (S)-2- aminobutanoyltransferase CntL TPM. 1 sTS TPM. 2 sTS TPM. 1 CSSC TPM. 2 CSSC 2.8 1.24 42.35 67.31 SAUSA300 _RS13040 SAUSA3 00_2361 putative metal homeostasis protein 2490 .1 1355 .85 51134 .91 41439 .61 SAUSA300 _RS13355 SAUSA3 00_2412 cntM staphylopine dehydrogenase CntM 1.51 0.99 16.89 38.39 SAUSA300 _RS13325 SAUSA3 00_2406 SAUSA300 _RS04580 SAUSA3 00_0848 SAUSA300 _RS06690 SAUSA3 00_1234 MFS transporter 24.5 7 23.5 5 310.4 9 534.5 3 FAD/NAD(P)-binding protein 6.41 3.17 29.4 171.1 5 rpsN 30S ribosomal protein S14 142. 38 80.8 8 991.6 4 2246. 49 DE Log2 FC - 5.084086 128 - 5.024874 151 - 4.831891 364 - 4.662309 168 - 4.368667 787 - 4.253496 035 DE Adjusted P-value 5.27E-26 4.53E-22 1.58E-29 9.76E-31 7.41E-18 2.38E-22 333 Table B-4 (Cont’d) SAUSA300 _RS01635 SAUSA3 00_0307 5'-nucleotidase, lipoprotein e(P4) family SAUSA300_RS03000 hypothetical protein 12.1 188. 34 13.5 1 169. 37 160.9 7 150.1 3 2775. 63 1589. 92 SAUSA300 _RS13340 SAUSA3 00_2409 ABC transporter permease 2.99 2.36 20.31 54.35 SAUSA300_RS03005 vraX C1q-binding complement inhibitor VraX 6898 .76 9175 .19 13009 8.29 60616 .24 SAUSA300 _RS05540 SAUSA3 00_1029 isdA LPXTG-anchored heme- scavenging protein IsdA 43.4 64.4 1 297.7 9 505.1 4 SAUSA300 _RS13330 SAUSA3 00_2407 ABC transporter ATP-binding protein 3.45 3.47 11.71 53.39 SAUSA300 _RS08880 SAUSA3 00_1628 SAUSA300 _RS13345 SAUSA3 00_2410 SAUSA300 _RS13350 SAUSA3 00_2411 cntA amino acid permease ABC transporter permease staphylopine-dependent metal ABC transporter substrate-binding protein CntA 21.6 6 18.6 1 33.4 9 12.4 7 113.0 6 312.0 7 59.12 15.3 2 11.3 6 38.2 204.0 3 205.1 1 SAUSA300 _RS09355 SAUSA3 00_1712 ribE 6,7-dimethyl-8- ribityllumazine synthase 54.4 4 42.9 8 240.5 9 334.7 3 334 - 4.227392 16 - 4.217603 633 - 4.199861 087 - 4.184492 053 - 3.514307 723 - 3.501991 93 - 3.435439 103 - 3.434548 452 - 3.386083 399 - 3.208032 845 1.39E-18 1.13E-14 4.75E-24 1.84E-13 4.09E-17 1.02E-14 1.97E-17 4.00E-15 7.58E-13 7.65E-13 Table B-4 (Cont’d) SAUSA300 _RS03315 SAUSA3 00_0618 metal ABC transporter substrate-binding protein SAUSA300_RS15375 hypothetical protein SAUSA300_RS15090 SAUSA300_RS15730 SAUSA300_RS15740 SAUSA300 _RS12980 SAUSA3 00_2351 adcA SAUSA300_RS15795 pepA1 SAUSA300 _RS03665 SAUSA3 00_0683 SAUSA300_RS15735 phenol-soluble modulin PSM-alpha-3 phenol-soluble modulin PSM-alpha-4 phenol-soluble modulin PSM-alpha-1 zinc ABC transporter substrate-binding lipoprotein AdcA type I toxin-antitoxin system Fst family toxin PepA1 DeoR/GlpR family DNA- binding transcription regulator phenol-soluble modulin PSM-alpha-2 SAUSA300 _RS00605 SAUSA3 00_0117 sirA staphyloferrin B ABC transporter substrate-binding protein SirA 258. 97 240. 53 512. 32 420. 56 417. 86 46.6 8 440. 19 206. 02 184. 21 700. 74 500. 1 484. 5 20.8 9 391. 97 41.8 38.4 520. 36 26.0 1 645. 79 28.7 8 519.6 8 2555. 86 1755. 08 300.1 4 1880. 69 4471. 81 1439. 61 3355. 4 1294. 02 3576. 07 56.86 466.5 5 2456. 95 1290. 29 151.4 7 239.3 9 1675. 5 4447. 18 74.07 209.8 - 3.002466 305 - 2.977529 774 - 2.959942 807 - 2.956950 307 - 2.955553 758 - 2.952860 287 - 2.952268 106 - 2.943524 601 - 2.931688 49 - 2.890004 889 7.55E-11 2.63E-05 1.13E-13 7.48E-14 7.92E-14 3.23E-07 4.30E-07 5.60E-12 1.13E-13 2.89E-13 335 Table B-4 (Cont’d) SAUSA300 _RS13045 SAUSA3 00_2362 2,3-diphosphoglycerate- dependent phosphoglycerate mutase 618. 45 SAUSA300 _RS13850 SAUSA3 00_2493 cwrA cell wall inhibition responsive protein CwrA 543. 67 564. 86 434. 08 1514. 2 4828. 17 3279. 08 941.3 SAUSA300 _RS02195 SAUSA3 00_0409 spn myeloperoxidase inhibitor SPIN 1033 .96 769. 97 4360. 22 3178. 19 SAUSA300 _RS10530 SAUSA3 00_1920 chemotaxis-inhibiting protein CHIPS 755. 37 SAUSA300 _RS07015 SAUSA3 00_1291 SAUSA300 _RS10275 SAUSA3 00_1878 rlmD amidohydrolase 23S rRNA (uracil(1939)- C(5))-methyltransferase RlmD SAUSA300 _RS05795 SAUSA3 00_1068 beta-class phenol-soluble modulin SAUSA300_RS10485 hypothetical protein SAUSA300 _RS07010 SAUSA3 00_1290 dapD SAUSA300 _RS01625 SAUSA3 00_0305 2,3,4,5-tetrahydropyridine- 2,6-dicarboxylate N- acetyltransferase formate/nitrite transporter family protein 19.3 8 17.2 3 736. 3 71.4 9 155. 11 142. 2 557. 66 18.2 6 22.9 5 1791. 56 4444. 32 63.58 90.07 86.77 64.72 1082 .15 3464. 54 3534. 23 73.8 7 130. 25 110. 14 375.5 9 160.5 7 455.4 9 710.3 2 429.3 9 544.9 9 - 2.887349 944 - 2.884622 592 - 2.826992 551 - 2.777613 179 - 2.727134 119 - 2.720040 553 - 2.714539 171 - 2.704770 409 - 2.702224 538 - 2.657219 054 1.17E-12 1.27E-05 2.52E-07 3.14E-11 9.87E-10 4.94E-07 4.36E-08 1.29E-05 7.26E-10 1.82E-08 336 Table B-4 (Cont’d) SAUSA300 _RS03320 SAUSA3 00_0619 metal ABC transporter permease 91.3 3 78.8 167.1 9 603.2 SAUSA300 _RS01620 SAUSA3 00_0304 DUF4064 domain-containing protein 16.7 SAUSA300 _RS05570 SAUSA3 00_1035 isdG staphylobilin-forming heme oxygenase IsdG SAUSA300_RS15490 type I toxin-antitoxin system toxin PepG1 22.0 2 477. 23 15.9 7 17.3 4 455. 28 41.54 89.51 65.99 76.77 2420. 73 678.5 4 SAUSA300 _RS05790 SAUSA3 00_1067 beta-class phenol-soluble modulin 1106 .64 1728 .55 4820. 71 4917 SAUSA300_RS11570 hypothetical protein SAUSA300 _RS09670 SAUSA3 00_1767 gallidermin/nisin family lantibiotic SAUSA300 _RS01970 SAUSA3 00_0370 selX staphylococcal enterotoxin- like toxin X 5681 .91 4107 .36 21374 .9 10774 .7 15.9 8 23.1 7 16.3 68.29 38.64 18.9 5 46.85 118.5 5 SAUSA300 _RS03960 SAUSA3 00_0736 raiA ribosome-associated translation inhibitor RaiA 4599 .5 3956 .35 17578 .62 9706. 41 SAUSA300_RS10555 hypothetical protein 414. 3 365. 63 1740. 21 716.3 3 - 2.611758 841 - 2.604771 522 - 2.579655 241 - 2.577771 179 - 2.563896 531 - 2.540979 248 - 2.534092 969 - 2.522882 501 - 2.512502 454 - 2.508266 241 1.04E-09 4.31E-10 1.85E-07 0.000116 514 3.54E-07 2.83E-05 2.65E-05 1.94E-09 2.11E-05 6.60E-05 337 Table B-4 (Cont’d) SAUSA300 _RS11560 SAUSA3 00_2099 czrB CDF family zinc efflux transporter CzrB SAUSA300 _RS12545 SAUSA3 00_2269 hypothetical protein SAUSA300_RS12715 hypothetical protein 141. 8 191. 99 33.0 8 SAUSA300 _RS04395 SAUSA3 00_0815 DUF4888 domain-containing protein 69.1 8 SAUSA300 _RS06940 SAUSA3 00_1278 pepF oligoendopeptidase F SAUSA300 _RS07025 SAUSA3 00_1293 lysA diaminopimelate decarboxylase 77.1 6 104. 32 SAUSA300 _RS04855 SAUSA3 00_0902 pepF oligoendopeptidase F 42.9 SAUSA300 _RS14010 SAUSA3 00_2525 fructosamine kinase family protein 24.5 7 SAUSA300 _RS12865 SAUSA3 00_2328 DUF4889 domain-containing protein 390. 68 424. 63 166. 1 30.0 7 67.7 4 80.3 8 105. 7 53.2 3 20.0 5 307. 86 759.5 6 1273. 14 573.5 1 583.6 9 113.6 5 82.31 84.03 531.8 9 207.7 2 271.2 2 195.4 7 534.7 87.93 218.1 4 62.37 62.14 1025. 36 853.3 6 SAUSA300 _RS05185 SAUSA3 00_0964 DUF5011 domain-containing protein 1344 .95 1198 .29 4004. 37 2605. 7 - 2.481461 542 - 2.466327 657 - 2.439049 512 - 2.419905 295 - 2.355707 981 - 2.355706 834 - 2.271933 875 - 2.268664 211 - 2.247029 225 - 2.240557 187 2.17E-06 9.67E-07 1.65E-05 1.88E-06 3.51E-07 6.34E-09 2.39E-08 1.21E-05 3.44E-05 9.67E-05 338 Table B-4 (Cont’d) SAUSA300 _RS00130 SAUSA3 00_0025 adsA LPXTG-anchored adenosine synthase AdsA 4.83 4.47 11.14 14.81 SAUSA300 _RS10185 SAUSA3 00_1865 vraR two-component system response regulator VraR SAUSA300 _RS02705 SAUSA3 00_0505 pdxT pyridoxal 5'-phosphate synthase glutaminase subunit PdxT SAUSA300 _RS04165 SAUSA3 00_0772 clfA MSCRAMM family adhesin clumping factor ClfA 111. 81 208. 5 20.7 9 117. 53 233. 65 30.1 7 277.5 9 350.5 367.7 4 991.6 5 40.34 118.9 3 SAUSA300 _RS13765 SAUSA3 00_2481 sterile alpha motif-like domain-containing protein 1124 4.19 9876 .44 34671 .29 16151 .83 SAUSA300 _RS02365 SAUSA3 00_0442 SAUSA300 _RS10525 SAUSA3 00_1919 SAUSA300 _RS04565 SAUSA3 00_0845 SAUSA300 _RS12480 SAUSA3 00_2259 YibE/F family protein 87.7 92.5 6 216.8 4 251.2 5 scn complement inhibitor SCIN-A M17 family metallopeptidase LCP family protein 3693 .57 3390 .7 7817. 15 11222 .06 44.8 2 48.3 1 378. 55 45.3 4 58.6 8 283. 98 93.39 150.8 5 113.1 8 157.6 3 422.2 3 1700. 83 SAUSA300 _RS03325 SAUSA3 00_0620 metal ABC transporter ATP- binding protein 339 - 2.224498 518 - 2.222222 326 - 2.197436 211 - 2.194119 213 - 2.168418 284 - 2.167103 753 - 2.164778 699 - 2.154971 493 - 2.101833 959 - 2.097167 098 2.82E-06 2.51E-06 7.09E-08 2.33E-07 0.000529 061 8.38E-06 2.32E-06 1.02E-06 5.48E-06 6.77E-06 Table B-4 (Cont’d) SAUSA300 _RS06485 SAUSA3 00_1201 glnA type I glutamate--ammonia ligase 341. 1 347. 9 459.6 2 1631. 59 NAD/NADP-dependent octopine/nopaline dehydrogenase family protein SDR family oxidoreductase YjzD family protein 22.2 6 24.6 3 33.52 102.1 8 15.3 9 740. 03 23.7 4 669. 63 31.72 75.47 1986. 21 1210. 13 hypothetical protein 5.84 7.06 16.36 14.43 SAUSA300 _RS12430 SAUSA3 00_2251 SAUSA300 _RS11550 SAUSA3 00_2097 SAUSA300 _RS04765 SAUSA3 00_0884 SAUSA300 _RS00215 SAUSA3 00_0040 SAUSA300 _RS13725 SAUSA3 00_2473 alpha/beta hydrolase SAUSA300 _RS13645 SAUSA3 00_2460 GNAT family N- acetyltransferase SAUSA300 _RS03025 SAUSA3 00_0565 DUF423 domain-containing protein 87.4 9 106. 56 70.7 1 105. 79 94.0 5 78.5 6 212.7 7 237.7 8 252.1 3 188.1 2 171.6 4 157.5 2 SAUSA300 _RS05995 SAUSA3 00_1107 TM2 domain-containing protein 2190 .76 1945 .41 5682. 01 2971. 77 SAUSA300 _RS14175 SAUSA3 00_2551 nrdD anaerobic ribonucleoside- triphosphate reductase 17.6 7 14.6 8 34.12 39.52 340 - 2.088957 463 - 2.074184 097 - 2.073058 115 - 2.072766 933 - 2.058583 378 - 2.035252 592 - 2.003367 465 - 1.990628 548 - 1.984113 099 - 1.976282 154 1.34E-06 8.89E-07 2.11E-06 0.000456 21 0.000317 197 3.94E-05 0.000411 997 0.000175 66 0.001351 833 8.36E-05 Table B-4 (Cont’d) SAUSA300 _RS01990 SAUSA3 00_0374 GlsB/YeaQ/YmgE family stress response membrane protein 2666 .59 3344 .13 6286. 49 7095. 9 SAUSA300 _RS11520 SAUSA3 00_2091 deoD purine-nucleoside phosphorylase 510. 44 622. 99 887.3 1889. 62 SAUSA300 _RS09015 SAUSA3 00_1652 universal stress protein SAUSA300_RS16020 hypothetical protein SAUSA300 _RS09140 SAUSA3 00_1674 trypsin-like peptidase domain-containing protein SAUSA300 _RS01265 SAUSA3 00_0237 SAUSA300 _RS03335 SAUSA3 00_0622 SAUSA300 _RS14565 SAUSA3 00_2621 SAUSA300 _RS06685 SAUSA3 00_1233 SAUSA300 _RS14075 SAUSA3 00_2537 nucleoside hydrolase M50 family metallopeptidase SMP- 30/gluconolactonase/LRE family protein rpmG 50S ribosomal protein L33 L-lactate dehydrogenase 1143 .03 989. 51 2394. 1 2210. 15 6646 .76 5039 .17 11754 .97 14607 .47 137. 65 25.1 6 61.6 6 11.0 4 170. 54 24.3 4 69.8 5 13.2 5 242.4 5 500.7 5 50.02 59.68 140.3 3 140.7 3 27.91 22.36 7750 .21 5201 .14 16477 .46 9467. 32 153. 26 133. 22 241.4 9 410.5 7 - 1.974138 146 - 1.958397 09 - 1.952065 351 - 1.945365 73 - 1.945099 422 - 1.944052 105 - 1.936603 756 - 1.926173 373 - 1.900792 019 - 1.898492 363 6.84E-05 2.99E-06 0.000256 582 9.04E-05 4.14E-06 8.11E-05 0.000179 051 0.000573 173 0.002186 41 2.42E-05 341 DUF3147 family protein 11.3 4.5 16.61 19.99 Table B-4 (Cont’d) SAUSA300 _RS14670 SAUSA3 00_2642 SAUSA300 _RS09800 SAUSA3 00_1790 peptidylprolyl isomerase 361. 94 SAUSA300 _RS13385 SAUSA3 00_2418 carboxymuconolactone decarboxylase family protein 109. 83 SAUSA300_RS08745 hypothetical protein SAUSA300 _RS13280 SAUSA3 00_2398 fetB iron export ABC transporter permease subunit FetB SAUSA300 _RS07195 SAUSA3 00_1321 SAUSA300 _RS13975 SAUSA3 00_2518 SAUSA300 _RS02890 SAUSA3 00_0541 bacilliredoxin BrxA alpha/beta hydrolase deoxynucleoside kinase SAUSA300 _RS05095 SAUSA3 00_0948 menB 1,4-dihydroxy-2-naphthoyl- CoA synthase SAUSA300 _RS10505 SAUSA3 00_1918 phospholipase 275. 34 44.2 1 193. 91 18.4 1 134. 85 245. 75 79.6 7 342 372. 49 138. 64 252. 35 61.6 4 182. 28 17.8 6 145. 95 209. 05 476.9 7 1357. 86 235.1 2 291.7 1 424.3 8 756.5 106.8 2 105.3 3 436.6 6 287.7 1 28.75 51.73 239.8 2 357.1 2 423.4 7 509.8 8 65.4 114.4 9 203.5 9 - 1.897774 308 - 1.894978 207 - 1.889078 501 - 1.865833 571 - 1.859001 088 - 1.856049 223 - 1.852317 746 - 1.849793 208 - 1.834799 054 - 1.828037 314 0.001578 65 6.32E-06 0.000103 575 2.68E-05 0.000434 876 0.001666 507 3.15E-05 5.42E-05 0.000234 875 6.60E-05 Table B-4 (Cont’d) SAUSA300 _RS04985 SAUSA3 00_0928 SAUSA300 _RS07460 SAUSA3 00_1366 SAUSA300 _RS08320 SAUSA3 00_1525 competence protein ComK 12.7 hypothetical protein glycine--tRNA ligase SAUSA300 _RS04185 SAUSA3 00_0776 thermonuclease family protein SAUSA300 _RS04760 SAUSA3 00_0883 MAP domain-containing protein SAUSA300 _RS01655 SAUSA3 00_0310 PTS sugar transporter subunit IIC SAUSA300 _RS00885 SAUSA3 00_0168 isdI staphylobilin-forming heme oxygenase IsdI SAUSA300 _RS14620 SAUSA3 00_2632 HdeD family acid-resistance protein 68.1 3 SAUSA300 _RS03030 SAUSA3 00_0566 SAUSA300 _RS00705 SAUSA3 00_0135 amino acid permease superoxide dismutase 45.4 3 265. 49 343 51.8 9 326. 21 42.2 5 70.8 8 215. 89 65.3 9 14.1 5 51.3 1 369. 09 32.9 3 89.4 2 191. 53 59.7 4 89.5 1 50.5 3 304. 7 19.18 40.77 99.36 460.2 55.04 105.2 2 1101. 39 109.1 5 121.1 1 211.7 1 354.3 5 455.8 2 108.8 1 139.8 6 130.2 9 182.1 6 58.42 156.5 4 528.3 7 543.4 6 - 1.819591 096 - 1.807348 439 - 1.805508 328 - 1.792396 052 - 1.782988 447 - 1.782003 764 - 1.779195 165 - 1.773810 679 - 1.767434 199 - 1.764391 635 3.36E-05 0.000634 316 1.56E-05 7.24E-05 6.24E-05 0.000262 324 0.000299 457 0.000190 5 2.70E-05 0.000670 455 Table B-4 (Cont’d) SAUSA300 _RS02710 SAUSA3 00_0506 NupC/NupG family nucleoside CNT transporter SAUSA300 _RS05245 SAUSA3 00_0976 purD phosphoribosylamine-- glycine ligase SAUSA300 _RS03405 SAUSA3 00_0635 iron ABC transporter permease 217. 63 45.1 3 75.5 3 218. 71 49.1 3 67.2 1 399.7 3 417.8 6 68 118.9 4 109.0 2 170.1 9 SAUSA300 _RS07035 SAUSA3 00_1295 cspA cold shock protein CspA 3550 5.41 2437 8.62 65893 .82 36585 .83 SAUSA300_RS03635 hypothetical protein SAUSA300 _RS07000 SAUSA3 00_1288 dapA SAUSA300 _RS14170 SAUSA3 00_2550 nrdG SAUSA300 _RS07160 SAUSA3 00_1314 SAUSA300 _RS05340 SAUSA3 00_0991 SAUSA300 _RS14595 SAUSA3 00_2627 4-hydroxy- tetrahydrodipicolinate synthase anaerobic ribonucleoside- triphosphate reductase activating protein YozE family protein def peptide deformylase anion permease 344 118. 33 21.4 4 50.4 8 228. 8 239. 26 120. 67 113. 91 207.1 8 212.5 2 27.9 33.36 64.28 46.1 2 284. 07 284. 4 108. 5 89.47 81 397.0 4 556.2 1 332.9 5 694.2 9 191.1 8 213.2 3 - 1.751889 824 - 1.717598 011 - 1.715867 328 - 1.706968 213 - 1.703052 969 - 1.692306 711 - 1.691215 066 - 1.686713 441 - 1.660757 846 - 1.653514 614 0.000704 809 0.000108 67 0.000223 999 0.007102 539 0.001262 424 0.000130 572 0.002137 734 0.000403 211 0.000108 67 0.001374 985 Table B-4 (Cont’d) SAUSA300 _RS04490 SAUSA3 00_0832 DUF86 domain-containing protein 44.8 8 45.7 3 67.64 97.59 SAUSA300 _RS04990 SAUSA3 00_0929 IDEAL domain-containing protein 1983 .26 903. 46 2511. 05 2756. 31 SAUSA300_RS10805 transcriptional regulator SAUSA300 _RS06775 SAUSA3 00_1248 HesB/YadR/YfhF family protein 30.1 3 263. 77 30.4 9 233. 96 43.65 67.39 470.6 9 336.4 3 SAUSA300 _RS06835 SAUSA3 00_1258 2-hydroxymuconate tautomerase 1764 .03 1415 .14 3231. 43 1800. 33 SAUSA300 _RS03035 SAUSA3 00_0567 threonine/serine exporter family protein SAUSA300 _RS01090 SAUSA3 00_0207 SAUSA300 _RS04400 SAUSA3 00_0816 SAUSA300 _RS13155 SAUSA3 00_2378 M23 family metallopeptidase CsbD family protein membrane protein SAUSA300 _RS10175 SAUSA3 00_1863 YtxH domain-containing protein 117. 79 48.2 6 120. 91 197.4 2 194.0 7 53.9 80.85 88.84 9469 .14 1265 7.73 14167 .41 25661 .71 85.5 3 868. 92 111. 63 894. 3 91.35 306.5 6 1264. 96 1649. 91 - 1.644470 596 - 1.640969 217 - 1.622088 691 - 1.620107 987 - 1.617697 923 - 1.587349 791 - 1.584196 156 - 1.584100 205 - 1.561080 472 - 1.540443 059 0.000573 173 0.004874 3 0.000887 659 0.006175 256 0.010331 72 0.003226 298 0.002339 682 0.000414 747 0.000496 699 0.001749 293 345 Table B-4 (Cont’d) SAUSA300 _RS09040 SAUSA3 00_1656 SAUSA300 _RS09440 SAUSA3 00_1726 universal stress protein 4000 .78 4549 .22 7380. 44 5519. 76 CrcB family protein 20.9 1 143. 51 35.2 9 177. 63 142. 88 35.2 6 64.7 1 21.8 9 169. 22 30.37 39.66 193.6 7 334.2 2 38.3 51.85 64.49 150. 33 151. 88 39.7 5 71.2 6 267. 74 369. 1 234.2 2 286.4 6 247.6 4 182.7 9 52.5 62.54 81.7 139 471.9 5 326.0 5 688.0 9 481.4 5 - 1.531998 422 - 1.515219 88 - 1.506316 27 - 1.491225 148 - 1.488899 692 - 1.485921 019 - 1.460960 702 - 1.453651 462 - 1.440472 531 - 1.426868 523 0.008598 472 0.002996 801 0.000902 686 0.003208 24 0.003891 948 0.011886 151 0.004533 387 0.001576 888 0.017451 703 0.019203 661 SAUSA300 _RS03590 SAUSA3 00_0669 undecaprenyl-diphosphate phosphatase SAUSA300 _RS14335 SAUSA3 00_2577 manA mannose-6-phosphate isomerase, class I SAUSA300 _RS04705 SAUSA3 00_0872 YisL family protein SAUSA300_RS04210 hypothetical protein SAUSA300 _RS01890 SAUSA3 00_0356 cyclase family protein SAUSA300 _RS05490 SAUSA3 00_1020 glycerophosphodiester phosphodiesterase SAUSA300 _RS12820 SAUSA3 00_2320 DUF2871 domain-containing protein 300. 47 SAUSA300 _RS07815 SAUSA3 00_1432 hypothetical protein 466. 74 346 Table B-4 (Cont’d) SAUSA300 _RS04685 SAUSA3 00_0868 lepB signal peptidase I SAUSA300 _RS12720 SAUSA3 00_2302 glycopeptide resistance protein TcaA 215. 3 67.2 7 SAUSA300 _RS05680 SAUSA3 00_1053 formyl peptide receptor-like 1 inhibitory protein 103. 34 SAUSA300 _RS14345 SAUSA3 00_2579 SAUSA300 _RS12500 SAUSA3 00_2262 sdpB SAUSA300_RS06715 amidase domain-containing protein CPBP family intramembrane glutamic endopeptidaseSdpB DNA damage-induced cell division inhibitor SosA 61.6 9 115. 62 54.1 7 244. 14 73.1 5 135. 89 65.6 9 131. 32 45.0 9 324.6 2 340.6 2 91.37 118.4 2 163.9 6 177.5 5 72.91 124.9 9 138.4 6 245.3 5 75.14 62.69 SAUSA300 _RS04720 SAUSA3 00_0875 metal-sulfur cluster assembly factor 2157 .52 1858 .45 3013. 21 2486. 78 SAUSA300_RS05060 SAR1012 family small protein SAUSA300 _RS01985 SAUSA3 00_0373 helix-turn-helix transcriptional regulator SAUSA300 _RS10205 SAUSA3 00_1869 map type I methionyl aminopeptidase 117. 31 528. 79 315. 99 122. 81 356. 82 346. 41 157.5 6 189.8 674.5 7 540.8 3 395.4 3 573.9 1 - 1.414555 238 - 1.408137 34 - 1.391949 937 - 1.391829 312 - 1.386155 028 - 1.382950 861 - 1.376334 008 - 1.374804 231 - 1.368071 835 - 1.354493 125 0.008097 902 0.004874 3 0.009159 035 0.002522 619 0.002424 322 0.019075 819 0.017956 927 0.008367 857 0.022823 811 0.005219 82 347 Table B-4 (Cont’d) SAUSA300 _RS03045 SAUSA3 00_0569 SAUSA300 _RS00580 SAUSA3 00_0112 SAUSA300 _RS03825 SAUSA3 00_0712 SAUSA300 _RS04710 SAUSA3 00_0873 heme-dependent peroxidase L-lactate permease peptide MFS transporter CoA-disulfide reductase SAUSA300 _RS02430 SAUSA3 00_0454 recR recombination mediator RecR SAUSA300 _RS01560 SAUSA3 00_0292 hypothetical protein 408. 44 80.3 1 361. 77 260. 29 280. 45 194. 91 464. 95 80.8 1 349. 19 284. 82 259. 58 162. 79 547.4 1 699.7 1 90.96 446.1 151.2 2 552.5 9 334.6 7 430.3 4 313.9 3 467.6 3 274.7 3 179.2 5 SAUSA300 _RS09770 SAUSA3 00_1784 traP signal transduction protein TRAP 1852 .02 1751 .65 2359. 68 2513. 71 SAUSA300 _RS02350 SAUSA3 00_0439 hypothetical protein SAUSA300_RS12530 hypothetical protein SAUSA300 _RS09165 SAUSA3 00_1678 formate--tetrahydrofolate ligase 338. 47 134. 68 44.6 1 329. 48 141. 35 61.7 4 489.0 6 365.5 3 174.8 2 202.0 7 45.4 123.5 5 - 1.353228 586 - 1.352592 372 - 1.331649 516 - 1.327296 927 - 1.324897 022 - 1.313861 922 - 1.313810 591 - 1.310934 537 - 1.310168 6 - 1.300306 08 0.007187 2 0.003891 948 0.009270 639 0.008508 348 0.006623 051 0.036533 213 0.014563 041 0.029178 439 0.012913 273 0.003965 203 348 Table B-4 (Cont’d) SAUSA300 _RS11360 SAUSA3 00_2063 atpE F0F1 ATP synthase subunit C 123. 74 133. 08 148.2 8 212.2 9 SAUSA300 _RS03010 SAUSA3 00_0562 thiD bifunctional hydroxymethylpyrimidine kinase/phosphomethylpyrimi dine kinase 261. 8 257. 99 271.9 7 490.4 9 SAUSA300 _RS10935 SAUSA3 00_1989 agrB accessory gene regulator AgrB 857. 02 1101 .23 1150. 16 1544. 07 SAUSA300 _RS05160 SAUSA3 00_0960 qoxD cytochrome aa3 quinol oxidase subunit IV 1183 .89 1388 .16 1833. 15 1402. 26 SAUSA300 _RS14020 SAUSA3 00_2527 hypothetical protein SAUSA300 _RS11310 SAUSA3 00_2054 fabZ 3-hydroxyacyl-ACP dehydratase FabZ SAUSA300 _RS06360 SAUSA3 00_1176 pgsA CDP-diacylglycerol--glycerol- 3-phosphate 3- phosphatidyltransferase SAUSA300_RS09030 hypothetical protein SAUSA300 _RS05470 SAUSA3 00_1017 DUF420 domain-containing protein SAUSA300 _RS05050 SAUSA3 00_0940 DoxX family protein 195. 43 266. 26 286. 65 97.1 9 216. 93 613. 49 178. 91 297. 19 259. 49 97.1 4 251. 59 450. 74 234.5 5 255.0 4 334.1 1 388.3 7 332.7 4 348.5 6 107.4 7 144.2 5 290.0 1 273.8 9 674.1 2 617.3 2 - 1.298009 33 - 1.297532 703 - 1.295519 826 - 1.292802 829 - 1.261693 849 - 1.230300 86 - 1.207979 469 - 1.196443 392 - 1.193227 67 - 1.188234 093 0.008599 868 0.004904 817 0.009638 217 0.030017 983 0.020068 907 0.019632 788 0.028796 897 0.021841 334 0.035325 655 0.043394 609 349 Table B-4 (Cont’d) SAUSA300 _RS13290 SAUSA3 00_2400 M42 family metallopeptidase SAUSA300 _RS14105 SAUSA3 00_2541 lqo L-lactate dehydrogenase (quinone) SAUSA300 _RS01870 SAUSA3 00_0353 ABC-2 transporter permease SAUSA300 _RS05075 SAUSA3 00_0944 1,4-dihydroxy-2-naphthoate polyprenyltransferase SAUSA300 _RS04575 SAUSA3 00_0847 SAUSA300 _RS04545 SAUSA3 00_0841 SAUSA300 _RS11600 SAUSA3 00_2104 glmS PaaI family thioesterase NAD(P)/FAD-dependent oxidoreductase glutamine--fructose-6- phosphate transaminase (isomerizing) SAUSA300 _RS10940 SAUSA3 00_1990 cyclic lactone autoinducer peptide SAUSA300 _RS11495 SAUSA3 00_2088 S-ribosylhomocysteine lyase 93.5 3 854. 76 127. 18 144. 04 271. 11 225. 3 88.9 702. 34 522. 71 102. 65 107.5 1 141.0 3 1163 .52 1221. 75 1179. 67 125. 67 152. 14 246. 89 213. 5 132. 72 960. 66 538. 77 153.1 7 147 165.2 3 195.8 8 284.2 1 333.5 6 235.1 2 287.7 96.78 188.6 2 732.3 5 1307. 69 545.3 9 657.2 - 1.185015 768 - 1.178605 822 - 1.165101 924 - 1.156657 21 - 1.122290 299 - 1.113804 911 - 1.111734 202 - 1.068465 88 - 1.054221 759 0.020256 411 0.037556 934 0.040283 358 0.029271 275 0.037490 909 0.035548 336 0.019413 764 0.026476 425 0.048463 664 350 Table B-5. Transporters differentially expressed in GSH, GSSG, and sTS. Locus Old locus Gene Product DE Log2 FC DE Adj. P-value gmpC Differentially expressed transporters in GSH SAUSA300_ RS00925 SAUSA300_ RS00915 SAUSA300_ RS02340 SAUSA300_ RS02330 SAUSA300_ RS00920 SAUSA300_ RS02335 SAUSA300_ RS02315 SAUSA300_ RS02035 SAUSA300_ RS13025 SAUSA300_ RS01440 SAUSA300_ RS01055 SAUSA300_ RS01060 SAUSA300_ RS10985 SAUSA300_ RS12890 SAUSA300_ 0176 SAUSA300_ 0174 SAUSA300_ 0437 SAUSA300_ 0435 SAUSA300_ 0175 SAUSA300_ 0436 SAUSA300_ 0432 SAUSA300_ 0382 SAUSA300_ 2359 SAUSA300_ 0268 SAUSA300_ 0200 SAUSA300_ 0201 SAUSA300_ 1998 SAUSA300_ 2333 ABC transporter permease ABC transporter ATP-binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein methionine ABC transporter ATP- binding protein ABC transporter substrate-binding protein 3.125833599 5.35039E-07 2.989014416 3.54407E-07 2.315679543 4.10697E-05 2.290401543 1.35302E-05 2.282409423 0.000547181 methionine ABC transporter permease 1.995195436 0.001769395 sodium-dependent transporter L-cystine transporter transporter substrate-binding domain- containing protein MFS transporter ABC transporter ATP-binding protein ABC transporter permease YeeE/YedE family protein nitrate/nitrite transporter 1.950671187 0.001418977 1.838262363 0.00898218 1.753694235 0.002677665 1.728656723 0.013484163 1.667394083 0.01218702 1.524008315 0.020157954 1.304988845 0.030246065 -3.275833554 7.74351E-10 351 Table B-5 (cont’d) fetB SAUSA300_ 0305 SAUSA300_ 2406 SAUSA300_ 2409 SAUSA300_ 1628 SAUSA300_ 2410 SAUSA300_ 2398 SAUSA300_ 2407 SAUSA300_ 2313 SAUSA300_ 2099 SAUSA300_ 1315 SAUSA300_ RS01625 SAUSA300_ RS13325 SAUSA300_ RS13340 SAUSA300_ RS08880 SAUSA300_ RS13345 SAUSA300_ RS13280 SAUSA300_ RS13330 SAUSA300_ RS12780 SAUSA300_ RS11560 SAUSA300_ RS07165 Differentially expressed transporters in GSSG SAUSA300_ RS00925 SAUSA300_ RS00915 SAUSA300_ RS02340 SAUSA300_ RS00920 SAUSA300_ RS02035 SAUSA300_ 0176 SAUSA300_ 0174 SAUSA300_ 0437 SAUSA300_ 0175 SAUSA300_ 0382 gmpC czrB formate/nitrite transporter family protein MFS transporter ABC transporter permease amino acid permease ABC transporter permease iron export ABC transporter permease subunit FetB ABC transporter ATP-binding protein L-lactate permease CDF family zinc efflux transporter CzrB PTS glucose transporter subunit IIA ABC transporter permease ABC transporter ATP-binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein ABC transporter substrate-binding protein L-cystine transporter -2.826851365 5.35039E-07 -2.73389307 1.70962E-06 -2.695416261 3.1823E-07 -2.497406008 1.33354E-06 -2.068669945 0.000155603 -1.989683824 0.001769395 -1.903394709 0.001594128 -1.51253439 0.015456886 -1.308531176 0.031701317 -1.275878242 0.03733 3.303313167 9.01551E-10 2.830762445 9.05749E-09 2.654314417 9.05749E-09 2.408938291 4.67518E-06 2.306320567 1.28398E-05 352 Table B-5 (cont’d) SAUSA300_ RS10985 SAUSA300_ RS01055 SAUSA300_ RS02315 SAUSA300_ RS02335 SAUSA300_ RS02330 SAUSA300_ RS13025 SAUSA300_ RS01060 SAUSA300_ RS01440 SAUSA300_ RS03880 SAUSA300_ RS13020 SAUSA300_ RS13015 SAUSA300_ RS13340 SAUSA300_ RS06690 SAUSA300_ RS07010 SAUSA300_ RS07015 SAUSA300_ 1998 SAUSA300_ 0200 SAUSA300_ 0432 SAUSA300_ 0436 SAUSA300_ 0435 SAUSA300_ 2359 SAUSA300_ 0201 SAUSA300_ 0268 SAUSA300_ 0721 SAUSA300_ 2358 SAUSA300_ 2357 SAUSA300_ 2409 SAUSA300_ 1234 SAUSA300_ 1290 SAUSA300_ 1291 YeeE/YedE family protein 2.196473104 7.51319E-07 ABC transporter ATP-binding protein 2.136824537 6.18049E-06 sodium-dependent transporter 2.108173714 2.0706E-06 methionine ABC transporter permease 1.986024342 5.44287E-05 methionine ABC transporter ATP- binding protein transporter substrate-binding domain- containing protein 1.982114069 7.69607E-06 1.868432696 5.3321E-05 ABC transporter permease 1.815353512 0.000587577 MFS transporter 1.732819023 0.000196649 siderophore ABC transporter substrate-binding protein 1.435417673 0.021157034 amino acid ABC transporter permease 1.377126022 0.040614674 amino acid ABC transporter ATP- binding protein 1.279374623 0.013517702 ABC transporter ATP-binding protein -2.76312879 3.44703E-09 rpsN ABC transporter permease -2.642568704 3.35575E-08 dapD amino acid permease -2.520651649 2.56794E-08 MFS transporter -2.326963795 6.21097E-07 353 Table B-5 (cont’d) SAUSA300_ RS01625 SAUSA300_ RS05195 SAUSA300_ 0305 SAUSA300_ 0966 SAUSA300_ RS13345 SAUSA300_ 2410 copZ SAUSA300_ 2495 SAUSA300_ 2537 SAUSA300_ RS13860 SAUSA300_ RS14075 Differentially expressed transporters in sTS SAUSA300_ RS00915 SAUSA300_ RS00925 SAUSA300_ RS02330 SAUSA300_ RS11605 SAUSA300_ RS02335 SAUSA300_ RS13025 SAUSA300_ RS01060 SAUSA300_ RS00920 SAUSA300_ RS02340 SAUSA300_ 0174 SAUSA300_ 0176 SAUSA300_ 0435 SAUSA300_ 2105 SAUSA300_ 0436 SAUSA300_ 2359 SAUSA300_ 0201 SAUSA300_ 0175 SAUSA300_ 0437 gmpC nitrate/nitrite transporter -2.081032901 1.90088E-05 purE formate/nitrite transporter family protein staphylopine-dependent metal ABC transporter substrate-binding protein CntA ABC transporter permease L-lactate permease ABC transporter ATP-binding protein ABC transporter permease methionine ABC transporter ATP- binding protein -2.066362184 7.22394E-06 -1.806048803 0.00029532 -1.799606649 0.004998035 -1.396183733 0.005316458 3.34231159 1.77E-13 3.098636848 1.70E-08 2.95296498 1.52E-11 2.47E-05 1.07E-07 PTS mannitol transporter subunit IICB 2.697969241 methionine ABC transporter permease 2.573132474 transporter substrate-binding domain- containing protein ABC transporter permease ABC transporter substrate-binding protein dipeptide ABC transporter glycylmethionine-binding lipoprotein 2.502235403 3.10E-09 2.500861509 1.50E-06 2.331970676 7.16E-06 2.298614243 2.06E-06 354 Table B-5 (cont’d) SAUSA300_ RS09895 SAUSA300_ RS01065 SAUSA300_ RS01055 SAUSA300_ RS13020 SAUSA300_ RS02035 SAUSA300_ RS10985 SAUSA300_ RS12845 SAUSA300_ RS02315 SAUSA300_ RS12670 SAUSA300_ RS10470 SAUSA300_ RS05255 SAUSA300_ RS04805 SAUSA300_ RS13325 SAUSA300_ RS13340 SAUSA300_ RS13330 SAUSA300_ 1808 SAUSA300_ 0202 SAUSA300_ 0200 SAUSA300_ 2358 SAUSA300_ 0382 SAUSA300_ 1998 SAUSA300_ 2324 SAUSA300_ 0432 SAUSA300_ 2293 SAUSA300_ 1913 SAUSA300_ 0978 SAUSA300_ 0891 SAUSA300_ 2406 SAUSA300_ 2409 SAUSA300_ 2407 ABC transporter permease subunit 2.268943961 1.29E-05 ABC transporter permease 1.888041935 0.000125814 ABC transporter ATP-binding protein 1.68588224 0.000393937 amino acid ABC transporter permease 1.590410837 0.003709448 L-cystine transporter 1.528200303 0.004872848 YeeE/YedE family protein 1.51993081 0.001033925 sucrose-specific PTS transporter subunit IIBC 1.456119968 0.004064768 sodium-dependent transporter 1.213364032 0.009638217 corA magnesium/cobalt transporter CorA 1.213160134 0.040617602 pmtA phenol-soluble modulin export ABC transporter ATP-binding protein PmtA 1.206438904 0.011021615 ABC transporter ATP-binding protein 1.128877854 0.031545527 peptide ABC transporter substrate- binding protein 1.115766728 0.030017983 MFS transporter -4.662309168 9.76E-31 ABC transporter permease -4.199861087 4.75E-24 ABC transporter ATP-binding protein -3.50199193 1.02E-14 355 Table B-5 (cont’d) SAUSA300_ RS08880 SAUSA300_ RS13345 SAUSA300_ 1628 SAUSA300_ 2410 SAUSA300_ RS13350 SAUSA300_ 2411 cntA adcA sirA czrB fetB SAUSA300_ RS03315 SAUSA300_ RS12980 SAUSA300_ RS00605 SAUSA300_ RS01625 SAUSA300_ RS03320 SAUSA300_ RS11560 SAUSA300_ RS03325 SAUSA300_ RS13280 SAUSA300_ RS01655 SAUSA300_ RS03030 SAUSA300_ RS02710 SAUSA300_ RS03405 SAUSA300_ 0618 SAUSA300_ 2351 SAUSA300_ 0117 SAUSA300_ 0305 SAUSA300_ 0619 SAUSA300_ 2099 SAUSA300_ 0620 SAUSA300_ 2398 SAUSA300_ 0310 SAUSA300_ 0566 SAUSA300_ 0506 SAUSA300_ 0635 amino acid permease -3.435439103 1.97E-17 ABC transporter permease -3.434548452 4.00E-15 staphylopine-dependent metal ABC transporter substrate-binding protein CntA metal ABC transporter substrate- binding protein zinc ABC transporter substrate- binding lipoprotein AdcA staphyloferrin B ABC transporter substrate-binding protein SirA formate/nitrite transporter family protein -3.386083399 7.58E-13 -3.002466305 7.55E-11 -2.952860287 3.23E-07 -2.890004889 2.89E-13 -2.657219054 1.82E-08 metal ABC transporter permease -2.611758841 1.04E-09 CDF family zinc efflux transporter CzrB metal ABC transporter ATP-binding protein iron export ABC transporter permease subunit FetB -2.481461542 2.17E-06 -2.097167098 6.77E-06 -1.859001088 0.000434876 PTS sugar transporter subunit IIC -1.782003764 0.000262324 amino acid permease -1.767434199 2.70E-05 NupC/NupG family nucleoside CNT transporter -1.751889824 0.000704809 iron ABC transporter permease -1.715867328 0.000223999 356 Table B-5 (cont’d) SAUSA300_ RS14595 SAUSA300_ RS00580 SAUSA300_ RS03825 SAUSA300_ RS01870 SAUSA300_ 2627 SAUSA300_ 0112 SAUSA300_ 0712 SAUSA300_ 0353 anion permease -1.653514614 0.001374985 L-lactate permease -1.352592372 0.003891948 peptide MFS transporter -1.331649516 0.009270639 ABC-2 transporter permease -1.165101924 0.040283358 357