ELUCIDATION OF THE COBALT DETOXIFICATION MECHANISMS OF GEOBACTER SULFURREDUCENS By Michael Hunter Dulay 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 hallmark of the physiology of Geobacter bacteria is their ability to couple their oxidative metabolism to the respiration of a broad spectrum of metals, including FeIII and MnIV oxide minerals. In nature, these metal oxides often coprecipitate with or adsorb various metal species such as the micronutrient Co. During the reductive dissolution of these metals by Geobacter cells, the divalent form of Co (CoII) may be released into the environment. While most organisms require CoII in the form of cobamide vitamins, the metal is toxic and is expected to exert a selective pressure on Geobacter and syntrophic partners to detoxify their local environment in order to remain viable and metabolically active. In this dissertation, I investigated the mechanisms that allow the model representative Geobacter sulfurreducens to tolerate CoII exposure and the role that its complex respiratory chains have in the reductive detoxification of the metal. Consistent with adaptation through regular exposure to CoII, G. sulfurreducens expresses a complex network of detoxification pathways to mitigate metal intoxication. In chapter 2, I demonstrate that this transcriptional response enables G. sulfurreducens to survive CoII concentrations typically used to enrich metal-resistant microorganisms. Among the most highly differentially expressed genes were cell envelope c-type cytochromes (CbcBA) that are involved in the reduction of low-potential electron acceptors. In concert with this, metal-stressed cells removed 25 µM of CoII from culture supernatants and accumulated Co nanoparticles on the cell surface, suggesting that extracellular mineralization plays a role in the detoxification response. In chapter 3, I investigated the role of CbcBA in CoII detoxification using a mutant carrying a deletion in the cbcBA genes and a genetically complemented strain. CbcBA was found to support acclimation of cells to CoII stress, but compensatory effects were often observed that minimized the impact of the cytochrome defect in metal detoxification. Loss of the cytochrome pathway stimulated vesiculation, a phenotype associated with increased membrane fluidity and permeability. As a result, CoII permeated into resting cells lacking the cytochrome pathway, impairing their growth recovery in fresh media. Hence, the CbcBA cytochrome pathway is part of a complex cellular response that contributes to CoII detoxification in G. sulfurreducens. In addition, I describe the optimization of the resting cell assay in the appendix of this dissertation and demonstrate the need for careful formulation of buffers involved in metal reduction and tolerance studies. Alternative to the numerous cytochromes for the extracellular reduction of metals, G. sulfurreducens also assembles conductive pili decorated with metal traps that can bind CoII and reduce it to Co0 nanoparticles. In chapter 4, I investigated a biological role for the pili in CoII detoxification via the extracellular reduction and precipitation of the metal. The study showed that cells that dynamically extend and retract their pili have a growth advantage in the presence of CoII which may be related to accumulation of metal nanoparticles along the filaments. These results indicate that the conductive pili play a major role in the mineralization of CoII and that this reaction is critical to avoid metal intoxication. The last chapter summarizes the major conclusions of this work and describes future research directions that can expand our understanding of the adaptive responses used by Geobacter bacteria to respire metals as a cellular protective mechanism. The ecological impacts of these reactions and applications in biotechnology are discussed. This dissertation is dedicated to my family, old and new. iv ACKNOWLEDGEMENTS I would first like to thank my two advisors, Dr. Gemma Reguera and Dr. Kaz Kashefi. I came to MSU with interest in your work as scientists but quickly found that your mentorship and friendship were equally as valuable. You have supported me through learning moments and victories alike and have been integral in developing my presentation and thinking skills. The wisdom you shared in the lab and the classroom will always stick with me. I have grown a lot since first arriving here and this development would not have been possible without your support. I hope to make you both proud as I journey out in pursuit of leading my own research. I would also like to thank my lab mates who have helped make the lab feel like a home. Marcela Tabares trained me in the difficult ways of culturing Geobacter and worked with me on the first few cobalt growth curves during my rotation. Our coauthored paper became the backbone of my dissertation and without her expertise with RNA Seq, I would have been at a loss. I want to thank the third member of Team Geo, Morgen Clark, who trained me on the AFM and always been a source of good conversations about philosophy and how we do science. I’ve deeply appreciated our conversations about data analysis, metals and microbiology, fantasy books, and the next national park we want to go to. The lab always felt special when all three of us were there, and dancing and singing were never far away. I would also like to thank Dr. Kristin Jacob who was a mentor since my first day of interviews. Thank you for supporting my coffee drinking habits and for helping me sort out the stresses of pursuing a PhD. I would also like to thank our most recent lab member, Dr. Emily Greeson. You are truly a force of nature, both in the realm of running a research project and in making sure your family and friends are cared for. v I would like to thank my support at the microscope. The staff at the Center for Advanced Microscopy were instrumental in my training with an electron microscope. Thank you, Dr. Alicia Withrow for teaching me the in’s and out’s of the TEM. It was a joy working closely and learning from you. Thank you to the late Dr. Xudong Fan for your guidance in performing EDS analysis. And thank you to Dr. Reza Loloee for your support with the AFM, even when the AFM tried its best to halt our progress. I must also thank my family who inspired me to pursue my love of science and learning and gave me the necessary background to make it through my PhD training. To my mom and dad, JoLynn Larsen and Michael Dulay, it was your encouragement of my potential that got me to where I am today. You showered me with love and support through difficult times in my youth and always reinforced that I could grow and become more. There was no goal too lofty or path too arduous for me in your eyes and I doubt I could have made it through my PhD training without the belief in myself that you fostered in me. To my brother Tanner Dulay, you have been my partner in all things imagination and mischief. You inspire me with your effort and passion, and I hope to collaborate in our shared love of science. To my brother Griffen Dulay and sister Peyton Dulay, it has been a joy to watch you grow up and learn about the world. Your insight on life has taught me to always take a fresh look at what I take for granted. To my grandmother, Carolyn Dulay, you were the rain and sunshine that began my growth as a scientist. You encouraged my early fascination with the world and went above and beyond as a grandparent and mentor. It was trips behind the scenes at the San Francisco Zoo to feed, watch, and care for the animals that instilled in me a sense of awe for the natural world. vi Together, the Flying Dulays have been a source of love, encouragement, and joy that I will be forever grateful for. I could not have asked for a more amazing family. It goes without saying that my success was deeply supported by my partner, Dr. Emily Gibson. Since meeting you in my first year of graduate school, we have grown together and made it through good times and bad. No matter the challenges we face, we are a team, a married couple, and always friends. Your excitement about the world is inspirational. You are gentle and empathetic in the face of strife, and righteous and passionate in the face of adversity. It was your personal care during long weeks of endless experiments that kept me energized and rested. It was your hugs and peptalks during bitter learning moments that kept me optimistic and bright. I am lucky, so lucky, to have found you in this world. vii TABLE OF CONTENTS Chapter 1: The Biogeochemistry of Cobalt ...................................................................... 1 BIOLOGY AND THE REQUIREMENT FOR METALS AND METALLOENZYMES ..... 2 THE COBAMIDE PROSTHETIC GROUP ................................................................... 4 GEOLOGY AND GEOCHEMISTRY OF COBALT AND ITS LIMITATION IN THE ENVIRONMENT .......................................................................................................... 7 METAL RESPIRATION AS A PART OF THE BIOGEOCHEMICAL CYCLE ............... 9 REFERENCES .......................................................................................................... 13 Chapter 2: Cobalt resistance via detoxification and mineralization in the iron-reducing bacterium Geobacter sulfurreducens ............................................................................ 22 ABSTRACT ............................................................................................................... 23 INTRODUCTION ....................................................................................................... 23 RESULTS .................................................................................................................. 27 DISCUSSION ............................................................................................................ 44 MATERIALS AND METHODS ................................................................................... 52 ACKNOWLEDGEMENTS .......................................................................................... 57 TABLES ..................................................................................................................... 58 REFERENCES .......................................................................................................... 60 Chapter 3: Contribution of the low-voltage CbcBA cytochrome respiratory chain to CoII detoxification by Geobacter sulfurreducens .................................................................. 69 ABSTRACT ............................................................................................................... 70 INTRODUCTION ....................................................................................................... 71 RESULTS .................................................................................................................. 73 DISCUSSION ............................................................................................................ 90 MATERIALS AND METHODS ................................................................................... 94 REFERENCES .......................................................................................................... 99 Chapter 4: Expression of electrically conductive pili as a mechanism for CoII tolerance............................................................................................................... 104 ABSTRACT ............................................................................................................. 105 INTRODUCTION ..................................................................................................... 105 RESULTS ................................................................................................................ 108 DISCUSSION .......................................................................................................... 114 MATERIALS AND METHODS ................................................................................. 118 REFERENCES ........................................................................................................ 121 Chapter 5: Conclusions and Future Directions ............................................................ 126 APPENDIX: EFFECT OF GROWTH MEDIA FORMULATIONS ON THE VIABILITY AND COII-DETOXIFICATION CAPACITY OF RESTING CELLS OF GEOBACTER SULFURREDUCENS .................................................................................................. 131 viii Chapter 1: The Biogeochemistry of Cobalt 1 BIOLOGY AND THE REQUIREMENT FOR METALS AND METALLOENZYMES Since the time of the last universal common ancestor, cells have used metals as catalytic centers for enzymatic activity. The metals predicted to be present in the early-Earth environment, such as Fe and Ni, could abiotically catalyze reactions such as H2-oxidation coupled to C-fixation 1-3 and N2-reduction 4,5. Incorporation of these metal catalysts as prosthetic groups in enzymes allowed for a more controlled, efficient, and diverse set of biotic reactions to occur 6,7. Indeed, geologically occurring Fe-S minerals and Fe-S catalytic centers of some metalloenzymes are highly similar in structure and facilitate critical activities for life 3,8,9. This parallelism has led some to suggest that early cells took up free metals prior to the development of sophisticated chaperone pathways in order perform these reactions away from mineral surfaces 6,9. Due to their ubiquity in biology, the distribution of various metals through geologic time is of key interest in understanding extant life and how it evolved to use specific metals as catalytic centers 10. Transition metals such as Fe and Mn 11-13, Ni and Co 14-17, Mo 18,19, Cu 20,21, Mg 22, Zn 23 and V 24 are all part of prosthetic groups in metalloenzymes that drive many biological reactions 25. All of these metals, with the exception of Zn, have a broad range of redox states that allow for the stabilization of the transition state of enzymatic reactions by either receiving or donating electrons with a substrate 25. While many metal cofactors have similar properties and catalytic activities 10 there is variation in the biological requirements for specific metals between different organisms 16,26. However, the essentiality of metal cofactors is clear as nearly half of all identified proteins to-date require at least one metal for proper functionality 27. 2 Cofactor selection by the metalloenzyme can be both highly selective and promiscuous. Briefly, specificity between a metal-binding site and the prosthetic group is driven by the electrochemical properties of the metal, such as its coordination geometry, charge radius, and position along the Irving-Williams series (binding affinities follow a trend of MnII < FeII < CoII < NiII < CuII > ZnII) 28. But it can also be influenced by the chemical properties of the binding site, such as the flexibility of the ligand and the presence of specific amino acids or other biomolecules 29. Greater thermodynamic specificity can be introduced through various metallochaperones, alterations to the metalloenzyme and modulation of the availability of metals to the metal-binding site 29. And yet, many native metalloproteins bind more than one metal 30. Replacement of the native metal with an alternative element can lead to changes in substrate specificity 31, rates of activity 32, and even the type of chemical reaction catalyzed by the enzyme 33. Of all the metals within metalloenzymes, Mg is the most commonly used (16%), followed by Zn (9%), Fe (8%), Mn (6%), Ca (2%), Co (1%), Cu (1%) and V, Mo, W, Na, Ni, and K (all falling below 1%) 27. Interestingly, the use of Cd as a cofactor has been reported in a single enzyme and suggests that nutrient limitations in various environments may drive the development of novel metal-enzyme interactions 34,35. Collectively, all of these metals participate in reactions of the enzyme classes EC1 oxidoreductases, EC2 transferases, EC3 hydrolases, EC4 lyases, EC5 isomerases, and EC6 ligases 27. While metalloenzymes often coordinate metal ions through sidechain interactions at the catalytic center, many metals assemble into large cofactors to which the metalloenzyme binds. Among the most common of these cofactors are those derived from bulky porphyrin rings including Fe-containing heme (~616 Da) and siroheme (~916 Da), Co-containing 3 cobamides (~1202-1578 Da), Mg-containing chlorophyll (~894-911 Da), and Ni- containing coenzyme F430 (~902 Da) 36-40. THE COBAMIDE PROSTHETIC GROUP While organisms can alter the structure of many prosthetic groups, selection for cobamide catalytic centers is highly dependent on structure and tightly regulated during biosynthesis and its secretion to support syntrophic partnerships 16,41-43. The first steps in cobamide synthesis, which are shared with the pathways for the synthesis of other porphyrin rings, involve the conversion of two molecules of 5-aminolevulinate into one porphobilinogen by HemB (EC 4.2.1.24), the polymerization of four molecules of porphobilinogen into the ring-like hydroxymethylbilane by HemC (EC 2.5.1.61) and the closure of the ring by HemD (EC 4.2.1.75) to produce uroporphyrinogen III. This tetrapyrrole precursor is then split off the biosynthetic path of other porphyrins and converted into precorrin-2 by CysG/CobA (EC2.1.1.130). Precorrin-2 can then enter a multi-step process of corrin ring biosynthesis, for which an aerobic (CobIGJMFKLHBNST) and anaerobic (CbiKXLHFGDJTECA) pathway exists (Fig. 1.1). A key difference in these pathways is the chelation of the Co ion at the beginning of the anaerobic pathway (CbiK/CbiX, EC 4.99.1.3) as opposed to the end of the aerobic pathway (CobNST, EC 6.6.1.2). The insertion of the Co ion early in anaerobic biosynthesis assists in subsequent steps to contract the precorrin ring through oxidoreductase activities in the absence of O2 38. The two pathways converge at cob(II)yrinic acid diamide which goes through adenosylation by BtuR/CobA/CobO (EC 2.5.1.17) and subsequent nucleotide loop assembly to produce a complete cobamide. 4 Figure 1.1 Abbreviated pathways for cobamide synthesis. Proteins (bold) involved in the anaerobic (left) and aerobic (right) contraction of the porphyrin ring are presented alongside key intermediate molecules (pink). Proteins involved in the final steps of cobamide synthesis follow below cob(II)yrinic acid diamide. Insertion of the Co ion is marked with a red circle. A separate biosynthetic pathway is responsible for producing the lower ligand that attaches to the nucleotide loop. The identity of the ligand varies broadly and alters the accessibility of the cobamide to different organisms 16,41,42,44. Indeed, many organisms specialize in using one type of cobamide over another and very few are able to alter the lower ligand once attached 16,43. For example, coculture studies measuring the dechlorination activity of Dehalococcoides mccartyi found that cobamides with 5’,6’- dimethylbenzimidazole, which are produced by Geobacter lovleyi, supported the growth of D. mccartyi via dechlorination 42. By contrast, cobamides containing 5- 5 hydroxybenzimidazole, which are produced by Geobacter sulfurreducens, did not 42. However, studies of D. mccartyi grown with cobamides alongside exogenous 5’,6’- dimethylbenzimidazole revealed that these cells can remodel the prosthetic groups to contain the desired lower ligand, rescuing growth and dechlorination activity 45. To this date, 15 cobamide-dependent functions have been identified that span a broad range of activities 14,16. These activities include the catabolism of carbon (glycerol/propanediol, propionate) and nitrogen (ethanolamine, D-ornithine, glutamate, beta-lysine) 46,47, one-carbon metabolism in the Wood-Ljungdahl pathway (production of acetyl-CoA) 48 among other methyltransfer reactions, reduction of nucleotide di- and tri- phosphates to their respective deoxynucleotide states 49, tRNA synthesis (production of queosine) 50, synthesis of bacteriochlorophyll 51, methionine synthesis 46, mercury methylation 52, and, as noted above, reductive dehalogenation 47. Bacterial genome analyses reveal that 86% of species have at least one cobamide-dependent enzyme though only 37% contain a complete cobamide biosynthesis pathway 16. In the skin microbiome, only 1% of species synthesize cobamides de novo although 39% of the species contain at least one cobamide-dependent enzyme 41. Thus, despite the ubiquitous nature of cobamide-dependent enzymes, there is a large disparity between cobamide producers and consumers in microbial communities. Notably, all cobamide producers require the prosthetic groups for their own metabolism and altruistic organisms (i.e., those that synthesize these metabolically costly prosthetic groups solely for others) are yet to be identified 16. Mutualistic interactions between cobamide producers and syntrophic partners, however, are well documented 43,53,54. While some cobamide- dependent enzymes reaction have counterparts that do not require the prosthetic group, 6 many species maintain both enzymes to broaden the range and efficiency of the desired reaction 16. For example, cells requiring ribonucleotide reductases can overcome O2 limitation associated with a requisite oxygen-dependent activation of the diiron enzyme through the alternative use of cobamide-dependent enzymes 55 and cobamide-dependent methionine synthase has a 100-fold greater turnover than the Zn-dependent form which can be more sensitive to oxidation and stress 56,57. Taken together, the availability of cobamides, as well as free Co, in the environment is critical for many individual and community processes 16,43. GEOLOGY AND GEOCHEMISTRY OF COBALT AND ITS LIMITATION IN THE ENVIRONMENT Concentrations of Co can vary broadly across important geological landscapes. Spectroscopic measurements of the Sun predict the solar system abundance of Co to be ~105 atoms per 1012 atoms of H (less than 0.00001% or 0.1 ppm) 58. On the other hand, concentrations in iron meteorites thought to represent the early Earth core suggest its enrichment (500-10,000 ppm) 58-60. However, Co concentrations in Lunar basalt (20-45 ppm) 58 and the average Earth crust (25 ppm) 61 are notably lower. Concentrations of Co can be much higher locally in segregations of metal particles from Lunar regolith (>10,000 ppm) or igneous rock (2,500-44,000 ppm) 62 as well as those found in the Earth crust (<65% or 650,000 ppm) 63. Notably, Co does not form a pure metal but instead is found in many S- and As-containing minerals (e.g., cobaltite, CoAsS) and regularly incorporates into Ni, Cu, Mn, and Fe minerals 58,63-65. Weathering processes can often dictate the Co concentration in soils, sediments, and water ecosystems 58. Parent rocks rich in Co, such as igneous ultramafic (100-200 7 ppm) and mafic (40 ppm) or metamorphic schists (40 ppm), can provide ecosystems with ample Co, whereas depleted rocks, such as shales (19 ppm), can produce ecosystems that are have limited availability of the micronutrient 58,63. Indeed, sheep raised on pastures over Co-depleted granite rock, whose soil has <0.39 ppm Co content, developed vitamin B-12 anemia (i.e. cobalamin deficiency) as opposed to those raised on richer soils with 1.67 ppm 58,66. Similar effects of Co-deficient soils on animal health have been reported in Kenyan animal reserves where lowland areas fall below a guideline 10 ppm Co for healthy soils as compared to highland areas that are less heavily flooded and grazed 58,67. Concentrations in aquatic bodies are also modulated by rock weathering. Measurements of Co levels in large rivers typically range from 0.02-0.148 ppm 68 but contamination can increase the amount up to 2.028 ppm 69. The relatively low concentrations of this metal in freshwater systems compared to soils are important for the viability of many aquatic organisms, as they are particularly susceptible to Co toxicity. Duckweed plants, for example, rapidly die at concentrations typically associated with healthy agricultural soils 70. While many metals solubilized by weathering are precipitated by humic substances in water, Co is readily dissolved when bound by organic substances resulting in only 10% Co precipitation in some river waters 71. As a result, near-shore buildups of Co have been reported that can raise the metal levels 30-fold compared to open ocean waters 58. Oceans may also experience regional differences in Co concentrations, though on average metal levels are very low (0.0003 ppm or 0.3 ppb) and in the form of CoSO4 and CoCl2 salts 58. In fact, open ocean levels of Co can fall low enough to limit biological activity. This is surprising because volcanism at ocean ridges 8 can release substantial amounts of Co into the waters but the metal accumulates locally in deep sea clays (12-16 ppm) 58 and in Mn-oxide minerals along deep seamounts (2,000 ppm) 72,73. Although Co can be abundant in many environments, it is rapidly sequestered via co-precipitation with metal oxides and S-containing minerals such as pyrite (FeS2) 58,65. Adsorption of Co to the mineral phases may also occur and follows standard cationic behavior as a function of pH: adsorption can be insignificant below pH 4 but substantial at >pH 7 65. Once sequestered in FeIII or MnIV oxides, Co is predicted to remain stably trapped 74. Furthermore, the longer Co is sequestered in metal oxides, particularly MnIV oxides, the more difficult it becomes for desorption to occur 74. Thus, despite its ubiquity and relative abundance in many environments, the bioavailability of Co has often been considered to be quite low. METAL RESPIRATION AS A PART OF THE BIOGEOCHEMICAL CYCLE For over a century, the ability of microbes to use extracellular metals as electron donors 75-77 and acceptors 78-80 for their metabolism has been of industrial 81,82, environmental 83,84 and astrobiological interest 6,85,86. Metal-reducing microbes use oxidized metals as electron acceptors to gain energy from the establishment of a proton motive force across the inner membrane, similar to how O2 is reduced by the respiratory chains of aerobic organisms. The ability of microbes to respire metals allows them to colonize a wide range of environments where stronger electron acceptors (e.g., O2 and nitrate [NO3 -]) may not be available, such as below the 1-cm crust of terrestrial soils, the ocean floor 87,88 and the subsurface 89. The energy derived from metal reduction is dependent on the half- or reduction-potential of the metal species, making MnIV reduction to MnII (half-potential of 9 1.23 V) more favorable than FeIII reduction to FeII (half-potential of 0.771 V), and so on 90. Metal respiration can be facilitated through numerous mechanisms. In general, metal-reducing bacteria use NADH dehydrogenase enzymes to split protons and electrons at the membrane using reducing molecules (e.g., NADH) generated during their oxidative metabolism. The protons provide the gradient needed for ATP synthesis, while the electrons are transferred to the membrane menaquinone pool and from there to c- type cytochromes of the inner membrane (quinol oxidases) and, in Gram-negative bacteria, to periplasmic and outer membrane c-type cytochromes, which reduce the metal on the cell surface 21,91,92. Metal-reducing bacteria in the genus Geobacter are unique in their ability to extend their redox-surface area via conductive protein appendages known as pili, which use tightly packed aromatic residues to discharge electrons from the periplasmic cytochromes onto terminal electron acceptors like FeIII oxides 83,93,94. Outer membrane cytochromes of Geobacter have also been reported to form conductive filaments in the extracellular matrix of biofilms 95 but planktonic cells, the most relevant state for metal reduction, primarily produce pili as nanowires 96. Metal- reducing bacteria may also secrete soluble electron shuttles such as flavins to transfer electrons to the extracellular minerals 78. All metal reducers may also stimulate their reductive activities with exogenous humic substances, which function as natural electron shuttles in environments where organic matter degradation is a significant process 97,98. Collectively, metal-reducing microbes play important roles, whether directly or indirectly, in the biogeochemical cycling of many metals. The bioreduction of FeIII and MnIV oxides dissolves the mineral phases and solubilizes the metals in their reduced 10 forms (FeII, MnII). Abiotic reactions between the free aqueous ions and the metal oxides may also occur concurrently, generating mixed valence metal minerals such as magnetite (Fe3O4) 93,99. The reductive dissolution of the FeIII and MnIV oxides supplies the essential metals to syntrophic partners for assimilation or for their use as electron donors (e.g. FeII oxidizers). The dissolution of the metal oxides also releases other metal species sequestered in the minerals such as Co 65,74. Free CoIII is readily chelated by naturally- occurring organic molecules such as citric acid 65, providing a soluble electron acceptor for respiration via its reduction to CoII 100,101. But CoII may also be sequestered from the metal oxides by the activities of metal-reducing microbes. As a result, CoII concentrations may increase locally in environments where metal oxides are actively being reduced by microbes. This is expected to exert selective pressure on metal reducers to become resistant to the metal. In support of this proposal, metal cycling bacteria are the dominant genera in Co-containing deep-sea Mn nodules, such as those of the Shewanella and Colwellia genera 87 . Dissolution of the nodules by the bacterial activities releases Co into the environment 87 and provides a source of this micronutrient for cobamide-dependent members of the nodule community. Thus, Co resistance is critical for metal-reducing microbes to solubilize the Co pool sequestered in the mineral phases, making the metal available for the synthesis of cobamides. 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PMID: 33324382; PMCID: PMC7726332. †Equal contribution *Corresponding author 22 ABSTRACT Bacteria in the genus Geobacter thrive in iron- and manganese-rich environments where the divalent cobalt cation (CoII) accumulates to potentially toxic concentrations. Consistent with selective pressure from environmental exposure, the model laboratory representative Geobacter sulfurreducens grew with CoCl2 concentrations (1 mM) typically used to enrich for metal-resistant bacteria from contaminated sites. We reconstructed from genomic data canonical pathways for CoII import and assimilation into cofactors (cobamides) that support the growth of numerous syntrophic partners. We also identified several metal efflux pumps, including one that was specifically upregulated by CoII. Cells acclimated to metal stress by downregulating non-essential proteins with metals and thiol groups that CoII preferentially targets. They also activated sensory and regulatory proteins involved in detoxification as well as pathways for protein and DNA repair. In addition, G. sulfurreducens upregulated respiratory chains that could have contributed to the reductive mineralization of the metal on the cell surface. Transcriptomic evidence also revealed pathways for cell envelope modification that increased metal resistance and promoted cell-cell aggregation and biofilm formation in stationary phase. These complex adaptive responses confer on Geobacter a competitive advantage for growth in metal-rich environments that are essential to the sustainability of cobamide-dependent microbiomes and the sequestration of the metal in hitherto unknown biomineralization reactions. INTRODUCTION Metal micronutrients such as nickel (NiII), cobalt (CoII), manganese (MnII) and iron (FeII) are essential for life yet toxic above relatively low concentrations 1. Not surprisingly, microorganisms have evolved numerous adaptive responses to import the essential 23 metals from the environment while preventing their excessive intracellular accumulation and intoxication 2. Metal homeostasis is primarily achieved by the antagonistic activities of metal importers and exporters 2. Cells often use high affinity transporters to import the metals with specificity and rely on specialized proteins and chaperones to integrate them into pathways dedicated to the synthesis of metalloproteins and enzyme cofactors 1. Collectively, biometals contribute to the synthesis of up to one third of the cell’s proteome and to metabolic functions essential to the growth and survival of the cell 1. Each of these metals must be available in just the right intracellular concentration (i.e., the cellular metal quota) to prevent intoxication 3. Thus, dedicated metalloregulatory systems monitor the intracellular metal levels and modulate the expression of transporters and other proteins essential for metal homeostasis 2. Metal exporters provide the primary mechanism to eliminate excess metal 2 but the cellular response to metal intoxication is often more extensive, as cells have to cope with the direct and indirect impacts of the reactive metals on proteins and DNA. For example, CoII can bind and inactivate numerous proteins non- specifically, displace other metals (particularly, FeII) from prosthetic groups and metal- binding sites, and generate free radicals 4. Its high affinity for thiol groups disrupts disulfide bonds in proteins, reduces the free thiol pool and can interfere with sulfur assimilation 5. Hence, CoII intoxication causes generalized damage in the cells, requiring extensive reprograming to cope with multiple stressors. The essentiality yet toxicity of metal micronutrients such as CoII exerts selective pressure on microorganisms to tune their metabolism to the fluctuating availability of the metal species from geochemical sources. Yet, many aspects of the biological cycling of metal micronutrients remain relatively obscure. This is particularly true for CoII, a metal 24 micronutrient that some microorganisms assimilate to produce enzyme cofactors (cobamides) in the cobalamin (vitamin B12) family that catalyze metabolic reactions essential to all living cells 6. Genes encoding cobamide-dependent enzymes are widespread in prokaryotes but only a fraction of surveyed genomes have complete pathways for de novo cobamide synthesis 6,7. As a result, most microorganisms need to salvage cobamides from the environment, a nutritional dependency that drives syntrophic interactions with cobamide producers 8. Cobamide-dependent microbiomes depend on the ability of cobamide producers to import and assimilate the soluble CoII cation. The divalent species, however, readily oxidizes to CoIII on the surface of MnIV oxide particles 9,10. CoII mobility in soil and sediment systems is also limited by the tendency of the metal to coprecipitate with FeIII and MnIV oxide minerals 11. Additionally, FeIII and MnIV oxides sorb large amounts of the metal cation, sequestering it in solid phases that reduce its bioavailability 12. The absorption and co-precipitation of most of the available CoII into FeIII and MnIV minerals gives FeIII and MnIV-reducing bacteria, such as those in the genus Geobacter, a competitive advantage for growth in cobamide-dependent microbiomes (Fig. 1). These bacteria gain energy for growth from the reductive dissolution of the metal oxides, which are reactions that could solubilize FeII and MnII and could remobilize CoII and CoIII 13. Geobacter species are also important drivers of organic matter degradation, a process that generates organic chelators with affinity for CoIII. This keeps the trivalent species in solution and available for use as an electron acceptor 13. The dissimilatory reduction of chelated forms of CoIII by Geobacter reduces CoIII to CoII 13. The low reduction potential of the divalent species (− 0.28 V versus standard hydrogen electrode, SHE) and its 25 toxicity to bacteria at relatively low concentrations have been assumed to prevent its biological reduction to Co0 14,15. Yet, Geobacter species, including the model laboratory strain Geobacter sulfurreducens, assimilate CoII to synthesize cobamides, which they secrete to sustain several syntrophic partners 16 (Fig. 2.1). These syntrophic interactions are favored in local epigenetic zones enriched in FeIII and MnIV oxides, which are the regions where CoII preferentially accumulates 17. This raises yet unexplored questions about the cellular tolerance of Geobacter species for CoII and the mechanisms that allow these microorganisms to survive and even thrive in CoII-rich environments. Figure 2.1 Known contribution of Geobacter species to the cycling of cobalt (Co). Geobacter bacteria reduce chelated and mineral forms of CoIII to CoII, whose bioavailability is limited by its tendency to adsorb and/or co-precipitate with FeIII and MnIV oxides. The reduction of FeIII and MnIV oxides by Geobacter bacteria solubilizes CoII for the synthesis of cobamides that support the growth of syntrophic partners. We gained insights into the environmental controls of Geobacter activities in cobamide-driven microbiomes by investigating the adaptive responses of G. sulfurreducens to growth and reproduction in the presence of CoII. Consistent with environmental exposure, we demonstrate high CoII resistance in this laboratory strain and describe pathways for protein and DNA repair, cell envelope modifications, and biofilm formation that allow the cells to effectively cope with CoII stress. Importantly, we show that metal acclimation activates respiratory chains that could participate in the reductive precipitation of the metal on the cell’s surface to alleviate toxicity. These adaptive 26 responses allow Geobacter species to grow in CoII-rich environments, sustaining the productivity of the native microbiomes and contributing to hitherto unknown reactions of the Co cycle. RESULTS Genomic determinants of CoII homeostasis in G. sulfurreducens Metal ions bridge the outer membrane by simple diffusion through nonselective pores 18 but require specific transporters to traverse the inner membrane (Fig. 2.2). We identified in G. sulfurreducens complete NikMNQO and CbiMNQO importers, the most widespread prokaryotic systems for NiII and CoII uptake 19. Although both systems can import NiII and CoII, specific amino acid signatures in the M subunits make CbiMNQO the high affinity importer of CoII 19. At high enough concentrations, however, CoII could selectively outcompete NiII and enter the cytoplasm via the NikMNQO system. These transporters are annotated as ATP-binding Cassette (ABC) transporters, but they are part of the prokaryotic family of energy-coupling factor (ECF) transporters that also transport water- soluble vitamins and cofactors 20. The metal ECF subclass has a distinct modular architecture (A, T and S components) to bind the substrate (S component) without assistance from extracytoplasmic solute-binding proteins 20. In most Nik/CbiMNQO systems, the S component is a heterodimer of M and N subunits 19 but these subunits are fused in a single gene in G. sulfurreducens (nikMN, GSU1279; cbiMN, GSU3004). The end result is the same: the assembly of a MN subcomplex (S component) that binds the metal and transports it across the membrane in a reaction energized by the O subunit dimer (A, or ATPase, component) and rate-modulated via interactions with the transmembrane Q subunit (T component) 20. 27 Figure 2.2 Genomic reconstruction of potential pathways for CoII transport and assimilation in G. sulfurreducens. The genes (A) and model (B) show transmembrane ECF importers (in blue) as well as two CDF proteins for transmembrane export and four RND systems for exporting periplasmic metal across the outer membrane (gold color). Panel B also shows generic outer membrane porins for the simple diffusion of metal cations. The genome of G. sulfurreducens also encodes for Cbi (letter designation), Cob (full name) and other enzymes needed for the anaerobic synthesis of cobamide (pink arrows). The pathway starts with the incorporation by the cobaltochelatase CbiX of the metal into Factor II, which is synthesized from heme intermediates such as uroporphyrinogen-III. The genome encodes Cbi proteins that convert the CoII-Factor II into cob(II)yrinic acid diamide, except for CbiJ (in gray) whose role in the pathway is yet to be experimentally validated. Cob (full name) and Cbi (only letters) proteins and other enzymes complete the synthesis of a cobamide with a 5-OHBza lower ligand (formula in C). As shown in Fig. 2.2, the cbiMNQO genes are part of a large cluster (GSU2989- 3010) encoding most of the enzymes needed for the anaerobic synthesis of a cobinamide intermediate (Cbi proteins) and its conversion into cobamide (Cob and Cbi proteins) 21. We identified in a separate genomic location two additional cobamide biosynthetic 28 enzymes, GSU1578 and CobA (GSU1577). Also unlinked were two genes encoding enzymes for the methylation (HemD, GSU3286) and oxidation (CysG, GSU3282) of uroporphyrinogen III, the common precursor of cobamide and heme biosynthesis 21. These two enzymes convert uroporphyrinogen III into Factor II, the preferred substrate for the anaerobic synthesis of cobamide 21. The anaerobic cobaltochelatase CbiX (GSU3000) incorporates the metal into Factor II, while several Cbi proteins methylate, contract, amidate, and decarboxylate the molecule to generate a cobyrinic acid diamide intermediate (Fig. 2.2C). All of the proteins needed for these reactions were annotated or had a clear homolog in the genome of G. sulfurreducens, except for the precorrin-6X reductase CbiJ (highlighted in gray in Fig. 2.2B). This enzyme is often assigned to this reaction based on its homology with the aerobic enzyme CobK, but its biological role has never been confirmed 21. The cob(II)yrinic acid diamide intermediate is then converted into adenosyl cobinamide in sequential reactions initiated by an adenosylcobamide- binding subunit of an (R)-methylmalonyl-CoA mutase (GSU1578). The step catalyzed by CobU (GSU3010) generates an adenosine-GDP-cobinamide substrate for attachment of the cobamide lower ligand. G. sulfurreducens produces a cobamide with a 5- hydroxybenzimidazole (5-OHBza) lower ligand (Fig. 2.2C) that is synthesized from 5- amino-imidazole ribonucleotide (AIR) by BzaF (GSU3005) 22. The bzaF gene, which is unique to the Geobacteraceae and other members of the order Desulfurococcales, is a functional homologue of the bzaA and bzaB genes that catalyze the synthesis of 5-OHBza in other bacteria 22. The attachment of the lower ligand to adenosine-GDP-cobinamide completes the synthesis of the cobamide (Fig. 2.2C). 29 At high enough concentrations, CoII can also enter the inner membrane non- specifically via magnesium (MgII) uptake systems 23. To compensate for the uncontrolled influx of the metal, cells express metal exporters 2. We identified in the genome two genes (GSU0487 and GSU2613) encoding the cation diffusion facilitator (CDF) proteins, DmeF and FieF (Fig. 2.2). These proton-driven antiporters export a broad range of divalent cations (CoII, ZnII, FeII, CdII and NiII) across the inner membrane 24. However, the preferred substrate for DmeF is CoII 25 while FieF specializes in the export of excess FeII 26. The intracellular accumulation of CoII can disrupt the homeostatic balance with FeII and allow FieF to move more CoII than FeII across the inner membrane. The genome also contains four tripartite metal efflux systems of the Resistance-Nodulation-Division (RND) superfamily 25 (Fig. 2.2). RND efflux pumps use the proton gradient to energize the export of cytoplasmic or periplasmic substrates 27. Some of these exporters function as multidrug efflux pumps 28 while others specialize in proton-dependent transport of divalent metal cations 29,30. As in other Gram-negative bacteria 28, the Geobacter metal RND systems contain a transmembrane pump, a periplasmic adaptor protein and an outer membrane porin. This trimeric configuration facilitates the export of periplasmic metals using the electrochemical gradient 28. Collectively, inner and outer metal exporters ensure that CoII does not accumulate to toxic levels inside the cytoplasm 2. High CoII tolerance in G. sulfurreducens suggests significant environmental exposure to the metal As metal resistance evolves under selective pressure, we determined the growth efficiency of G. sulfurreducens in the presence of CoII (Fig. 2.3). For these experiments, we inoculated cells at low densities (OD660, ~0.03) in a medium optimized for growth of 30 G. sulfurreducens (DB medium) 31 with acetate and fumarate (DBAF medium) and supplemented with various concentrations of CoCl2. We measured growth with up to 1 mM CoCl2 (Fig. 2.3A), a concentration commonly used to enrich for metal-resistant bacteria from soils and industrial wastes contaminated with heavy metals 32. Generation times increased (Fig. 2.3B) and planktonic biomass yields (maximum OD660 during entry in stationary phase) decreased (Fig. 2.3C) in a dose-dependent manner, as cells coped with higher levels of metal toxicity. For example, G. sulfurreducens cells doubled every 4.58(0.05) hours in the untreated cultures, which we estimated to have approximately 27 µM of CoII using an assay we developed for the colorimetric detection of CoCl2 in the culture medium. Supplementation with an additional 100 or 250 M CoCl2 increased generation times to 5.41(0.25) and 10.13(0.54) hours, respectively (Fig. 2.3B). Generation times increased even more at higher CoCl2 concentrations (500 and 1000 M) and, on average, one out of three replicate cultures did not resume growth after a week (Fig. 2.3B). Furthermore, cultures that resumed growth did so after extended phases of acclimation (long lag phases before entering exponential phase) and reached lowest biomass yields (Fig. 2.3C). 31 Figure 2.3 Effect of CoII on G. sulfurreducens growth. (A) Cell growth (absorbance at 660 nm) in acetate-fumarate cultures with or without CoCl2 supplementation (up to 1,000 µM). Data points for 0-250 µM CoCl2 treatments show average and standard deviation of triplicate cultures. Treatments with 500 and 1,000 µM CoCl2 show average and standard error of the only two replicates that resumed growth within a week. (B-C) Effect of CoII toxicity on growth efficiency. Panel (B) shows generation (doubling) times for each of the replicates in the untreated (0 µM) and treated (100-1,000 µM) cultures shown in (A). Panel (C) shows the effect of the CoCl2 treatment in reducing the culture’s growth yields (OD660 in early stationary phase relative to the untreated 0 µM cultures) or in extending the lag phase (time before entry in exponential phase). The trendlines in (C) are the polynomial fits for the average data points of relative growth yields (R2=0.999) and lag phases (R2=0.994) from the cultures shown in (A). Transcriptomic analysis reveals multiple mechanisms for CoII detoxification We gained insights into the mechanisms that allow G. sulfurreducens to cope with CoII stress by comparing the transcript abundance of mid-log phase cells grown with or without 250 µM CoCl2 supplementation (Fig 2.4). CoII intoxication led to the differential expression of 47 genes. Of them, 32 were upregulated (Table 2.1) and 15 were downregulated (Table 2.2). This is approximately 0.9% (upregulated) and 0.4% (downregulated) of the genes annotated in the genome of G. sulfurreducens. Most of the upregulated genes encoded proteins with predicted roles in metal detoxification such as efflux pumps, protein and DNA repair enzymes, cell envelope modification pathways, and transcriptional regulation (Table 2.1). We also identified among the upregulated genes pathways for extracellular electron transfer that could provide a mechanism for energy transduction and CoII 32 mineralization on the cell surface. By contrast, most of the downregulated genes coded for non-essential proteins with metal-binding domains or amino acids that CoII is known to bind strongly (Table 2.2). Thus, their downregulation reduces the burden of CoII retention in the cell’s proteome. Figure 2.4 Transcriptional response of G. sulfurreducens to CoII. (A) Heatmap of the transcriptional response in two replicates for the untreated ( CoII) and treated ( CoII) cultures. The datasets and statistical analyses of the expression data are provided in Supplementary file 1. (B) Dispersion plot of transcript abundance (log fold change [logFC] versus log counts per million [logCPM]) identifying the significantly upregulated (yellow) and downregulated (blue) genes. (C) Heatmap of genes differentially expressed with excess CoII or FeII. The latter was calculated as the inverse ratio of the log2 fold transcriptional changes reported for G. sulfurreducens cultures growing with sufficient versus excess FeII 33. The asterisk shows genes under Fur control 33. The calculations and data comparisons are provided in Supplementary file 2. Periplasmic detoxification of CoII. The diffusion of CoII through non-selective outer membrane porins 18 leads to its rapid accumulation in the periplasmic space and risks disruption of essential cellular functions such as protein secretion and respiration. CoII toxicity in the periplasm is consistent with the upregulation of two periplasmic cytochromes (GSU1538 and GSU2513) with predicted roles in hydrogen peroxide (H2O2) detoxification (Fig. 2.5). This suggests that CoII accumulated in the periplasm at levels high enough to catalyze Fenton-chemistry 33 reactions yielding reactive oxygen species (ROS) 5. GSU1538 has the conserved domain of di-heme cytochrome c peroxidases (PF03150), a group of periplasmic enzymes that reduce H2O2 to prevent oxidative stress 34. Bacterial cytochrome c peroxidases can receive electrons from small monoheme cytochromes 34. The upregulation of GSU2513, a periplasmic monoheme cytochrome c protein, suggests a similar redox partnership with the GSU1538 peroxidase. Figure 2.5 Transcriptional response of G. sulfurreducens during growth under CoII stress. The figure illustrates pathways (upregulated, gold; downregulated, blue) differentially expressed under CoII stress. Proteins in gray represent proteins predicted to participate in the detoxification pathways that did not undergo differential expression. Additional information for the proteins and encoding genes is available in Tables 2.1 and 2.2. Abbreviations: bCyt: Cytochrome b; CasABCD: CRISPR-Cas complex; b/cCyt: Cytochrome b or c; CydAB: Cytochrome bc oxidase complex; DGC: diguanylate cyclase; FdhT: formate dehydrogenase chaperone; FdnGHI: formate dehydrogenase; GRX, glutaredoxin; HgtR: hydrogen-dependent growth transcriptional regulator; HK: heme- binding histidine kinase; Hydr: Hydrolase; LP: lipoprotein; MauG: MauG-like diheme peroxidase; Mhc: monoheme cytochrome c; Mtd: mannitol dehydrogenase; PII: P-II family nitrogen regulator; Pcc: Porin-cytochrome complex; RNAP: RNA polymerase; UvrD: UV repair protein D (DNA helicase of the nucleotide excision repair pathway); Xrt: exosortase. CoII-stressed cells also upregulated GSU2812, a glutaredoxin-family protein (glutaredoxin motif, PF00462) containing a signal peptide (amino acids 1-27) for export to the periplasm. Glutaredoxins, like thioredoxins, are thioldisulfide oxidoreductases that 34 reduce or oxidize disulfide bonds depending on the redox potential of the cellular compartment (cytoplasm or periplasm) where they operate 35. For example, E. coli secretes several thioredoxin proteins (e.g., DsbA, DsbC) to the periplasm to form disulfide bonds and fold proteins 36. A periplasmic monothiol glutaredoxin (glutaredoxin 3, Grx3) complements the activities of DsbA and DsbC in reactions dependent on the glutathione biosynthetic pathway 35. The high affinity of CoII for thiol groups in cysteines leads to the rapid oxidation of the amino acid and the formation of non-native disulfide bonds, which glutaredoxins can resolve to prevent protein inactivation 37. We also identified among the upregulated genes an operon containing the three subunits of one of the four RND systems (GSU2135-2137) identified in the genome of G. sulfurreducens (Fig. 2.2). This RND transporter has a membrane-bound metal pump (GSU2135) homologous to CzcA from Cupriavidus metallidurans strain CH34 and CusA from E. coli 27. The pump binds the metal in the periplasm and undergoes conformational changes that move one proton into the cytoplasm and translocate the metal through a channel formed by the B and C subunits (Fig. 2.5). CusABC complexes often work coordinately with periplasmic metal chaperones (CusF) to transport monovalent cations (CuI and AgI). The lack of CusF chaperones in the G. sulfurreducens genome suggests that the CoII RND transporter is a CzcABC system. Indeed, CzcABC transporters receive their name for their ability to mobilize the divalent cations cobalt, zinc and cadmium 27. Furthermore, this metal efflux system plays roles in CoII detoxification and resistance in other bacteria 38. Thus, we designated the GSU2135-2137 genes as czcABC (Table 2.1). 35 Cytoplasmic detoxification of CoII. The first gene in the czcABC operon (GSU2134) codes for a protein with the conserved PII domain (PF00543) of nitrogen regulatory proteins. These proteins form homotrimers to bind metabolites signaling the energy (ATP, ADP), carbon (2-oxogluratate) and nitrogen (glutamine and 2-oxoglutarate) levels inside the cell 39. GSU2134 belongs to a phylogenetically distinct clade of proteobacterial PII proteins (PII-NG) that evolved from canonical nitrogen regulators GlnB and GlnK 40. Like most of the PII-NG proteins 40, GSU2134 clusters in the genome with the genes encoding a proton-cation CzcABC antiporter. Furthermore, PII-NG is a structural homolog of the metal-binding protein CutA1 of E. coli 41. CutA1 binds the divalent copper cation (CuII) at a site structurally equivalent to the ATP binding site of PII-NG proteins 41 and uses metal binding to regulate genes involved in CuII tolerance 42. The structural homology of PII-NG and metal sensors and its cytoplasmic location are consistent with a role in intracellular CoII sensing and modulation of the regulatory cascade needed for cell acclimation to metal stress. Indirect effects of CoII stress on FeII homeostasis. In G. sulfurreducens, the operon encoding the PII-NG regulator (GSU2134) and CzcABC proton/metal antiporter (GSU2135-2137) is also upregulated under FeII limitation via the master regulon of FeII homeostasis, Fur 33. To test if CoII intoxication could indirectly limit the availability of FeII, we used published transcriptomic data for G. sulfurreducens grown with sufficient versus excess FeII 33 to identify genes differentially expressed under FeII intoxication. More than half of the genes responding to CoII stress (24 in total) were also differentially expressed during FeII intoxication (Fig. 2.4C). Most of the genes had opposite patterns of expression, supporting the idea that CoII intoxication limits FeII 36 availability. For example, a cluster of Fur-regulated genes comprised of the PII-NG- czcABC operon (GSU2134-2137) and upstream genes (GSU2129, GSU2131-33) were upregulated by CoII intoxication but downregulated in cells growing with excess FeII. We also identified a protein (GSU1639) with the conserved Rrf2 domain (PF02082) of FeII-dependent transcriptional regulators 43,44 that was downregulated under CoII stress but upregulated during FeII intoxication (Fig. 2.4C). The Rrf2 domain ligates Fe or Fe-S clusters via redox-sensitive cysteine residues to tune the protein’s DNA specificity to FeII homeostasis 45. For example, the Rrf2 domain of E. coli IscR has three cysteines and one glutamic acid that bind Fe-S clusters, changing its DNA recognition to regulate genes involved in Fe-S cluster to FeII availability 44. GSU1639 shares 55% similarity (33% identity) with IscR and has the conserved cysteines and glutamic acid needed for Fe-S cluster coordination at the Rrf2 domain. Furthermore, it is under direct control of Fur, the master regulator of FeII homeostasis 33. This suggests that GSU1639 binds Fe-S clusters to co-regulate the cluster’s biosynthetic pathways to FeII homeostasis. CoII infiltration in Fe-S clusters and/or its high affinity for the cysteines in the Rrf2 binding site could prevent the regulator from sensing the Fe-S cluster signal and impair the ability of the cells to sense FeII availability. CoII but not FeII intoxication upregulated the hydrogen-dependent growth transcriptional repressor HgtR (GSU3364), a master regulator of central metabolism (Fig. 2.4C). HgtR downregulates genes involved in energy generation and biosynthesis such as gtlA (citrate synthase in TCA cycle), atpG (ATP synthase F0 ’ subunit), and nuoA (NADH dehydrogenase I, A subunit) to tune growth rates to the cell’s nutritional status 46. The overexpression of the repressor provides a mechanism to adjust growth to the energy 37 demands of cells coping with CoII intoxication and low FeII availability. FeII limitation may have also triggered the induction of GSU0356, a heme-binding sensor histidine kinase that could regulate the cellular response to the accumulation of metal-free or CoII- impacted heme groups (Table 2.1). The heme sensor lacks a signal peptide but contains three internal helices for insertion in the inner leaflet of the inner membrane, a subcellular localization optimal for cytoplasmic sensing. In addition to phosphoacceptor (HisKinA, PF00512) and ATPase (HATPase, PF02518) domains, GSU0356 has a domain of unknown function (DUF3365, PF11845) with a heme-binding site (CXXCH sequence). Heme-responsive histidine kinases typically bind the heme group reversibly 47. This sensory capacity allows the proteins to prevent the toxic build-up of metal-free hemes 48. The upregulation of the heme sensor during CoII and FeII intoxication (Fig. 2.4C) suggests that both conditions may have resulted in heme toxicity. Evidence for DNA damage. CoII-stressed cells upregulated components of one of the two Type I CRISPR loci (CRISPR2) in G. sulfurreducens (GSU1385 and GSU1387) (Table 2.1). The CRISPR2 locus (GSU1384-1393) contains 8 CRISPR-associated (Cas) proteins and an array with 143 spacers. The locus lacks a Cas4 protein but has Cse1 and Cse2 (named after the CRISPR system of E. coli) components, meeting the criterion for classification as a subtype I-E CRISPR 49. CoII-stressed cells upregulated Cse1 (GSU1385, also known as CasA), the large subunit of the antiviral defense Cas complex (Cascade) that facilitates RNA-guided recognition of complementary DNA 50. Cse1 recognizes a short protospacer adjacent motif (PAM) in the crRNA and discriminates self from foreign DNA targets 51. A Zn-finger motif in Cse1 binds ZnII to control interactions with the target DNA 52. This 38 structural motif is sensitive to infiltration by CoII, a metal that changes the selectivity of the Cas complex for the target DNA and stimulates its nicking activity 53. CoII also upregulated Cse4 (GSU1387, also known as CasC or Cas7), a protein that polymerizes as a hexameric arch along the spacer region of the crRNA within the Cascade complex 54. Cse4 has a ferredoxin-like fold in its RNA recognition motif 50 with a conserved metal- binding βαββαβ fold that could bind CoII 55,56. The final result is a CoII-compromised Cascade complex with increased nicking activity that could lead to DNA damage. CoII can also infiltrate the DNA helix and cause structural changes and breaks in the strands 57. Consistent with the need to repair damaged DNA, cells upregulated a UvrD helicase (GSU0763) of the nucleotide excision repair pathway. UvrD can also bind RNA polymerase (RNAP) stalled on the DNA lesions and backtrack the enzyme to expose the damaged site to DNA repair proteins 58. This mechanism allows the RNAP to resume transcription as soon as the repair has concluded. Downregulation of non-essential metalloproteins. Most of the downregulated genes encoded non-essential proteins with prosthetic groups, metal-binding motifs and amino acids sensitive to CoII inactivation (Table 2.2). Almost all of the targets where cytoplasmic or periplasmic redox-active proteins with FeII-prosthetic groups (e.g., hemes and Fe-S clusters) or proteins with thiol-containing cysteines that CoII readily binds and inactivates (Fig. 2.5). For example, metal-stressed cells downregulated the two subunits (CydAB) of the cytochrome bd complex (GSU1640- 1641), a respiratory quinol:O2 oxidoreductase widespread in prokaryotes that conserves energy from the transfer of electrons from the menaquinone pool to O2 59. Thus, the CydAB complex is not needed under the strictly anaerobic conditions used in our study. 39 Similarly, cells downregulated two subunits (GSU0778-0779) of the trimeric formate dehydrogenase enzyme, FdnGHI, and the associated secretory protein FdnT (GSU0781), which are only needed when growing with formate as electron donor. Another example of a downregulated protein is Prx-2 (GSU3246), a cytoplasmic thioredoxin peroxidase of the 2-cysteine peroxiredoxin subfamily 60. These thiol-based peroxidases scavenge the low levels of H2O2 produced intracellularly during normal growth and transduce the H2O2 signal to control cellular homeostasis 61. When hyperoxidized, however, the enzymes aggregate and become chaperone holdases to protect proteins from denaturation 61. The affinity of CoII for the thiol groups of peroxiredoxins could impair these functions. Thus, cells downregulate its expression to minimize negative impacts of metal inactivation on the cellular stress response. Reductive precipitation of CoII as a detoxification mechanism We identified among the most upregulated genes two cytochrome-encoding genes (GSU0593 and GSU0594) that could participate in the extracellular reduction of CoII. GSU0593 and GSU0594 are the cytochrome b (CbcB) and cytochrome c (CbcA) subunits, respectively, of the Cbc5 menaquinol:ferricytochrome c oxidoreductase, a pentasubunit complex that expressed during the reduction of FeIII oxide minerals 62. The cytochromes (b and c1) and Fe-S proteins in this type of cytochrome bc complexes transfer electrons from the menaquinone pool via a proton motive Q-cycle pathway 63. To complete the Q cycle, Cbc5 catalyzes two “redox turnovers” that consume two protons in the cytoplasm and release four protons in the periplasm. Thus, each Q cycle transfers two electrons and contributes two protons to the transmembrane proton gradient. The Cbc5 complex could electronically connect with extracellular electron acceptors through 40 Geobacter outer membrane porin-cytochrome c complexes (Pcc) 64. Given the known role of Pcc complexes in the reductive precipitation of other divalent cations to their elemental, metallic form 65, we examined untreated and CoII-treated cells by transmission electron microscopy (TEM) for the extracellular formation of metal nanoclusters (Fig. 2.6A). To prevent artifactual mineralization of heavy metal salts used to negatively stain cells for TEM 66, we examined unstained cells. This approach allowed us to visualize numerous electron dense nanoparticles on the surface of CoII-stressed cells that were absent in untreated samples (Fig. 2.6A). The homogenous dispersal of the nanoclusters is consistent with the distribution of outer membrane cytochrome foci in G. sulfurreducens 64. Further, X-ray energy dispersive spectroscopy (EDS) analyses confirmed the presence of Co on the outer surface of the treated cells but not in cell-free controls, consistent with the immobilization of the metal on the cell surface (Fig. 2.6B). Using a colorimetric assay based on the color response of CoII when bound by 2-beta- mercaptoethanol (Fig. 2.6B), we estimated that CoII-stressed cells had removed from the solution an average of 25 M of the metal (Fig. 2.6C). These results suggest that the detoxification response of G. sulfurreducens also included pathways for CoII mineralization, as reported for the uranyl cation 67. 41 Figure 2.6 CoII mineralization at the cell outer surface. (A) Transmission electron microscopy (TEM) images of unstained cells from untreated (0 µM CoCl2) and treated (250 µM CoCl2) cultures. Scale bar, 500 nm. (B) Representative X-ray energy dispersive (EDS) spectra of cells from 250 µM CoCl2 cultures (black) and cell-free control areas examined by TEM identifying Co energy signatures from the cells. The inset shows the average energy intensity (counts) detected for the primary X-ray Co emission peaks (Lα, 0.776 keV; Kα, 6.924 keV) for four different cells (maroon) and control (gray) samples. Pairwise comparisons (t-test) between the cell and cell-free Lα and Kα average intensities produced p values below 0.0001 (***). (C) CoII removal by cells in cultures with or without 250 µM CoCl2 supplementation. The initial and final (early stationary phase) CoII concentrations in culture supernatant fluids were calculated colorimetrically after complexation with 2-beta-mercaptoethanol and using a standard curve (linear fit from 0 to 500 µM CoCl2; R2 = 0.9947). The difference between the final and initial concentration of CoII was the amount of metal removed by the cells. Cell envelope modifications to prevent CoII infiltration and form biofilms The transcriptomics analyses identified lipoproteins (GSU2133 and 3576) and EPS- associated proteins (GSU1079 and GSU1994) that could have modulated the properties of the cell surface to prevent metal infiltration and promote its extracellular immobilization 68. At least one of the lipoproteins was predicted to be targeted to the outer membrane, the other one could only be confirmed as non-cytoplasmic (Table 2.1). Lipid modification of exported hydrophilic proteins facilitates their anchoring to the inner leaflet of the outer membrane yet most, if not all, of the lipoproteins get translocated to the outer leaflet 69,70. Surface-exposed lipoproteins control the permeability of the cell to soluble substrates and can also mediate cell adhesion 69. Additional modifications to the cell envelope are expected from the upregulation of two proteins (GSU1079 and GSU1994) carrying the 42 PEP-CTERM motif (PF07589) of EPS-associated proteins 71. The motif comprises a carboxy-terminal (CTERM) Pro-Glu-Pro (PEP) recognition peptide, a transmembrane helix and an arginine-rich cluster 71. The protein sorting signal is recognized and cleaved by a dedicated exosortase (Xrt) in the inner membrane and the mature protein is then exported to the EPS matrix by yet unknown secretory pathways 71. Except for the presence of the conserved sorting signal, PEP-CTERM proteins have little homology to other proteins. Most are, however, rich in serine and threonine, suggesting they are glycosylated during export 72. The genome of G. sulfurreducens encodes 5 PEP-CTERM proteins, an EpsH family Xrt exosortase (GSU1979) and a two-component system (PrsK histidine kinase, GSU1941; PrsR response regulator, GSU1940) predicted to regulate export 71. The widespread presence of PEP-CTERM proteins in Gram-negative bacteria that form biofilms suggests a role for these proteins in the development of surface-attached communities 71. In support of this, the expression of EPS-associated proteins in CoII- stressed cells of G. sulfurreducens preceded the formation of thick biofilms at the bottom of the tube once the cultures reached stationary-phase (Fig. 2.7A). We did not identify in the transcriptome any of the genes that encode proteins required for the synthesis of the biofilm EPS, Xap 73. This is not unexpected, because G. sulfurreducens expresses the xap genes during exponential growth in acetate-fumarate cultures 73. The Xap EPS anchors outer membrane cytochromes 73 and provides a mechanical and redox barrier to the permeation of soluble divalent metal cations in biofilms 68. To test for a similar protective effect by the EPS produced by planktonic cells, we challenged mid-log phase cultures with up to 1 mM concentrations of CoCl2 and monitored the effect of the metal 43 treatments in growth (OD660). As shown in Fig. 2.7B, metal shock had little effect on growth efficiency in any of the cultures. Such high levels of metal resistance are consistent with the role of the EPS matrix in preventing metal permeation. Furthermore, planktonic cultures challenged with the metal did not form thick biofilms in stationary phase (Fig. 2.7A). Thus, biofilm formation appears to be an adaptive response to persistent metal toxicity. Figure 2.7 Adaptive responses of cells to CoII stress. (A) Biofilm formation in stationary phase cultures growing with 250 µM CoCl2 compared to the lack of biofilms in cultures growing without CoCl2 supplementation (0 µM) or challenged with the CoCl2 in mid-log phase (~0.3 OD660) (250*). Statistically significant differences in pairwise comparisons (t-test) are highlighted with asterisks (p<0.0002 [**] or p<0.0001 [***]). (B) Planktonic growth of cultures challenged with increasing concentrations of CoCl2 (arrow) in mid-log phase (~0.3 OD660), including the ones used for the biofilm assays shown in panel (A) (250*). DISCUSSION The high CoII tolerance and complex acclimation response of G. sulfurreducens is consistent with selection mechanisms during long-term environmental exposure to the metal. FeIII and MnIV oxides form heterogenous mixes with natural organic matter and metal micronutrients 74 that provide optimal conditions for the growth of Geobacter species 13. The high reactivity of the FeIII and MnIV (hydr)oxides sequesters CoII and other 44 metal cations in the mineral phases 11,12, concentrating them in the metal oxide-rich epigenetic zones 17. The reductive dissolution of the metal-bearing minerals mobilizes the metal cations 74 and increases their concentration in the pore-water to toxic levels 40. CuII, for example, can be mobilized to levels (~20 M) above the minimum concentration (10 M) that inhibits the growth of G. sulfurreducens in the laboratory 75. Yet, this bacterium grew from low cell densities, albeit with trade-offs in growth efficiency, in the presence of up to 1 mM CoCl2 (Fig. 2.3). Furthermore, it was relatively unaffected when exposed to the same metal concentrations during the exponential phase of growth (Fig. 2.7). We attributed this to the expression in exponentially-growing cells of the biofilm EPS 73, which can shield the cells from metal infiltration. Cell density can also affect cellular metabolism and the secretion of metabolites that change the chemical speciation, bioavailability, and toxicity of metals 76. Furthermore, increases in cell numbers activate stress responses that acclimate the population and increase tolerance to a number of stressors 77. By contrast, cells inoculated at low cell densities must first reprogram their physiology to acclimate to the presence of the stressor. Acclimation is evident in the extended periods of growth arrest (lag phase) that G. sulfurreducens cultures experienced when growing with sublethal concentrations of CoCl2 (Fig. 2.3) and in the multiple cellular pathways that were activated to couple growth to CoII detoxification (Fig. 2.4). The transcriptomic studies provided insights into the extensive transcriptional reprogramming that allowed G. sulfurreducens to cope with CoII stress (Fig. 2.5). Transcript levels for CoII importers remained constant, consistent with the absence in G. sulfurreducens of transcriptional regulators (e.g., CzrA and CoaR) that other bacteria use to directly control CoII uptake for metal homeostasis 56. Instead, G. sulfurreducens 45 acclimation involved metal (PII-NG) and heme (GSU0356 histidine kinase) sensors and a transcriptional regulator of central metabolism (HgtR) (Fig. 2.5). Cells also upregulated a CzcABC pump for proton-driven export of metal traversing the outer membrane, a canonical mechanism used by other Gram-negative bacteria to increase metal resistance 38. In addition, CoII upregulated a periplasmic glutaredoxin, which repairs and rejoins cysteines oxidized by CoII to refold proteins to their native and functional conformation 78. The activation of a periplasmic MauG-like di-heme cytochrome c peroxidase (GSU1538) suggested that CoII accumulated in the periplasm at levels sufficiently high to generate H2O2 5. Di-heme cytochrome c peroxidases detoxify H2O2 in the periplasm by reducing it to two H2O molecules 34. This reaction receives electrons from a dedicated electron donor such as the monoheme cytochrome GSU2513, which was also upregulated by CoII (Table 2.1). Without the peroxidase-cytochrome pair, H2O2 would oxidize solvent-exposed [4Fe- 4S]2+ clusters in proteins, producing inactive [3Fe-4S]3+ species that abolish the redox activity of the metalloprotein 79. The detoxification of H2O2 is also important to prevent Fenton-like reactions that generate highly toxic •OH radicals and exacerbate oxidative stress 80. Despite mechanisms for periplasmic detoxification, CoII may have infiltrated the cytoplasm and damaged essential macromolecules. The presence of cytoplasmic chelators such as glutathione facilitates reactions between CoII and H2O that generate ROS and oxidatively damage DNA 80. CoII can also bind components of the CRISPR Cascade complex that mediates antiviral defense, changing its specificity for target DNA and stimulating its RNA-independent DNA cleavage activity 53. To cope with DNA damage, G. sulfurreducens activated the expression of UvrD, a helicase of the nucleotide 46 excision repair pathway 81 and transcription-coupled repair 58. The latter is particularly important to maintain the transcriptional activity of the cell during metal intoxication. This is because UvrD associates with NusA to backtrack RNAP when stalled at a DNA lesion. The helicase then recruits the UvrAB repair complex to the damaged site 58. This intervention allows the RNAP to resume transcription as soon the lesion is repaired 81. The Irving-Williams series (MnII ZnII) predicts greater stability for CoII than FeII or MnII complexes independently of the ligand 82. As a result, CoII intoxication preferentially impacts FeII and MnII metalloproteins. To prevent the retention of the toxic metal in the metalloproteome, G. sulfurreducens downregulated non- essential proteins with FeII prosthetic groups (Fig. 2.5). Nearly all of the downregulated proteins contained Fe-S clusters or metallocenters coordinating FeII atoms (Table 2.2). The chemical similarities with FeII facilitate the infiltration of CoII into Fe-S clusters but the greater electron density of CoII alters the coordination of the metal with the enzyme and its activity 56,83. CoII is also able to compete with FeII for binding to the porphyrin ring of heme groups such as those in cytochromes 83. This could be catastrophic in the periplasm, where heme-containing respiratory chains are particularly abundant. CoII- hemes are weaker transporters of charges than the native FeII-hemes 84, impairing, or even abolishing, respiratory growth. To compensate for this, G. sulfurreducens downregulated non-essential heme-containing proteins such as the cytochrome bd oxidase subunits CydAB required for aerobic respiration (Fig. 2.5). Similarly, cells downregulated genes encoding the formate dehydrogenase complex (the Fe-S cluster protein FdnH and the cytochrome b FdnI) and the secretory accessory protein FdnT, as these proteins are only needed for formate-dependent growth. Cells also downregulated 47 an Rrf2 protein (GSU1639), which uses cysteine residues to bind Fe-S clusters and co- regulate Fe-S cluster biosynthesis and FeII homeostasis 44. The high affinity of CoII for cysteines may prevent Rrf2 protein from sensing Fe-S cluster availability in the cytoplasm. To prevent further deregulation of FeII homeostasis, cells downregulated the rrf2 gene. The principles of the Irving-Williams series 82 also explain the high affinity of CoII for FeII-heme. Downregulating non-essential proteins with FeII-hemes can provide some partial relief (Table 2.2). However, CoII can also infiltrate the FeII-hemes during their biosynthesis and prevent their incorporation into proteins. This leads to the accumulation of free CoII-hemes in the cytoplasm and cytotoxicity 85. The upregulation of a heme- containing histidine kinase (GSU0356) (Table 2.1) could provide a mechanism to sense the impact of CoII on the heme pool and coordinate the heme detoxification response, as reported in other bacteria 86. The advantage of this heme-sensing mechanism is that cells can simultaneously co-regulate heme biosynthesis to CoII and FeII homeostasis 48. We initially reasoned that CoII infiltration in the free hemes could have increased the intracellular levels of FeII and exacerbate metal toxicity 85,86. For example, free FeII, like CoII, can generate ROS via Fenton chemistry and cause intracellular damage 87. However, although CoII and FeII intoxication had overlapping transcriptional responses, most of the shared gene targets were reversely regulated (Fig. 2.4C). Thus, cells faced conditions of FeII limitation during CoII intoxication. The accumulation of CoII in the periplasm could competitively exclude FeII from import across the inner membrane, reducing its intracellular availability. Furthermore, once removed from metalloproteins and prosthetic groups, FeII can be sequestered non-specifically by cytoplasmic chelators, effectively reducing its intracellular availability. 48 In addition to mechanisms for metal detoxification in the periplasm and cytoplasm, G. sulfurreducens induced pathways that could have promoted the extracellular immobilization of the metal. For examples, cells upregulated outer membrane lipoproteins that could have modulated the permeability of the outer membrane 18 and/or function as adhesins to promote cell-cell aggregation 69. Additionally, CoII triggered the expression of EPS-associated proteins (PEP-CTERM proteins) typically expressed by biofilm-forming bacteria 71. The synthesis by planktonic cells of G. sulfurreducens of the biofilm EPS (Xap) precedes biofilm formation and allows the cell to anchor to the Xap matrix cytochromes needed for metal reduction 73. This redox activity could allow the planktonic cells to reductively precipitate CoII on the cell surface, generating the metal nanoclusters visualized by TEM (Fig. 2.6A). The mineral particles resolved by TEM formed on discreet foci on the cell surface, similarly to the distribution of outer membrane cytochromes of the Pcc complexes 88. Furthermore, the Pcc outer membrane cytochromes can bind and reductively precipitate divalent metal cations to their elemental form (e.g., HgII to Hg0) 65. A similar reaction could allow the cytochromes to reductively precipitate CoII to Co0 on the cell surface. The Pcc outer membrane cytochrome complexes contain periplasmic and extracellular c-type cytochromes within an outer membrane porin to electronically connect periplasmic carriers to extracellular electron acceptors 64. The upregulation by CoII of a respiratory cytochrome bc complex (Cbc5) could provide a mechanism for energy conservation from the reduction of CoII at the Pcc foci (Fig. 2.5). The Cbc5 complex is anchored to the inner and outer membranes and could interact with the periplasmic cytochrome of the Pcc complex to complete the electron transfer pathway to CoII (Fig. 2.5). We did not detect any of the Pcc genes in the CoII transcriptome but confirmed the 49 upregulation of the Pcc outer membrane c-cytochrome OmcC (GSU2731) when the false discovery rate (FDR) threshold was increased from 0.05 to 0.08. This could indicate that some cells may be upregulating the PccC cytochrome. Experimental testing of this hypothesis is warranted. The expression of lipoprotein adhesins and a redox-active EPS could also have allowed cells to aggregate and form biofilms (Fig. 2.7A), an adaptive response that confers on G. sulfurreducens increased resistance to soluble, toxic metals 68. The downregulation of a cytoplasmic diguanylate cyclase (DGC) with a canonical GGDEF domain (GSU1643) (Table 2.2) in CoII-stressed cells may have reduced the intracellular levels of c-di-GMP in order to regulate the planktonic-to-biofilm transition. Most DGC enzymes contain sensory domains that modulate the synthesis of the bacterial second messenger bis-(3′ ,5′ )-cyclic dimeric guanosine monophosphate (c-di-GMP) to specific input signals, including metals. For example, ZnII reversibly binds the subunits of the E. coli DgcZ dimer (formerly YdeH) to allosterically regulate the synthesis of c-di-GMP 89. The Geobacter DGC enzyme does not have metal-binding domains but has instead the N-terminal phosphoreceiver (REC) domain of DGCs in the PelD superfamily (Table 2.2). The best studied PelD-like DGC is WspR, the response regulator of the Wsp chemosensory pathway, which regulates cell-cell aggregation and biofilm formation in Pseudomonas aeruginosa 90. Phosphorylation of the receiver domain in the WspR dimer activates the synthesis of c-di-GMP and autoaggregative/biofilm phenotypes 91. MgII cations bind near the receiver’s active site of the WspR dimer and contribute to its activity 92. The downregulation in CoII-stressed cells of the DGC enzyme could reflect a feedback mechanism to the infiltration of CoII in the protein 56. Alternatively, CoII-stressed cells may 50 have downregulated the WspR-like DGC to reduce GTP demand for c-di-GMP and increase the availability of the nucleotide triphosphate for EPS synthesis 93. The EPS matrix can then promote cell-cell aggregation and biofilm formation as a protective mechanism against metal toxicity 68. Biofilm formation in G. sulfurreducens embeds the cells in an electroactive biofilm matrix of cytochromes and conductive pili that effectively immobilizes metals outside of the cells 68. The conductive pili are particularly important to overcome metal toxicity because they provide a large redox surface area for the extracellular immobilization and reductive precipitation of toxic metals 67,68. The pilus surface is decorated with specialized motifs optimal for the coordination of divalent metal cations 94. These metal traps have high affinity for CoII and, at high enough potentials, can reductively precipitate it as Co0 nanoparticles 14. Furthermore, the conductive pili are retractable appendages 95, a dynamic feature that allows cells to detach the minerals and recycle the structural peptides in the membrane for a new cycle of pilus polymerization and metal reduction 96. We did not identify in the CoII transcriptome any of the genes encoding proteins of the pilus biosynthetic apparatus (Table 2.1) nor did we observe pilus filaments by TEM (Fig. 2.6). This was not unexpected because we used growth temperatures (30oC) that prevent pilus assembly in planktonic cells 67,97. Under these conditions, cytochrome respiratory chains involving Pcc cytochromes provided the primary pathway for extracellular electron transfer in CoII-stressed cells. Thus, Pcc cytochromes could have promoted the mineralization of CoII on discreet surface foci as a detoxification mechanism (Fig. 2.6). The presence of metal nanoclusters on the surface of CoII-treated cells suggests that hitherto unknown biological reactions could contribute to the geochemical cycling of 51 this important metal. We estimated that, on average, cells removed from the solvent 25 M concentrations of CoII (Fig. 2.6C). As a comparison, the intracellular CoII quota is in the low to sub-μM range and typically below the limits of detection of mass spectrometry assays 3. CoII biomineralization may be more significant in biofilms, thanks to the concentration in the biofilm matrix of conductive pili 68,98 with high affinity motifs for CoII binding and reduction to Co0 14. These adaptive responses confer on Geobacter a competitive advantage for growth in metal-rich environments despite the mobilization of CoII during the reductive dissolution of metal oxide mineral phases. The ability of Geobacter bacteria to reductively precipitate CoII could also alleviate metal stress on syntrophic partners that depend on interspecies cobamide transfer to sustain their metabolism. Furthermore, the formation of Co0 nanoparticles effectively metallizes the cell surface and could allow Geobacter cells to gain energy from the reduction of low potential electron acceptors and to transfer respiratory electrons with syntrophic partners. Hence, CoII mineralization may help define the niche space of Geobacter-driven microbiomes and provide molecular markers to predict the impact of their activities in the fate of this and other essential elements. MATERIALS AND METHODS Genomic reconstruction of pathways for CoII transport and assimilation We performed a literature survey and used the KEGG database and BLAST searches to identify genes in the G. sulfurreducens genome with a predicted role in CoII homeostasis and assimilation into cobamide synthesis. The subcellular localization of the protein products was predicted with PSORTb 3.0 99. 52 Bacterial strains and culture conditions G. sulfurreducens strain PCA was obtained from our laboratory culture collection and routinely maintained in anaerobic mineral medium DB with 20 mM acetate and 40 mM fumarate, as described elsewhere 31. The cultures were incubated at 30oC while periodically monitoring growth as optical density at 660 nm (OD660). Unless otherwise indicated, culture transfers to fresh medium were in mid-log phase (OD660, 0.3-0.4) and diluting the cells to an initial OD660 of 0.03. When indicated, the media was supplemented with CoCl2 from stock anaerobic solutions of 5 and 50 mM CoCl2 prior to cell inoculation. For some experiments, CoCl2 was added to mid-log phase cultures (~0.3 OD660) incubated at 30oC. All growth experiments were performed in triplicate cultures. Cultures that did not initiate growth for 7 days were discarded (routinely one out of three replicates grown from low cell densities with 500 and 1,000 M CoCl2, as shown in Fig. 2.3). Growth curves (OD660) for each of the replicate cultures were analyzed to calculate the length of the lag phase (time before start of exponential growth), generation (doubling) time in exponential phase, and biomass yields (OD660 reached once the cultures entered stationary phase). The latter was expressed as relative growth yield in the treated (with 100-1,000 M CoCl2) versus the untreated (0 M CoCl2) cultures. RNA Extraction and sequencing (RNAseq) Cells were grown to mid-log phase (~0.3 OD660) in the presence or absence of sublethal concentrations of CoCl2 (250 M) before adding 1 ml of water saturated phenol (5% [v/v] Ambion® water saturated phenol, pH 6.6 in ethanol) to stop transcription. We harvested the cells by centrifugation (3,800 rpm, 8 min, 4oC) extracted their RNA with the QIAGEN RNeasy kit (QIAGEN) following manufacturer’s recommendations. DNA digestion in the 53 RNA samples used the QIAGEN RNase-free DNase Set and was confirmed by Reverse Transcriptase (RT)-PCR (Verso 1-step kit, Ambion). After assessing RNA integrity in a BioAnalyzer 2100 (Agilent), we selected the samples with the best RNA quality (two biological replicates from each treatment, 0 or 250 M) for RNA sequencing at Michigan State’s Research Technology Support Facility (RTSF, Genomic core). The facility uses validated procedures for rRNA depletion, library preparation, and Illumina Hi-Seq 4000 sequencing. Briefly, rRNA depletion used an Illumina TruSeq Total RNA Library Preparation kit with QIAseq FastSelect – 5S/16S/23S rRNA depletion (QIAGEN). Libraries were quantified using Qubit and Advanced Analytical Fragment Analyzer High Sensitivity DNA NGS assays. The libraries were then pooled in equimolar amounts for multiplexed sequencing and the pool was quantified using the Kapa Biosystems Illumina Library Quantification qPCR kit. Sequencing was in one lane of the Illumina HiSeq 4000 flow cell in 1x50bp single read format and using SBS reagents. Base calling was with the Illumina Real Time Analysis (RTA) v2.7.7 software while demultiplexing and conversion to FastQ format was with Illumina Bcl2fastq v2.19.1 package. We analyzed the RNAseq data from the CoII-treated and untreated samples with the SPARTA pipeline 100, using FastQC and Trimmomatic tools for quality control and trimming and used Bowtie to align the sequences to the reference genome (GCA_000007985.2 Geobacter sulfurreducens PCA). Gene-level transcript level abundance was calculated with the HTSeq software while the edgeR tool provided the differential expression values. Data filtering used a false discovery rate FDR< 0.05, log CPM>5, and a log2 FC<-1 (downregulated genes) or > 1 (upregulated genes) 100. We used the R software (www.r-project.org) with pheatmap function to draw clustered 54 heatmaps of differentially expressed genes. Individual searches in BioCyc 24.0 101 predicted the operon organization of the genes and identified one gene in an RND efflux pump operon (GSU2137) that did not make the maximum FDR value yet met the log CPM and fold-change thresholds. We added this gene to Table 2.1. We also searched each of the differentially expressed genes in the UniProtKB 102 and KEGG databases to assign functional roles. The subcellular localization was predicted using the sequence analysis tools at UniProtKB (SignalP), PSORTb 3.0 99 and CELLO v.2.5 103. Predictions about the domain organization of each protein and identification of metal-binding motifs used the Pfam 33.1 104 tool available at the UniProtKB database. The RNAseq data (Supplementary file 1) has been deposited in the Gene Expression Omnibus (GEO) functional genomics data repository (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE157146. We also identified in the RNAseq data genes differentially regulated in G. sulfurreducens under FeII intoxication, which were calculated as the ratio between Fe excess and homeostatic transcript abundance reported elsewhere 33. The transcriptomic data comparisons used to generate a heatmap of CoII versus FeII intoxication (Fig. 2.4C) are provided as a Supplementary file 2. Transmission Electron Microscopy (TEM) and X-ray Energy-Dispersive Spectroscopy (EDS) Mid-log phase cells from untreated or treated (0 or 250 M CoCl2 supplementation, respectively) cultures were fixed with 2.5% glutaraldehyde before deposition for 5 min on Formvar-coated grids (150 square-mesh Ni, Electron Microscopy Sciences). After three washes with ddH2O (30 sec each), we side-blotted the excess liquid and stored the samples at room temperature until ready for TEM examination. The cells were unstained 55 to prevent stain and mineral artifacts during examination with a JEOL 1400 Flash 120kV transmission electron microscope. For EDS elemental analyses, we deposited CoII- stressed cells on a PELCO NetMesh™ copper grid coated with a lacey formvar/carbon support film and examined the samples with a JEM-2200FS ultra-high resolution TEM instrument equipped with an EDS detector. To minimize interference during EDS detection, we collected energy spectra from cells exposed in the holes of the support film and, as controls, from similar areas within the grid that had no cells. We used the two primary X-ray signatures of Co around 0.776 keV (Lα peak) and 6.924 keV (Kα peak) to compare the average intensity of the cell-associated CoII to the cell-free controls. Because the detection limit of the EDS system is 0.001 keV, we averaged the counts detected at 0.76, 0.77, and 0.78 keV to calculate the intensity of the Lα peak and of 6.91, 6.92, and 6.93 keV for the Kα peak. The peak intensities from cells and cell-free samples on the grid were compared with the unpaired, unequal variance t-test function of the Excel software. Colorimetric detection of CoII We developed a colorimetric assay for the detection of CoII in culture supernatant fluids based on the color response of the metal after complexation with 2--mercaptoethanol (BME). The reducing agent, BME, replaces the water molecules in the cobalt hexaaqua complex (Co[H2O]6)2+, turning the solution brown and permitting the spectrophotometric detection of CoII at 475 nm. Prior to the assay, we grew G. sulfurreducens in DBAF medium at 30oC with or without 250 M CoCl2 supplementation, collected 200-l samples periodically, and recovered the culture supernatant fluids after centrifugation at 20,000 rcf for 3 min. To initiate the complexation reaction, we added 10 l of BME (from a 100 mM 56 aqueous stock) to 190 l of supernatant sample. After mixing the solution by aspiration with a pipette, we incubated the reactions at 30ºC for 20 min to reach the maximum color response and measured the absorbance at 475 nm against CoCl2 standards (0 to 500 M CoCl2) prepared in DBAF medium. Biofilm assay Biofilm formation in stationary phase cultures was measured with a crystal violet assay 105. Briefly, we poured out the liquid culture once the cells reached stationary phase, gently rinsed the tubes with ddH2O and added 1 ml of 0.1% crystal violet to stain for 15 min the biomass of biofilms formed at the bottom of the tube. After 15 min, we poured out the crystal violet solution, rinsed the tubes with ddH2O and left them to dry overnight. We used 1 ml of 30% acetic acid to solubilize the biomass-associated crystal violet for 15 min and measured its absorbance at 550 nm to estimate the biofilm biomass. ACKNOWLEDGEMENTS This work was supported by Grant EAR1629439 from the National Science Foundation and Hatch project 1011745 from the USDA National Institute of Food and Agriculture to GR. 57 Table 2.1 Upregulated genes in cobalt transcriptome TABLES Function (no. of genes) Transmembran e transport (3) Electron transfer (4) Locus Gene Gene product GSU2135 czcA CusA/CzcA heavy metal GSU2136 GSU2137 GSU0593 efflux RND transporter czcB Efflux RND transporter, periplasmic adaptor subunit czcC Outer membrane pore/TolC family protein cbcB Cytochrome b, putative GSU0594 cbcA Cytochrome c, heptaheme GSU1538 Cytochrome c peroxidase GSU2513 Cytochrome c, monoheme Metal-binding motif (Pfam) log(2) FC 2.47 1.75 1.69 3.70 Prokaryotic cytochrome b561 (PF01292) 3.68 Doubled CXXCH motif (PF09699) 2.19 Di-haem cytochrome c peroxidase (PF03150) 1.85 Cytochrome c oxidase, cbb3-type (PF13442) Subcellular localization Inner membrane Periplasm Outer membrane Inner membrane Periplasm (membrane- bound) Periplasm Periplasm Type-I CRISPR-Cas system (2) Cell redox homeostasis (1) Cell envelope (4) Signal transduction (3) DNA repair (1) Transposon functions (1) Unknown function (13) GSU1385 cse1 CRISPR processing 1.56 CRISPR_Cse1 Cytoplasm complex protein CasA (PF09481) GSU1387 cse4 CRISPR processing GSU2812 GSU1079 GSU1994 GSU2133 GSU3576 GSU0356 GSU2134 GSU3364 GSU0763 GSU2772 GSU0468 GSU0919 GSU0959 GSU2129 GSU2131 GSU2132 GSU2773 GSU3410 GSU3489 GSU3502 GSU3503 GSU3520 GSU3559 complex protein CasC glutaredoxin family protein PEP motif-containing protein, putative exosortase substrate PEP motif-containing protein, putative exosortase substrate Lipoprotein Lipoprotein, putative Sensor histidine kinase, heme-binding P-II family nitrogen regulator hgtR Hydrogen-dependent growth transcriptional repressor Helicase, putative Transposase of ISGsu3, IS5 family Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein/ATP- dependent Clp protease proteolytic subunit Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein 1.69 1.44 1.32 3.37 3.07 2.06 1.27 Heme-binding (PF11845) 2.93 1.49 1.82 1.46 2.30 3.67 1.61 3.49 3.05 2.99 2.74 3.48 3.35 3.64 3.81 3.11 3.49 Cytoplasm Periplasm Extracellular Extracellular Non-cytoplasmic Outer membrane Inner membrane Cytoplasm Cytoplasm Cytoplasm Inner membrane Unknown Cytoplasm Non-cytoplasmic Non-cytoplasmic Unknown Unknown Inner membrane Inner membrane Inner membrane Cytoplasm Inner membrane Non-cytoplasmic 58 Table 2.2 Downregulated genes in CoII transcriptome Function (no. of genes) Folding, secretion, and degradation (1) Carbohydrate metabolism (3) Locus Gene Gene product GSU0781 fdnT Twin-arginine translocation pathway protein, TatA/TatE family GSU0778 fdnH Periplasmically oriented, GSU0779 fdnI membrane-bound formate dehydrogenase, iron-sulfur cluster-binding subunit Periplasmically oriented, membrane-bound formate dehydrogenase, b-type cytochrome subunit, putative log(2) FC -1.95 Metal-binding motif (Pfam) Subcellular localization Inner membrane -2.23 Two [4Fe-4S]-binding (PF13247, PF12800) Inner membrane -2.02 NrfD, polysulphide Inner membrane reductase (PF03916) GSU3125 mtd mannitol dehydrogenase -2.90 ZnII-binding Cytoplasm Energy metabolism (2) GSU1640 cydA cytochrome bd menaquinol oxidase, subunit I GSU1641 cydB cytochrome bd menaquinol oxidase, subunit II GSU3126 oxidoreductase, aldo/keto reductase family Cell redox homeostasis (2) GSU3246 prx-2 Hydrolases (2) GSU1159 GSU3122 peroxiredoxin, typical 2-Cys subfamily Intracellular protease, PfpI family, putative/type 1 glutamine amidotransferase Metal-dependent hydrolase, beta-lactamase superfamily dehydrogenase (PF00107) -3.13 Cytochrome bd terminal oxidase subunit I (PF01654) cytochrome bd terminal oxidase subunit II (PF02322) -3.19 -3.08 -2.63 -2.03 -2.14 Inner membrane Inner membrane Cytoplasm Cytoplasm Cytoplasm Cytoplasm Signal transduction (2) DNA binding, replication, repair (1) Unknown function (2) GSU1639 rrf2 Winged helix-turn-helix -3.45 FeIIdependent Cytoplasm transcriptional regulator (PF02082) GSU1643 GSU3245 GSU0208 GSU1160 transcriptional regulator, Rrf2 family Response receiver- modulated diguanylate cyclase DNA polymerase II, putative Hypothetical Protein/DUF4350 domain- containing protein Hypothetical protein -1.99 -2.69 -2.97 -2.14 Cytoplasm Cytoplasm Inner membrane Non-cytoplasmic 59 1 2 3 4 5 6 7 8 9 10 11 12 REFERENCES Buccella, D., Lim, M. 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Transcript abundance and protein expression profiles in CoII-stressed cells confirmed the higher expression of not only CbcBA but also several periplasmic and outer membrane cytochromes involved in extracellular electron transfer. Consistent with increased sensitivity to CoII, a mutant carrying a deletion of the cbcBA genes (ΔcbcBA) agglutinated more than the wild type (WT) cells and a genetically complemented strain when challenged with non-lethal concentrations of CoCl2 in mid-exponential phase. However, given sufficient time to acclimate to non-lethal concentrations of the metal, the mutant cells overcame CoII intoxication and grew similarly to the WT cultures. . Furthermore, CoII penetrated more rapidly in the mutant cells, exacerbating intoxication and slowing down the growth recovery of the cells. Additionally, the CbcBA deficiency exacerbated the release of outer membrane vesicles under CoII stress, a compensatory effect associated with the detoxification of toxic metals bound to the outer membrane lipopolysaccharide and increased membrane permeability. These findings demonstrate that the CbcBA cytochromes are needed for optimal detoxification of CoII and highlight the important role of complementary pathways in CoII detoxification. 70 INTRODUCTION Metal respiration supports the growth of many microorganisms in redox environments where O2, oxidized N-species and other electron acceptors have been depleted 1,2. This metabolic process produces large scale transformations to the local environment and is pivotal in the biogeochemical cycling of elements essential for life 2,3. The reductive dissolution of the abundant insoluble iron (FeIII) oxides by dissimilatory metal reducing (DMR) bacteria, such as those in the genus Geobacter, cycles metals essential to ecosystem function 1. Indeed, these bacteria solubilize Fe and Mn from the abundant metal oxide minerals as species (FeII and MnII) that cells can assimilate for the synthesis of heme and metalloenzymes 1,4. Studies with the model DMR bacterium Geobacter sulfurreducens have identified several pathways for extracellular electron transport to metal oxides. Respiratory electrons from the quinone pool reduce membrane-bound quinol oxidases (ImcH, CbcL and CbcBA), which then transfer the electrons to the abundant periplasmic cytochromes (Ppc) of Geobacter species 5. Several porin cytochrome complexes (Pcc) across the outer membrane then transfer electrons from the Ppc to the extracellular electron acceptors 6. To expand the redox-active surface of the cell, Geobacter species also assemble electrically conductive pili monolaterally 7. The pili electronically connect the periplasmic cytochromes to soluble and insoluble metal acceptors and to other cells in biofilms 1,8,9. The dissolution of FeIII and MnIV oxides by DMR bacteria also releases metals that coprecipitate with and/or adsorb to the mineral phases 10. Micronutrients such as cobalt (CoIII or CoII species) are solubilized from the minerals and made bioavailable in this manner 11. Through their role in the Co cycle, DMR bacteria supply a metal that is 71 essential for the synthesis of the catalytic center of the cobamide-class of vitamins 12,13. Geobacter species are in fact among the few known bacteria with genomes encoding complete pathways for cobamide synthesis, which they secrete to support the activities of syntrophic partners 13,14. To continue to secrete cobamides while respiring FeIII oxides, Geobacter cells must overcome the toxic effects of Co on cellular metabolism, the formation of free radicals, and its infiltration into the active sites of metalloenzymes 15,16. As I described in Chapter 2, a complex detoxification response that includes the expression of RND efflux pumps, protein repair pathways, hydrogen peroxide detoxification, and membrane remodeling allows Geobacter cells to grow in the presence of otherwise toxic concentrations of CoII 12. Metal-stressed cells of G. sulfurreducens also upregulate the genes encoding the inner membrane cytochrome complex CbcBA 12. The two cbc genes are part of a cluster of cytochromes and redox proteins proposed to assemble as a large protein complex (Cbc5) anchored in the inner membrane and spanning the periplasmic space 17. The predicted organization of the Cbc5 complex in the cell envelope could facilitate the transfer of electrons from the menaquinone pool in the inner membrane to outer membrane Pcc, which serve as electron donors to various extracellular electron acceptors 6,18,19. Hence, the Cbc complex could provide a pathway for the extracellular reduction of CoII, explaining the accumulation of Co nanoparticles on the outer surface of G. sulfurreducens cells grown under CoII stress 12. In support of this, the cbcBA genes are required for the reduction of extracellular electron acceptors with potentials (-0.21 to -0.28 V vs Standard Hydrogen Electrode or SHE) 5 as low as the standard redox potential (E0) of the half reaction Co2+ + 2e– → Co0 (E0 = –0.277 V vs SHE). 72 The upregulation of the low-voltage CbcBA pathway in metal-stressed cells and the deposition of Co-containing nanoparticles on their surface12 led us to hypothesize that cells may express CbcBA to detoxify CoII by reductively mineralizing it to Co0 nanoparticles outside of the cell 12. To test this hypothesis, I reconstructed the CbcBA cytochrome pathway for the extracellular transfer of electrons to CoII from the transcriptomic data (RNAseq) of CoII-stressed cells of G. sulfurreducens 12 and investigated the CoII detoxification response of a ΔcbcBA strain and a genetically complemented strain. The results support the involvement of the low-voltage CbcBA reductive pathway in the Co detoxification response of G. sulfurreducens. However, the extent of its contribution may be masked by compensatory effects of complementary pathways involved in CoII detoxification. These findings further define the mechanisms that allow G. sulffureducens to thrive in Co-contaminated environments and the role that cytochrome respiratory chains play in metal detoxification. RESULTS Transcriptional upregulation of cytochrome respiratory chains under CoII stress Previous transcriptomic analysis (RNAseq) of mid-exponential phase cultures (DBAF medium) of G. sulfurreducens identified cbcBA among the most differentially upregulated genes by non-lethal concentrations of CoII (provided as 250 µM CoCl2) 12. The two cbc genes encode two cytochromes of a large periplasmic protein complex (Cbc5) anchored in the inner membrane 17 that could transfer electrons from the menaquinone pool in the inner membrane to outer membrane Pcc, and from them to CoII (Fig. 3.1A). Consistent with this model, transcripts for cytochromes (OmcB and OmcC) exposed on the outer membrane by the Pcc conduits were among the most abundant in CoII-stressed cells (Fig. 73 3.1B). Together, the Cbc5 and Pcc complexes could enable the reductive precipitation of CoII as a cellular detoxification mechanism 12. Figure 3.1 Cell envelope pathways for CoII Detoxification. (A) Illustration of cell envelope proteins predicted to detoxify CoII via metal export, modulation of membrane composition, and reductive mineralization on the cell surface. (B-D) Transcript levels (normalized to the recA gene; n = 2) of genes encoding cell envelope c-type cytochromes (B), components of the Type IV pilus apparatus (C), and efflux pumps and outer membrane proteins differentially upregulated under CoII stress (D). Shown are expression levels in the transcriptomes sequenced from mid-exponential phase DBAF cultures incubated at 30oC without CoII supplementation (-CoII, in black) or with 74 Figure 3.1 (cont’d) 250 µM CoCl2 (+CoII, in pink). Statistical differences (FDR < 0.05) are marked with an asterisk. Abbreviations: CbcAB: b- and c- type cytochrome complex; CbcL: low-potential cytochrome with a b- and c- type domain; CzcABC: CoII, ZnII, CdII RND-efflux pump; ImcH: high-potential inner membrane cytochrome; LP: membrane-bound lipoprotein; NADH1: NADH dehydrogenase; OmaB/C: periplasmic-facing cytochrome of the PccB or PccC conduits; OmbB/C: porins of the PccB or PccC conduits; OmcB/C: extracellular outer membrane cytochromes of the PccB or PccC conduits; PEP-C: C-terminal Pro-Glu-Pro recognition peptide (PEP-CTERM); PilABCMNOPQT: structural proteins of the Type IV pilus apparatus; PpcA/B/D/E: periplasmic cytochromes. Data previously published 20. The transcriptomic data also suggests that the expression of a cytochrome pathway for extracellular electron transfer to CoII is redox-controlled. The expression of cbcBA in cultures grown with low potential electron acceptors such as crystalline forms of FeIII oxides and electrodes poised at a sufficiently low reduction potential is controlled by the transcriptional regulator BccR 5. The bccR gene was also expressed during CoII treatment (Fig. 3.1B), albeit at much lower levels than the cbcBA genes. By contrast, quinol oxidases required for the reduction of electron acceptors with medium (CbcL, – 0.15 to –0.2 V vs SHE) or high (ImcH, above –0.15 V vs SHE) half potentials, had a lower relative transcript abundance in CoII-stressed cells (Fig. 3.1B). This is consistent with the redox-controlled regulation of quinol oxidases by BccR 5. Like CbcBA, the relative transcript abundance for periplasmic cytochromes (PpcA, B, D and E), which receive electrons from the quinol oxidases 21, was high in CoII-treated cultures (Fig. 3.1B). However, the genes the PpcA-E cytochromes were not differentially expressed under CoII stress compared to untreated cultures. This result is in agreement with previous studies reporting high but constitutive levels of expression of these cytochromes independent of the electron acceptor 22. Transcript levels were also high for the exposed cytochromes (OmcB and OmcC) of the Pcc conduits that transport electrons across the outer membrane (Fig. 3.1A). G. sulfurreducens upregulates these cytochromes during growth 75 with extracellular metals such as FeIII 18,23 and in stationary-phase via the sigma factor RpoS 24. As shown in Fig. 3.1B, transcript levels for omcC were particularly high though highly variable in the CoII-treated cultures (20 ± 13 relative to recA, compared to 5.2 ± 0.7 in the untreated controls). The high expression of genes encoding CbcBA, periplasmic cytochromes and outer membrane Pcc conduits support the notion that G. sulfurreducens upregulates a cytochrome pathway for the transport of respiratory electrons from the menaquinone pool in the inner membrane to the extracellular CoII. G. sulfurreducens can also assemble conductive pili of the Type IVa class to reduce extracellular electron acceptors such as FeIII oxide minerals 7 and the soluble uranyl cation 25. The anionic metal traps that decorate the pilus surface 8 can also bind and reductively precipitate CoII as Co0 nanoparticles 26,27. Any contribution by pili to the reductive detoxification of CoII in the metal-stressed cultures is unlikely because the incubation temperature (30oC) used for these studies prevented pilus assembly 7,25. Further supporting this, none of the structural genes of the Geobacter pilus apparatus were differentially upregulated by CoII (Fig. 3.1C). Rather, CoII stress significantly upregulated pathways for metal export (Czc metal pump) and surface chemistry modification (lipoproteins and exopolysaccharide (EPS)-associated PEP-CTERM proteins) (Fig. 3.1D). While lipoproteins and EPS-associated proteins may modulate the permeability of the outer membrane in ways that could prevent the permeation of the metal, the CzcCBA pump extrudes any CoII traversing the outer membrane 28,29. The transcriptional upregulation of the genes encoding CzcCBA in G. sulfurreducens 12 emphasizes the essential role that RND-efflux pumps play in metal detoxification in bacteria 30. CzcA, one of the three proteins of the CzcCBA pump, confers substrate 76 specificity and functions as a proton-cation antiporter to translocate the metal across the cell envelope 31. CzcA transcripts were also among the most significantly upregulated genes in the CoII transcriptome (Fig. 3.1D). CzcB, on the other hand, is not essential for metal translocation but can enhance substrate binding by CzcA by forming a complete pump-channel complex 31. In support of this, relative transcript abundances of czcB were only slightly elevated in CoII-treated cultures and did not reach statistical significance (0.63 ± 0.44 relative to recA compared to 0.19 ± 0.06 in untreated controls, p = 0.4). Role of CbcBA in CoII detoxification during exponential growth CoII detoxification is critical during the exponential phase of growth, when cells must invest most of their energy gains into growth-supporting processes and cell division. We thus hypothesized that the CbcBA deficiency would exacerbate growth defects in cells challenged with CoII. To test this, I grew the wild-type (WT) and a CbcBA-deficient (ΔcbcBA) mutant strain in freshwater medium (FW) with acetate and fumarate (FWAF) and challenged the cells with non-lethal concentrations of CoII (200 µM CoCl2) during the exponential phase of growth. For these experiments, cell growth (absorbance at 660 nm, A660) was monitored before and after CoCl2 addition and in reference to untreated controls (Fig. 3.2A). A genetically complemented strain, ΔcbcBA::cbcBA, was also included as a control. WT cells initially grew like the untreated cultures after the metal challenge but agglutinated and settled as a biofilm at the bottom of the tube (0.169 ± 0.060 A660), a phenotype associated with metal intoxication 12. Agglutination is a protective mechanism against metal stress 32,33 that G. sulfurreducens triggers through changes in the cell surface chemistry 12, such as those resulting from the production of the redox-active Xap exopolysaccharide of G. sulfurreducens 34. The agglutination response was exacerbated 77 in the ΔcbcBA mutant cultures (0.302 ± 0.029 A660, p = 0.005 vs the WT cells) (Fig. 3.2B). Complementation of the cbcBA deletion in the ΔcbcBA::cbcBA strain partially rescued the agglutination phenotype of the mutant (0.236 ± 0.035 A660, p = 0.06 vs the WT cells). By contrast, the untreated culture controls had no measurable agglutination (Fig. 3.2A). The agglutination phenotypes revealed in these experiments are thus consistent with the need of cells to express CbcBA to overcome CoII intoxication. 78 Figure 3.2 Role of CbcBA in CoII detoxification by exponentially-growing cells. (A) Growth (A660) of WT (black), ΔcbcBA mutant (white), and the genetically complemented ΔcbcBA::cbcBA (gray) strains in FWAF medium with (circles) or without (lines) CoII addition (200 µM CoCl2, arrow) in mid-exponential phase (A660 = 0.3-0.4). (B) Agglutination (A660) of CoII treated cultures, measured as the A660 difference between the undisturbed and gently resuspended cultures at the end of the incubation period. Symbols show agglutination for each replicate culture and the horizontal lines, the average (n = 5). Significant differences were calculated using a student’s T-test and are shown as p values. Any outliers identified using Grubb’s test are indicated as an ‘X’ and were excluded from statistical analyses. (C) Growth (A660) of viable cells recovered from the exponential CoII treatment in panel (A). (D) Length of the lag phase (estimated as the amount of time before cultures exceeded A660 = 0.05) and (E) the generation times of the recovering cultures. 79 Further supporting a higher level of CoII intoxication in the CbcBA-deficient cells, the ΔcbcBA mutant cultures challenged with the CoCl2 (Fig. 3.2A) did not readily resume growth once transferred to fresh medium without the metal (Fig. 3.2C). Metal toxicity requires repair of cellular damage and can delay, if not impair, the ability of metal-stressed cells to resume growth in standard culture media 25. This phenotype manifests as an extended lag phase prior to growth initiation that is proportional to the initial degree of metal intoxication of the cells 25. Consistent with this, the ΔcbcBA cultures had extended lag phases compared to the WT and ΔcbcBA::cbcBA cultures (Fig. 3.2D). Variability in the length of the lag phase was however high for the mutant cultures (18.7 ± 8.7 h), which reduced the statistical significance of the data compared to the WT (11.3 ± 2.9 h, p = 0.2) and genetically complemented (9.2 ± 3.4 h, p = 0.2) strain (Fig. 3.2D). Phenotypic variability is not uncommon in metal-stressed cultures, where cells stochastically respond to toxicity via multiple pathways to extrude the metal, repair damage, and coordinate protective responses that minimize metal permeation 12. Hence, the higher variability observed during the growth recovery of metal-stressed ΔcbcBA cells (Fig. 3.2A) is also consistent with a higher degree of intoxication resulting from the past exposure to the CoII (Fig. 3.2C). However, once exponential growth was initiated, the mutant cells grew at rates (generation or doubling times, Fig. 3.2E) similar to the WT and the genetically complemented cells. This is because CoII stress also triggers in G. sulfurreducens several pathways for the repair of metal damage 12. Furthermore, the true contribution of the cytochrome pathway to metal detoxification may be masked by the many additional pathways (metal stress, repair, cell surface chemistry changes, etc.) that cells can activate to overcome CoII stress. 80 The CbcBA respiratory chain is dispensable for growth after acclimation to CoII stress The metal challenge to mid-exponential grown cells and post-recovery experiments described above (Fig. 3.2) supported a role for CbcBA in metal detoxification and highlighted the important role that agglutination plays in rapidly protecting cells from CoII intoxication. Acclimation to metal stress is however possible when starting G. sulfurreducens cultures at low cell densities in the presence of sub-lethal concentrations (200 µM) of CoCl2 12. After a variable lag phase to activate pathways for metal detoxification, cells initiate exponential growth 12. To test if the activation of other detoxification pathways could compensate for the CbcBA deficiency, we investigated the effect of CoII in the acclimation of the WT and the ΔcbcBA mutant cells to metal stress. For this experiment, untreated cultures were transferred in mid-exponential phase to FWAF at low cell densities (starting A660 ~0.05) before adding CoII (200 µM CoCl2) and growth was monitored spectrophotometrically in reference to the untreated controls. As previously reported 12, the WT strain underwent a phase of acclimation characterized by an extended lag phase (54.3 ± 6.0 h with CoII compared to 5.60 ± 0.02 h without the metal, p = 0.005) (Fig. 3.3A). Additionally, CoII stress slowed down exponential growth in the WT cultures (8.9 ± 6.3 h with CoII vs 4.03 ± 0.13 h without the metal, p = 0.3) (Fig. 3.3A, inset) as energy is diverted towards metal extrusion, repair and other detoxification pathways. Cultures of the ΔcbcBA strain supplemented with CoCl2 also had an extended lag phase (57.8 ± 9.8 h compared to 5.37 ± 0.71 h without CoII, p = 0.01) and grew more slowly than the untreated cultures after acclimation (Fig. 3.3A). However, generation times after acclimation were similar in the WT and mutant strains (Fig. 3.2A, inset) as 81 were growth yields (0.37 ± 0.10 WT vs 0.35 ± 0.13 ΔcbcBA; p = 0.8). Additionally, the CoII-stressed WT and mutant cultures agglutinated once they reached stationary phase (0.19 ± 0.10 A660 and 0.14 ± 0.10 A660 in the WT and ΔcbcBA cultures, respectively; p = 0.5). Hence, given sufficient time to acclimate, cells can overcome CoII intoxication in a CbcBA-independent manner. Figure 3.3 Growth from low cell densities with non-lethal CoII concentrations. (A) Growth (A660) of the WT (black) and ΔcbcBA (white) in FWAF cultures in the absence (lines) or presence (solid symbols) of 200 µM CoCl2. Data show the average and standard errors for duplicate (untreated) and triplicate (treated) cultures for each strain. Inset shows the individual data points (circle) and average (line) generation times (G) in the untreated (-Co) and treated (+Co) cultures for both strains. (B) Growth (A660) recovery in metal-free FWAF medium of cultures from (A). The average generation time (G) in the post-recovery cultures is shown in the inset. As in previous assays (Fig. 3.2), we also measured the degree of metal intoxication in the CoII-stressed cells by recovering them in metal-free medium (Fig. 3.3B). Both strains resumed growth after a similar lag phase (12.6 ± 1.4 h WT vs 13.4 ± 3.0 h ΔcbcBA, p = 0.7). Generation times were also comparable in both strains (5.02 ± 0.39 h and 4.25 82 ± 0.17 h in the WT and mutant strains, respectively; p = 0.06). These results support our earlier conclusion that cells do not absolutely require the CbcBA pathway to acclimate to and grow in the presence of CoII. Contribution of CbcBA to CoII immobilization We also investigated a role for CbcBA in preventing the permeation of CoII inside the cells in assays developed to study the enzymatic immobilization and reduction of metals by resting (non-growing) cells of G. sulfurreducens 25. Resting cell suspensions of the WT and ΔcbcBA strains were prepared in a reaction buffer that preserved cell viability for at least 6 h 25. Appendix 1 describes the optimization of this buffer and the importance of identifying buffer formulations that provide sufficient osmoprotection to cells maintained in the resting state for several hours. Unable to actively extrude the metal, resting cells are more vulnerable to CoII permeation and more readily intoxicated, which slows down their recovery once grown in fresh medium without the metal 25. Recovery of cells from assays performed at 30oC as previously described 25 were highly variable among the triplicates for each strain, preventing comparisons. However, lowering the temperature of the resting cell assay to 25oC reduced some of the variability among the replicates and unmasked the linearity of CoII removal during the first 1 h of incubation (R2 = 0.99 and 0.94 for the WT and ΔcbcBA, respectively) (Fig. 3.4A). From the linearity of the reaction, we calculated WT rates of CoII removal of 29 µM/h and much higher (49 µM/h) for the ΔcbcBA mutant, consistent with increased permeability of CoII in the mutant cells. Despite the more rapid kinetics of removal by the resting mutant cells during the first hour of exposure to CoII, yields of metal removed after 6 h (41.4 ± 5.7 µM) were not significantly different than in the WT (30 ± 19 µM CoII,, p = 0.4) (Fig. 3.4A). This is consistent with 83 previous growth studies that found that WT and ΔcbcBA cultures removed comparable amounts of CoII (24.6 ± 0.57 µM and 27.6 ± 2.8 µM CoII, respectively) when allowed to acclimate to the metal. Figure 3.4 Role of CbcBA in CoII removal by resting cells (A) and growth recovery in metal-free medium (B-C). (A) Amount (µM) of CoII removed by resting cells of the WT (solid line) and ΔcbcBA (dashed line) cells over 6 hours. The resting cells were suspended in an osmotically balanced buffer with 200 µM CoCl2 at 25oC. Shown are the average removal by triplicate WT (solid line) and duplicate ΔcbcBA (dashed line) resting cell suspensions and the standard error (shaded areas). (B-C) Growth (A660) recovery in metal-free FWAF medium of WT (B) and ΔcbcBA (C) resting cells exposed (pink) or not exposed (black) to CoII. Lines are the average of triplicate cultures, except for the CoII- treated WT cultures, which were tested in duplicates. Shaded areas represent the standard deviation. The deletion of cytochromes required for the extracellular immobilization of metals in G. sulfurreducens often increases metal removal rates, because more metal penetrates inside the cell envelope 35. As a result, mutant cells are more intoxicated and recovered from the resting state in metal-free growth medium more slowly 25. Consistent with this, the ΔcbcBA cells recovered, on average, more slowly than the WT cells once transferred 84 to metal-free growth medium (Fig. 3.4B-C). The WT resting cells, for example, recovered from the CoII treatment after a lag phase (23.1 ± 2.1 h) nearly identical to that of untreated controls (23.0 ± 1.4 h, p = 0.96) (Fig. 3.4B). By contrast, the resting mutant cells recovered required a longer lag phase (35 ± 10 h) to initiate growth (Fig. 3.4C). Variability was also higher in the mutant cultures, as expected of cells with a much higher level of intoxication. Notably, untreated controls of the mutant also needed variable but extended lag phases (32 ± 15 h) to recover from the resting state (Fig. 3.4C). Thus, not only is CbcBA needed to reduce CoII intoxication, but for growth activation after the resting state. Vesiculation as a mechanism for CoII detoxification in the absence of the CbcBA pathway Atomic Force Microscopy (AFM) images of WT, ∆cbcBA, and ∆cbcBA::cbcBA cells deposited on the surface of highly oriented pyrolytic graphite (HOPG) revealed increased (albeit variable) vesiculation in the mutant and phenotypic rescuing of WT vesiculation in the complemented strain (Fig. 3.5). Hypervesiculation is typically associated with changes in the fluidity and permeability of the outer membrane 36,37, which matches well with the increased permeability of the ∆cbcBA cells to CoII (Fig. 3.4A) and the increased sensitivity of the mutant cells during osmotic shifts from the resting state (Fig. 3.4C). Hypervesiculation in G. sulfurreducens has also been proposed to promote the detoxification of LPS-bound metals 28. Geobacter cells synthesize a rough (no O-antigen) LPS that sequesters toxic divalent cations such as the uranyl cation to prevent them from traversing the outer membrane 28. Once saturated, the cell sheds the metal-containing LPS via outer membrane vesicles (OMVs), preparing the outer membrane for further metal binding 28. Consistent with this, CoII-stressed WT cells were hypervesiculated (14.3 85 ± 2.6 OMVs/µm2) compared to untreated controls (3.8 ± 1.4 OMVs/µm2, p < 0.001) (Fig. 3.5B). Vesiculation was also greater in the ΔcbcBA and ∆cbcBA::cbcBA cells grown with CoII (18.2 ± 5.6 OMVs/µm2 in ΔcbcBA compared to 4.9 ± 2.9 OMVs/µm2 in untreated controls, p = 0.003; 9.4 ± 1.8 OMVs/µm2 in ∆cbcBA::cbcBA compared to 2.4 ± 1.2 OMVs/µm2 in untreated controls, p = 0.001) (Fig. 3.5B). Complementation of the ∆cbcBA mutation in the ∆cbcBA::cbcBA cells rescued the hypervesiculation phenotype during CoII treatment (p = 0.02) to levels lower than WT cells (p = 0.01). The hypervesiculated phenotype of the CbcBA-deficient cells provides a pathway for CoII detoxification via the rapid release of OMVs with metal- sequestered LPS. Such a compensatory mechanism may have masked the true contribution of the CbcBA cytochrome pathway to CoII detoxification in some of the metal tolerance assays used in this study and may have contributed, at least partially, to the high phenotypic variability of CoII-stressed mutant cells. 86 Figure 3.5 Effect of CoII treatment on vesiculation. (A) Representative topographic AFM images of WT, ΔcbcBA and ΔcbcBA::cbcBA cells from FWAF cultures grown to late- exponential phase (A660 = 0.4-0.5) with or without 200 µM CoCl2. (B) Quantification of the OMVs produced by treated (pink) or untreated cultures normalized to A660 0.5. Individual micrographs (circles) and averages (lines) are presented. Cytochrome abundance is affected by CoII treatment Metal exposure 12,17 and mutations in genes encoding cell envelope proteins 25,38,39 can significantly impact the type and levels of cytochromes expressed by G. sulfurreducens. To test for similar effects by CoII and the ∆cbcBA mutation, we used an enhanced chemiluminescence (ECL) based heme stain to visualize the cytochrome profile of whole- cell and supernatant samples of the WT, ∆cbcBA, and ∆cbcBA::cbcBA strains in untreated versus CoII-treated cultures (Fig. 3.6). Untreated controls revealed five dominant bands corresponding to heme-containing proteins with molecular mass of roughly 10, 22, 30, 50, and 75 kDa. These estimated masses matched well with the molecular weights reported for some of the most abundant c-cytochromes of G. sulfurreducens, mainly the periplasmic PpcA-E c-cytochromes 21 and the outer membrane c-cytochromes OmcE (~30 kDa) 40, OmcS (~50 kDa) 40, and OmcB (~76 kDa) 87 6 and the secreted form of OmcZ (~30 kDa) 41. These bands may be attributed to either extracellular or intercellular cytochromes as the growth medium was not separated from the cell pellets in this bulk measurement of heme-containing proteins. Deletion of cbcBA did not affect cytochrome expression (Fig. 3.6). However, the genetically complemented strain had a marked decrease in the expression levels of the four largest heme-containing bands, particularly the 22 and 75 kDa bands. Hence, although the ∆cbcBA::cbcBA strain expresses the cbcBA gene from its native promoter, its relocation to a different genome location (downstream of glmS (GSU0270) encoding a glutamine fructose-6-phosphate aminotransferase) 5 may have pleitropically affected the expression of other cytochromes. This difference helps explain why some of the phenotypic defects of the ∆cbcBA mutant were only partially rescued (Fig. 3.2) in the genetically complemented strain. 88 Figure 3.6 Heme-containing proteins in whole cells from untreated and CoII-treated cultures. WT, ∆cbcBA, and ∆cbcBA::cbcBA cells were grown to stationary phase with (treated) or without (untreated) 200 µM CoCl2 and harvested by centrifugation before resuspension in Laemmli buffer without β-mercaptoethanol and boiling. Samples were loaded into a polyacrylamide gel and separated by electrophoresis at 300 V. The gels were blotted to a membrane before staining the heme-containing proteins by detecting their peroxidase activity with an ECL stain. Whole cell samples were normalized to A660 0.3 and ran alongside a 250 kDa protein ladder. Despite differences noted in the expression of heme-containing proteins in the complemented strain, all of the strains produced brighter bands when grown with CoCl2 (Fig. 3.6). The most notable increases in expression were for the bands (22, 30, 50, and 75 kDa) predicted to correspond to outer membrane c-cytochromes such as OmcE (~30 kDa) 40, OmcS (~50 kDa) 40, and OmcB (~76 kDa) 6 and the secreted form of OmcZ (~30 kDa) 41. This result aligns well with previous reports of increased cytochrome expression in metal-exposed cells 12,22. Most of the heme-stained bands were also present and had 89 comparable intensities in the complemented strain, consistent with distinct regulatory pathways for the activation of c-cytochromes during the CoII detoxification response. Importantly, the higher expression under CoII-stress of heme-containing proteins supports the notion that cell envelope respiratory chains are needed to detoxify the metal. This is again consistent with the predicted role of the CbcBA cytochrome pathway in extracellular electron transfer and reductive mineralization to CoII. DISCUSSION Our previous transcriptomic studies of metal-stressed cells of G. sulfurreducens 12 revealed three main mechanisms for CoII detoxification, all localized to the cell envelope: 1) extracellular mineralization of CoII (cytochrome path); 2) periplasmic metal export (RND-pump); and 3) outer surface remodeling (lipoproteins and exopolysaccharide- associated proteins) (Fig. 3.1). While export and surface remodeling are conserved pathways for metal detoxification in other bacteria 42, the surface immobilization of CoII as nanoparticles is a novel adaptive response specific to Geobacter. The differential upregulation of the CbcBA-encoding genes with CoII 12 and the known role for these cytochromes in electron transfer to extracellular electron acceptors with reduction potentials (between –0.28 and –0.21 V) as low as the CoII/Co0 pair 5 suggested a similar reductive pathway for the extracellular mineralization of the metal at the thermodynamic edge. Enabling this reaction requires a respiratory chain for the transfer of electrons from the menaquinone pool to extracellular cytochromes in the outer membrane. However, CbcBA is predicted to be part of a periplasmic complex anchored to the inner membrane 5,12 and, thus, lacks the exposure needed for the reductive precipitation of CoII on the outer surface. The transcriptomic data (Fig. 3.1) confirmed the high expression of 90 periplasmic (Ppc) and outer membrane (Pcc conduits) c-cytochromes that promote extracellular electron transfer in G. sulfurreducens 6,17,21,43. These redox carriers provide a likely pathway for the reductive mineralization of CoII at the cell surface. Consistent with this, the sparse distribution of Co nanoparticles on the surface of metal-stressed cells 12 matches well with the localization of Pcc cytochromes on the outer membrane 19. Unmasking the contribution of the CbcBA cytochromes to CoII resistance via mineralization proved challenging given the many redundant pathways that exist for extracellular electron transfer 21 and CoII detoxification 12 in G. sulfurreducens. Additionally, we observed a higher phenotypic variability in CoII-stressed ΔcbcBA than any other culture due to higher susceptibility of the mutant cells to intoxication, which could have masked the true contribution of CbcBA to CoII detoxification. Despite these challenges, we reproducibly measured higher levels of cell-cell agglutination in the CbcBA-deficient mutant after challenging exponentially growing cells to sub-lethal concentrations of CoCl2 (Fig. 3.2), a mechanism often employed to mitigate metal intoxication 33. Exponentially grown cells of G. sulfurreducens synthesize an EPS (Xap) that anchors cytochromes involved in extracellular electron transfer and promotes cell- cell agglutination 34,44. The agglutinated cells form biofilms at the bottom of the culture vessel, an adaptive response that enhances metal tolerance 32 and increases catalytic rates and metabolic activity 45,46. Average growth yields were also lower in the ΔcbcBA mutant after the mid-exponential metal challenge, as expected of a mutant that diverts more energy to detoxification and repair of metal-induced damage 12. While the agglutination phenotype of the ΔcbcBA strain was effective at minimizing intoxication (Fig. 3.2C) the heightened variability in the length of the lag phase suggests that this 91 mechanism only partially shielded mutant cells (Fig. 3.2D). Although the genetic complementation of the ∆cbcBA had pleiotropic effects in the expression of heme- containing proteins (Fig. 3.6), these defects did not impair the ability of the cells to agglutinate once exposed to CoII (Fig. 3.2). This is consistent with a generic response to metal detoxification that does not require the redox activity of the extracellular matrix. It was, however, possible for the CbcBA-deficient mutant to acclimate to metal stress and grow in the presence of sub-lethal concentration of CoCl2 from low cell densities (Fig. 3.3). After a phase of acclimation (~ 50 h lag phase), both the WT and mutant strains grew at similar rates and to comparable growth yields (Fig. 3.3). Acclimation to CoII stress leads to the activation of a complex detoxification response involving macromolecular repair, metal export and surface chemistry modifications 12. These complementary pathways could have compensated for the CbcBA deficiency. The high redundancy of cell envelope cytochromes in G. sulfurreducens 21, including other Cbc complexes 5,17, could have also provided alternative pathways for extracellular electron transfer to the Xap cytochromes and compensate, at least in part, for the genetic disruption of CbcBA. Indeed, growth with CoII increases the expression on heme- containing proteins with molecular masses matching well those of outer membrane cytochromes (Fig. 3.6). This is consistent with a significant contribution of extracellular electron transfer chains to CoII detoxification. Taken together, the results support a model whereby the extracellular immobilization of CoII via reductive pathways provides an additional, but dispensable, layer of protection from metal toxicity. The low-voltage reductive pathway involving the CbcBA quinol oxidase is central to this process and, for this reason, cells transcriptionally 92 upregulated the encoding genes to grow under metal stress (Fig. 3.1). The upregulation of the cbcBA genes and the accumulation of Co nanoparticles on the cell surface of metal- stressed cells suggested that the two processes are linked 12. In support of this, resting mutant cells removed CoII faster than the WT cells and showed greater signs of intoxication and reduced viability once recovering in metal-free medium (Fig. 3.4). This, and the faster CoII removal by the mutant strain, suggests a greater degree of metal permeation in cells unable to express CbcBA. Outer membrane permeability changes can also explain the delayed growth recovery of the mutant cells from the resting state even in the absence of metal pressure (Fig. 3.4C) and their hypervesiculation (Fig. 3.5). The data also suggests that the CbcBA cytochrome pathway is integrated into the unique adaptive responses used by G. sulfurreducens for metal respiration. The rough LPS of G. sulfurreducens sequesters metal cations to prevent them from traversing the outer membrane 28. The cells shed the metal saturated LPS in OMVs to replenish the LPS in the outer membrane and maintain its metal sequestration capacity. Like other bacterial OMVs 37,47, Geobacter vesicles carry periplasmic and membrane-bound proteins including c-type cytochromes 48. The high abundance of cytochromes in the cell envelope of G. sulfurreducens also makes their OMVs redox-active and able to catalyze the extracellular reduction of metals 48. The increased vesiculation of WT cells grown under CoII stress (Fig. 3.5) suggests that this pathway is also important for CoII detoxification. Metal stress also exacerbated the hypervesiculation phenotype of the ΔcbcBA strain (Fig. 3.5). This result supports the idea that the respiratory chains and outer membrane remodeling pathways are tightly integrated in G. sulfurreducens. As such, vesiculation can be upregulated to compensate for the loss of the cytochrome 93 pathway and ensures that CoII permeation is minimized. This compensatory effect may have masked the true contribution of the CbcBA cytochrome pathway to CoII detoxification and contribute to the phenotypic variability observed in the metal toxicity assays. If so, the CbcBA contribution to metal detoxification in G. sulfurreducens may be more significant than predicted from the laboratory experiments and may be most relevant for the survival of these bacteria in metal-rich environments. MATERIALS AND METHODS Transcriptomic analysis We used previously published RNAseq data 12 to estimate the transcript abundance of cytochromes, structural components of electrically conductive pili, and several CoII detoxification pathways measured in treated (250 µM CoCl2) and untreated cultures of G. sulfurreduences. The transcript abundance was then normalized to the expression levels of the housekeeping gene, recA, under each culture treatment. The average and standard deviation of the replicates was calculated using Excel and statistical significance was identified using edgeR with cutoffs of a false discovery rate FDR < 0.05, log CPM = 5, and a log2 FC > 1 49. Bacterial strains and culture conditions Cultures of G. sulfurreducens strain PCA were obtained from our laboratory culture collection. The ∆cbcBA and ∆cbcBA::cbcBA strains 5 were generously donated by Dr. Daniel Bond (University of Minnesota). All strains were grown anaerobically (80:20 v/v CO2:N2 atmosphere) in fresh water (FW) medium (a minimal medium used for metal reduction studies 25) supplemented with 15 mM acetate as electron donor and 40 mM fumarate as electron acceptor and incubated at 30oC. Growth in the cultures was 94 monitored spectrophotometrically as absorbance at 600 nm (A600). Cultures were routinely grown to an optical density of 0.5-0.6 (late-exponential phase) before being transferred to fresh medium to an initial A600 of 0.05. CoII challenge to exponentially growing cells The growth of strains in FWAF medium was monitored at 660 nm (A660) until reaching mid-exponential phase (A660 0.3-0.4). Cell growth was monitored at 660 nm (A660) instead of 600 nm to ensure lack of colorimetric interference from the CoCl2 in the medium. Cells were then treated with 200 µM CoCl2 as previously described 12 or an equivalent volume of sterile, anaerobic ddH2O (untreated). Growth was monitored until cells reached stationary phase (A660 0.6-0.7). Agglutination of the stationary phase cultures was estimated as the difference in A660 between disturbed and undisturbed cultures. Culture supernatant fluids were harvested by centrifugation immediately after the CoII addition and before measuring agglutination to assay the change in CoII concentrations 12. The Excel® software was used to test for similar variances using an F-test and significant differences using a student’s t-test. In addition, GraphPad was used to perform a Grubb’s test to identify outliers using α = 0.05 as a cutoff. CoII acclimation assays Strains were grown in FWAF medium in the presence (metal-treated) or absence (untreated controls) of 200 µM CoCl2, as previously described 12. The cultures were inoculated with cells previously grown in metal-free FWAF medium (A600 ~0.5-0.6) at a starting A600 of 0.05. All incubations were at 30oC. Growth phenotypes (length of lag phase, generation time) were estimated with the Growthcurver R script (https://github.com/sprouffske/growthcurver). Briefly, growth curve data was matched to 95 the best sigmoidal curve fit to calculate the maximum generation time achieved, the maximum cell density (carrying capacity or growth yields), and initial population density of the culture. The length of the lag phase was calculated as the time it took cultures to rise above an absorbance of 0.05 using the standard equation for logarithmic growth. Vesiculation measurements OMV production was measured in culture samples deposited on the surface of a highly oriented pyrolytic graphite (HOPG) stage and imaged with an atomic force microscope (AFM), as previously described 28. Briefly, samples were taken from mid-exponential cultures using a 1,000 µL pipette tip (slightly sliced with a razor blade to increase the size of the opening and minimize mechanical shearing of the cells). The culture droplets (~20 µl) were left to adsorb to the HOPG stage for 20 min before wicking away excess culture with absorbent paper. The adsorbed samples were then washed for 30 sec with ddH2O three times and dried in a sealed container under the gentle flow of a N2 gas stream. The stages were loaded into an Asylum Research Cypher S system equipped with an AC240TS tip (Asylum Research) and the samples (cells and OMVs) were imaged in tapping mode at a scan rate of 0.3 Hz in 15x15 µm scans. OMVs in the scanned fields were counted using the freehand tool in the ImageJ software (https://imagej.nih.gov/ij/index.html) and normalized to the A660 of the sample and µm2 of scanned field. CoII permeability assays with resting cells Resting cells were prepared as previously described 25, with some modifications. Unless otherwise indicated, all procedures were performed inside an anaerobic chamber (COY Labs). Mid-exponential cultures (A600 ~0,4) were dispensed into 50 mL conical tubes and 96 pelleted by centrifugation to remove growth media (10 min, 3,000 g). The cell pellets were then resuspended with a Pasteur pipette in a high salt (30 mM NaCl) wash buffer optimized to preserve the viability of G. sulfurreducens cells 25,35, pelleted again, and resuspended in the wash buffer. The washed cells were resuspended into 30 ml of reaction buffer to an A600 of 0.1 before adding 20 mM acetate as an electron donor and 200 µM of CoCl2 as an electron acceptor and incubating at 30oC or 25oC. Samples were taken at different intervals to spectrophotometrically measure the concentration of CoII with a colorimetric assay, as described elsewhere 12. The CoII concentrations were used to calculate the amount of CoII removed by the resting cells. CoII removal rates were calculated from the linear portion of metal removal by the resting cells (typically between 1 and 2 h). A 1-ml sample was taken at the end of the assay to inoculate 9 ml of fresh FWAF media and monitor the recovery of the resting cells as a function of their viability 25. Denaturing Polyacrylamide Gel Electrophoresis (PAGE), blotting and heme stain Changes in cytochrome expression were visualized using an enhanced chemiluminescence (ECL) method developed for the rapid staining of c-type cytochromes on filters 50. Briefly, cells were grown in FWAF with or without 200 µM CoCl2 until reaching stationary phase and their exact A600 was measured. Culture samples were then deposited into 15 mL falcon tubes and stored in a -20oC freezer. Upon thawing, the samples were normalized to an A660 0.3 based on the growth yield measured at stationary phase. Normalized samples were mixed with equal volumes of 2X Laemmli buffer lacking reducing agents to preserve heme peroxidase activity. The samples were then incubated at 95oC for 10 minutes, cooled to room temperature and loaded into a 12% Mini-Protean 97 TGX precast gel (Bio-Rad). Proteins in the samples were then separated electrophoretically at 300 V for roughly 30 min in a Mini-Protean Tetra Vertical Electrophoresis Cell. The proteins in the gel were then transferred to a nitrocellulose membrane using a Turbo Transfer System and stained with a 1:1 mixture of the ECL peroxide and ECL luminol/enhancer solutions from the Clarity Western ECL Substrate Kit (Bio-Rad) for 5 minutes. Antibodies containing horseradish peroxidase are not required for protein detection with this stain because the peroxidase activity of the hemes promotes the breakdown of H2O2 in the ECL solution and the visualization of heme-containing proteins from the fluorescence of the ECL molecule. Membranes were then rinsed with sterile ddH2O and photographed with a Bio-Rad Gel Doc XR+ set to chemiluminescent mode (detection of the heme-stained bands) and white light (detection of the ladder). 98 1 2 3 4 5 6 7 8 9 10 11 12 REFERENCES Reguera, G. & Kashefi, K. The electrifying physiology of Geobacter bacteria, 30 Adv years (2019). https://doi.org/10.1016/bs.ampbs.2019.02.007 Physiol Microb 1-96 74, on. Nealson, K. H., Belz, A. & McKee, B. Breathing metals as a way of life: geobiology Antonie (2002). in https://doi.org/10.1023/a:1020518818647 Leeuwenhoek 215-222 action. Van 81, Hansel, C. M. Manganese in marine microbiology. 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Anal Biochem 209, 323-326 103 Chapter 4: Expression of electrically conductive pili as a mechanism for CoII tolerance This chapter presents data (growth experiments at 30oC versus 25oC and WT versus ΔpilB and ΔpilT3/4 strains) generated as part of a collaborative effort with Marcela Tabares. 104 ABSTRACT The use of electrically conductive pili for extracellular respiration and mineralization of metals is a hallmark of the physiology of Geobacter bacteria. In vitro studies with the model representative, Geobacter sulfurreducens, suggested that the pili can also bind CoII and, at sufficiently low potentials, they can reductively precipitate it to Co0. To test this hypothesis, we compared the effect of CoCl2 on the growth of G. sulfurreducens at temperatures that induce (25oC) or prevent (30oC) pili assembly. Temperature-induced piliation rescued the intoxication effects of the metal on cell growth (reduced lag phase, generation times, growth yields). Mutants unable to assemble pili (∆pilB) were also more sensitive to CoII toxicity than the wild-type (WT) strain but the hyperpiliation of a pilus retraction-defective mutant (∆pilT3/4) rescued the growth defects and accelerated the recovery of CoII-stressed cells compared to WT cells. Transmission electron micrographs revealed extensive accumulation of metal nanoparticles along the ∆pilT3/4 pili, in contrast to the modest and localized deposition of metal on the surface of the non-piliated cells. These results demonstrate that pili expression confers a growth advantage on G. sulfurreducens during and after metal stress. The rapid mineralization of the metal along the pili provides an effective mechanism for its extracellular detoxification that also mitigates CoII permeation of the membrane. INTRODUCTION Bacteria in the genus Geobacter have long been studied as model organisms of metal respiration, particularly for the dissimilatory reduction of FeIII and MnIV oxides that solubilizes the metals as FeII and MnII 1-3. By coupling the catabolism of fermentation byproducts to the storage of electrons in periplasmic cytochromes, Geobacter cells build 105 a large cell envelope capacitance that allows for the rapid discharge of electrons upon contact with metal acceptors 4-6. Outer membrane c-cytochromes provide a path for the discharge of periplasmic electrons onto extracellular electron acceptors 4,7. To increase the redox-active surface of the cell during the reduction of metal oxide minerals, Geobacter bacteria assemble electrically conductive protein filaments (pili) of the Type IVa pilus class 8. The expression of Geobacter pili is required for the cells to grow with FeIII oxide minerals 8 and facilitates access to the solid metal phases through the narrow pores of soils 9. Antagonistic cycles of pilus assembly and disassembly via ATPases of the Type IV pilus biosynthetic apparatus (PilB and PilT, respectively) ensure that reduced mineral byproducts are shed off and clean fibers are produced for new electron discharges 10,11. Additionally, the dynamic pili promote electron transfer across biofilms 10,12 and are the primary mechanism for the reductive mineralization of the soluble uranyl cation (UO2 2+), a reaction that simultaneously generates energy for growth and detoxifies the toxic UVI species 12,13. Like other Type IV pili, the Geobacter pilus fibers are assemblies of a peptide subunit (the PilA pilin) 13. The Geobacter pilins lack the conserved globular domain of other bacterial pilins, which makes them shorter and predominantly helical in structure 8,14. They also contain several aromatic residues that cluster close to each other in the pilin assembly to transport charges along the pilus fiber 8,14. The formation of salt bridges between neighboring subunits during polymerization aligns the aromatic residues such that charge hopping is facilitated along the pilus and directed towards electron acceptors bound to the fiber’s surface 14,15. Indeed, alanine replacements of these aromatic residues do not prevent pilus assembly but drastically reduce their conductivity and the ability of 106 the cells to form electroactive biofilms 10. The assembly of pilin subunits in the fibers exposes the peptide’s C-terminal region on the pilus surface and forms motifs for metal binding and reduction 15,16. Such metal traps can also be recreated in planar configurations of pilin assemblies onto electrodes 15 and their metal affinity probed electrochemically 16. Such electrochemical studies revealed a high affinity of the pilin metal traps for the divalent cobalt cation, CoII, which was reductively mineralized to Co0 nanoparticles 16. This finding suggested that Geobacter may use the pili to reductively precipitate CoII outside the cell, effectively preventing the permeation of the toxic cation inside the cell envelope. Given the tendency of FeIII and MnVI oxides to co-precipitate and adsorb CoII 17-19, the mineralization of CoII by Geobacter pili could provide an effective mechanism to detoxify the substantial amounts of toxic metal released during the reductive dissolution of the minerals 20. CoII detoxification by the pili would also impact the microbial communities that depend on Geobacter bacteria for the synthesis and secretion of Co- containing vitamins (cobamides) 21-23. Indeed, the extracellular mineralization of CoII by the pili would alleviate metal toxicity, at least locally, to Geobacter cells as well as their cobamide-dependent syntrophic partners. This way, Geobacter cells can assimilate the metal into the cobamide biosynthetic pathways 24 and secrete them to support the growth of syntrophic partners 21,25. In this chapter, I explored the contribution of the pili to CoII tolerance in G. sulfurreducens. For these studies, I used temperature switches that trigger or prevent pilin assembly (growth at 25oC and 30oC, respectively) 8,26 and tested the response to CoII stress of mutants carrying deletions in the ATPases that power pilin assembly (PilB) or disassembly (PilT) 10,11. The findings support the role for the electrically 107 conductive pili of Geobacter in overcoming CoII toxicity via extracellular mineralization and expand the known reactions used by Geobacter bacteria to cycle this important metal. RESULTS Temperature-induced piliation partially rescues CoII intoxication Pilin assembly by G. sulfurreducens can be triggered in the absence of FeIII oxides by dropping the temperature from 30oC (standard laboratory conditions) to 25oC, a temperature that presumably lowers growth rates to levels similar to those of cells during the respiration of FeIII oxides 8,13. We took advantage of this temperature effect in piliation to assess the contribution of pili expression to CoII tolerance in cultures of G. sulfurreducens grown in DBAF medium in the presence or absence of non-lethal concentrations of CoII (provided as 250 µM CoCl2). As we previously reported 24, non- piliated cells (30oC cultures) grown with the metal required a phase of acclimation and doubled slower than in untreated cultures (Fig. 4.1A). This response was stochastic as one of the replicate cultures grew similar to untreated cells. However, the non-piliated cultures acclimated quickly (Fig. 4.1A), albeit slower (1.73 ± 0.64-fold longer lag phase than untreated controls, p = 0.2). Once acclimated to metal stress, the non-piliated cells doubled at highly variable rates, another sign of stochasticity in culture tolerance (4.8 ± 3.3-fold slower than the untreated controls, p = 0.2) (Fig. 4.1C). This is consistent with metal-stressed cells diverting more energy from growth-sustaining processes towards metal detoxification. Another symptom of metal stress was the higher variability among replicates of the metal-treated cultures compared to the untreated controls (Fig. 4.1A), 108 which results from the stochastic activation of multiple detoxification pathways by the non- piliated cells under CoII pressure 24. Figure 4.1 Growth response of G. sulfurreducens to CoCl2 treatment at pili- inhibiting and -inducing temperatures. (A-B) Growth (A660) of piliated (A) or non- piliated (B) cells in DBAF medium at 30oC (white) and 25oC (gray), respectively, in the absence (black circles) or presence (pink circles) of 250 µM CoCl2. Shown are average (line) and standard deviation (shaded area) of triplicate cultures for each condition. (C) Average (line) and individual (circle) generation times of Co-treated cultures relative to the untreated controls from A-B. Variability was even higher in the piliated cultures (25oC) (Fig. 4.1B). This result is not surprising, considering that cells must acclimate to both lower temperatures and metal stress. Consistent with this dual cause, some replicates had lag phases within the ranges of untreated controls while others required extended incubation (~48 h) before cells started to grow exponentially (Fig. 4.1B). The presence of at least one replicate with severely delayed growth at 25oC in the presence of CoII was in fact a routine occurrence. Despite this variability, once the piliated cells resumed growth they doubled in the presence of CoII at rates similar to the untreated controls (1.69 ± 0.84-fold slower, p = 0.2) (Fig. 4.1C). The partial rescue of the CoII-triggered growth rate defects when growing 109 under pili-inducing conditions supports a role for pili in mitigating CoII intoxication in exponentially growing cells. Genetic disruption of pilus assembly and retraction reduces CoII tolerance The confounding effects of cold and metal stress adaptation in the temperature-controlled experiments (Fig. 4.1) masked a potential role for piliation in the acclimation to metal stress. To bypass this limitation, we used a genetic approach to investigate the effect of piliation on the acclimation of cells to CoII stress. For these experiments, we grew the wild type (WT) strain at 30oC (non-piliated conditions) in the presence of CoCl2 concentrations (500 µM) that require a long phase of acclimation 24 and compared the phenotype to mutants carrying deletions in the genes encoding the ATPases that power pilus assembly (PilB) 10 and retraction (PilT3/4) 11 (Fig. 4.2). Fig. 4.2A shows typical results for the WT and mutant cultures, where two out of three replicates acclimated after an extended lag phase (Fig. 4.2A). (One replicate culture, which did not resume growth after 6 days of incubation, was discarded.) The average lag phase for the acclimated WT cultures was 37.5 ± 1.3 h but longer (and more variable) in the ∆pilB mutants (91.4 ± 55.4 h) (Fig. 4.2B). These phenotypes (extended and variable lag phase) are hallmarks of CoII acclimation in G. sulfurreducens 24 and are consistent with the need for cells to induce pili assembly in order to acclimate to CoII stress and resume growth in the presence of the metal. Two out of three ΔpilT3/4 cultures also resumed growth after a long phase of acclimation (70.2 ± 1.0 h) (Fig. 4.2B). The extended acclimation phase of the ΔpilT3/4 may be interpreted as supporting a role for pilus retraction in CoII detoxification. Pilus depolymerization in G. sulfurreducens is mediated by the main PilT ATPase, PilT4, and assisted by PilT3 11. Together, PilT4 and PilT3 coordinate pili retraction to shed off 110 reduced mineral particles that remain attached to the fibers during the respiration of FeIII oxides 11. A similar model could describe the role of PilB in pilus protrusion to reductively precipitate CoII at the pilus surface and the need for PilT3/4 to retract the pili to detach the Co0 nanoparticles and enable a new cycle of pilus protrusion and CoII mineralization. Figure 4.2 Contribution of pili assembly and retraction to CoII tolerance. (A) Growth (A660) of WT, pilus assembly mutant (ΔpilB) and pilus retraction mutant (ΔpilT3/4) in DBAF at 30oC in the absence (black symbols) or presence (pink symbols) of 500 µM CoCl2. (B- C) Average (lines) and individual (circles) lag phase duration (B) and generation times (C; relative to untreated cultures) of the cultures shown in A. While important for rapid acclimation to metal stress, the pili detoxification pathway was dispensable once acclimated cells resumed exponential growth (Fig. 4.2A). Indeed, the doubling times for the acclimated cultures of the ∆pilB and ∆pilT3/4 strains were 111 similar to the untreated cultures (Fig. 4.2C). Transmission electron microscopy (TEM) images of unstained metal-stressed cells from the exponentially growing cultures revealed phenotypes consistent with different responses to CoII intoxication by the WT and mutant cells (Fig. 4.3). WT cells were surrounded by a thick, electron-dense substance consistent with the reported production of Xap exopolysaccharide (EPS) during the exponential phase of growth 24. Application of additional stains, such as uranium, were unnecessary given the ability of the CoII salts to react with the amino groups of basic amino acids to form insoluble complexes that enhance the contrast of biological samples for TEM visualization 27. This staining suggests that CoII penetrated through the EPS barrier and into the cells. The EPS layer of WT cells was absent in the mutant strains (Fig. 4.3). Furthermore, the pili-deficient ∆pilB mutant cells accumulated Co nanoparticles sparsely on the cell surface, a phenotype associated with the reductive mineralization of the metal by outer membrane c-type cytochromes 24. By contrast, the hyperpiliated pilT3/4 strain 11 had substantial extracellular mineralization along the pili on one side of the cell (Fig. 4.3). Hence, although pili are not absolutely required for growth under CoII stress (Fig. 4.2), they provide an efficient strategy for the detoxification of the metal. The experimental evidence also suggests that EPS and pili pathways may be co- regulated such that EPS production prevents pili assembly (WT cells in Fig. 4.3). The production of a thick EPS layer provides a physical and chemical barrier to the permeation of metals 28 and also promotes cell-cell aggregation and biofilm formation 29, thereby protecting the cells from metal intoxication. Pili production can also provide an effective mechanism for CoII detoxification (∆pilT3/4 in Fig. 4.3) albeit on one side of the cell only. To protect the non-piliated side, cells coat the outer membrane with a rough (no O- 112 antigen) lipopolysaccharide (LPS), which sequesters metal cations, and vesiculate extensively to release the metal-saturated LPS 30. Figure 4.3 Transmission electron microscopy (TEM) images of CoII-treated cells. Representative TEM micrographs of WT, ∆pilB, and ∆pilT3/4 cells grown to mid- exponential phase (A660 ~0.3-0.4) in DBAF with 500 µM CoCl2. Samples are unstained and fixed onto formvar-coated grids with 2.5% glutaraldehyde prior to imaging. Scale bars are 500 nm. Expression of pili reduces CoII toxicity in resting cells Metal removal by resting (non-growing) cells and their consequent growth recovery in metal-free medium provide a suitable assay to assess the contribution of cellular appendages to mineralization and detoxification 13,31 without the interference of growth- supporting pathways 32. Hence, we exposed resting cells of the WT and hyperpiliated ∆pilT4 strains to 200 µM CoCl2 for 2 h and tested the extent of intoxication by recovering the resting cells in growth medium (FWAF) without the metal (Fig. 4.4A). As previously reported in Chapter 3, the WT cells recovered from the CoII challenge after a longer lag phase than untreated cell controls (Fig. 4.4B). However, once exponential growth started, the WT cells doubled at rates comparable to the untreated controls (Fig. 4.4C). The hyperpiliated ΔpilT4 strain recovered from the CoII challenge as rapidly as the untreated controls and without substantial variability among the replicates (Fig. 4.4A and B). 113 Furthermore, it grew at the same growth rates as untreated cultures (Fig. 4.4C). Hence, the mutant cells have a growth advantage after CoII exposure. The constitutive production of the pili by the mutant cells bypassed the need that WT cells have in inducing their biosynthesis, while their retraction defect increases piliation and their ability to detoxify CoII via its extracellular precipitation (Fig. 4.3). As a result, resting ΔpilT4 cells readily resumed growth after CoII exposure while the non-piliated WT cells required acclimation to induce the metal detoxification pathways (Fig. 4.4). Figure 4.4 Effect of piliation on viability after CoII exposure in a resting state. (A) Growth (A660) of WT (non-piliated) and ∆pilT4 (hyperpiliated) cells in metal-free FWAF medium after a 2-h resting cell assays in the presence (pink) or absence (black) of 200 µM CoCl2. (B-C) Average (line) and individual (symbols) length of the lag phase (B) and generation times (C), relative to the average of the untreated controls of the cultures shown in panel A. DISCUSSION The electrochemical demonstration that the pilin’s metal traps can bind CoII with high affinity and reductively precipitate it as Co0 nanoparticles 16 suggested a biological role for the Geobacter pili in the mineralization of this essential metal. In support of this hypothesis, temperature-induced piliation in G. sulfurreducens 8,13 partially rescued the 114 growth defects (longer lag phases and generation times) of cultures supplemented with non-lethal concentrations of CoII (Fig. 4.1). The growth rescue by piliation is similar to that reported for the uranyl cation, a toxic radionuclide that the pili reductively precipitate outside of the cell to prevent its permeation and non-specific reduction inside the cell envelope 13. The fact that pili are also required to reductively dissolve the FeIII- and MnIV- oxides 8,9, which trap significant amounts of CoII in the environment 17-20, suggests a dual role for the conductive appendages in supporting growth with the metal oxides and tolerance of the solubilized CoII cation. The sudden exposure of the cells to CoII exerts selective pressure to rapidly acclimate to metal toxicity (adapt or die). Genetic studies with a mutant unable to assemble the pilins (∆pilB) 10 suggested an important role for the pili in acclimation to metal stress as well. In support of this function, the pili-deficient mutant cells required extended lag phases to acclimate to potentially lethal concentrations of CoII (500 µM CoCl2) (Fig. 4.2). A mutant (ΔpilT3/4) carrying deletions in the primary and secondary ATPases (PilT4 and PilT3, respectively) that energize pili depolymerization 11 required a longer phase of acclimation, albeit shorter than the ∆pilB (Fig. 4.2). Pili retraction sheds off pilus-bound minerals and recycles the pilin subunits for new rounds of polymerization 11. Antagonistic cycles of pilus protrusion and retraction may thus be needed for G. sulfurreducens to acclimate to CoII stress and resume growth in the presence of the metal. However, other explanations are possible. The ΔpilT3/4 mutation also leads to the constitutive production of pili and, by preventing their retraction, the mutant cells become hyperpiliated 11. Hence, the delayed acclimation of this mutant to CoII stress compared to the WT cells may have been the result of these additional phenotypes. For example, the hyperpiliation phenotype, rather than the pilus retraction 115 defect, of a ∆pilT mutant of Pseudomonas aeruginosa interferes with secretory functions 33. Similar pleiotropic effects of the retraction mutant could have delayed the activation of pili-independent pathways for CoII detoxification. Piliation may also provide a first layer of defense to the cells, reducing intoxication so sufficient energy may be allocated towards the synthesis of efflux pumps, protein repair, and ROS detoxification 24. The transcriptional activation of these pathways allows the cells to grow and divide under metal stress 24. These pathways can compensate for the lack of piliation and pilus retraction, as indicated by the ability of acclimated cultures of the ∆pilB and ∆pilT3/4 mutants to grow under metal stress at WT rates (Fig. 4.2). The extensive mineralization along the pili of the hyperpiliated ∆pilT3/4 strain (Fig. 4.3) suggests that pili-mediated precipitation of CoII outside of the cell minimizes metal diffusion into the cell, as reported for the uranyl cation 13. The pili are indeed the primary mechanism for the extracellular mineralization of the uranyl cation in G. sulfurreducens, a reaction that prevents the toxic cation from traversing the outer membrane and mineralizing in the periplasmic space 13. As a result, the uranyl cation readily penetrates and gets reductively mineralized into the cell envelope of non-piliated cells while in the resting state and delays their ability to reinitiate growth once in metal-free medium 13. Similarly, resting cells exposed to CoII for a few hours were able to resume growth in metal-free medium more rapidly when piliated (Fig. 4.4). This fast response is consistent with piliation protecting the cells from metal penetration, which reduces intoxication and facilitates growth recovery in metal-free medium. The presence of c-cytochromes on the cell surface of G. sulfurreducens provides an alternative path for the reductive immobilization of CoII in non-piliated cells. These 116 cytochrome conduits could have contributed to the sparse mineralization of Co observed in TEM images of the non-piliated ∆pilB cells (Fig. 4.3). The distribution of mineral particles over the ∆pilB cells matches well with the pattern of deposition of Co nanoparticles on the surface of non-piliated WT cells (grown at 30oC). Non-piliated WT cells grown with CoII upregulate cell envelope cytochromes that could provide a pathway for the reductive precipitation of the metal on the outer surface 24. However, the contribution of the cytochrome pathway to CoII detoxification is likely small compared to that of pili, which provide a more extensive mineralization capacity (Fig. 4.3). The pili- mediated reaction is however not needed once the cells acclimate to metal stress, as alternative detoxification pathways are activated that can support growth and metal detoxification. Notably, non-piliated WT cells produced a thick, protective EPS layer under CoII stress (Fig. 4.3). Such a passive barrier to CoII permeation may offer advantages over the pili to exponentially growing cells. The low reduction potential of the CoII/Co0 pair (-0.27 V vs SHE) makes the reductive detoxification of CoII a barely energy-yielding reaction. ATP expenditure on the polymerization and depolymerization of the pili could make the reaction an energy intensive process. By contrast, once synthesized, EPS passively immobilizes CoII before it reaches the cell envelope. Furthermore, the EPS layer of G. sulfurreducens anchors outer membrane cytochromes, which could provide some reductive capacity 34. Importantly, EPS promotes cell-cell agglutination and biofilm formation, a process that enhances tolerance to metals 35 and other biocide compounds 36. The heterogenous phenotypes of biofilm cells may further increase metabolic rates and catalytic activities that are required for detoxification and survival during metal stress 35-37. 117 The biomineralization of Co via the conductive pili of G. sulfurreducens affords new opportunities for mining and reclaiming this critical metal from environmental and industrial sources. As a strategic resource, Co is a key component of many technologies that society relies on, including rechargeable batteries 38, superalloys 39, and super conductors 40,41. The biomineralizing properties of Geobacter bacteria and their pili could be harnessed to boost Co recovery from metal-impacted sites 42-44. Microbial electroreclamation technologies may also be developed to recover Co and other metal cations from spent products such as those used in the cathodes of electric vehicle batteries and other consumer electronics 41,45. The availability of platforms to mass- produce recombinant pilins 15 and assemble them on electrodes 16 or as protein nanowires 46 are also significant, as they provide sustainable solutions for the extraction of metals from a variety of sources and the circularization of manufacturing processes critical to advance the climate economy 15,16. MATERIALS AND METHODS Bacterial strains and culture conditions Cultures of G. sulfurreducens strain PCA 47, ΔpilB 10, ΔpilT3/4 and ΔpilT4 11 were obtained from our laboratory culture collection. All strains were grown anaerobically in a minimal medium, DB or FW, supplemented with 20 or 15 mM acetate as the electron donor, respectively, and 40 mM fumarate as the electron acceptor 48. Growth was monitored spectrophotometrically as absorbance at 600 nm (A600) and cultures were routinely grown to late exponential phase (A600, 0.5-0.6) prior to transfer to fresh medium. When indicated, an A660 was used to minimize CoII interference with growth measurements. 118 CoII tolerance assays Cells of the WT strain were grown in metal-free DBAF medium at 30oC (non-piliated cells) or at 25oC to induce piliation 8,13. The cultures were then transferred to fresh medium supplemented with 250 µM CoCl2 or an equal volume of anaerobically prepared sterile ddH2O (untreated controls). The starting A600 of the cultures was always 0.05, as previously described 24. Experiments with the pili-deficient (ΔpilB) and pilus retraction- deficient (ΔpilT3/4) mutant strains used higher concentrations of CoCl2 (500 µM) and were performed at 30oC. Growth in all cultures was monitored spectrophotometrically at 660 nm (A660) to minimize the influence of CoII on absorbance measurements. Growth phenotypes (length of the lag phase, generation time) were estimated using the Growthcurver R script (https://github.com/sprouffske/growthcurver). Briefly, growth curve data were matched to the sigmoidal curve of best fit to calculate the maximum generation time achieved, the maximum A660 achieved (carrying capacity), and initial population density of the culture. The length of the lag phase was defined as the time needed for a culture to rise above A660 of 0.05 using the standard equation for logarithmic growth. Transmission electron microscopy (TEM) Strains of WT, ΔpilB and ΔpilT3/4 were grown at 30oC to mid-exponential phase (A660, 0.3-0.4) in DBAF media treated with 500 µM CoCl2 and fixed with 2.5% glutaraldehyde prior to deposition for 5 min on Formvar-coated grids (150 square-mesh Ni, Electron Microscopy Sciences). The grids with the samples were washed with double deionized (dd)H2O three times (30 sec each), side-blotted to remove excess liquid and stored at room temperature until examination using a JEOL 1400 Flash 120kV transmission 119 electron microscope. A microscopy stain was purposedly omitted to prevent stain and mineral artifacts. CoII resting cell assay Resting cell assays were performed as previously described 13 with some modifications. Unless otherwise indicated, all procedures were performed inside an anaerobic chamber (COY Labs). Cultures of WT or ΔpilT4 cells were grown in FWAF medium at 30oC to mid- exponential phase (A600 ~0.4) and dispensed into 50-ml conical tubes. Samples were then pelleted by centrifugation to remove growth medium (10 min, 3,000 g), resuspended in a salt wash buffer using a Pasteur pipette, pelleted a second time, and resuspended in wash buffer, as previously optimized for extended viability of Geobacter resting cells in metal reduction assays 13,31. 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In Chapter 2, I described a collaborative effort with lab member Marcela Tabares that revealed a high CoII tolerance by G. sulfurreducens cells, comparable to what is typically seen in metal-resistant organisms isolated from highly contaminated sites. The cells underwent an extended lag phase to acclimate to metal stress. During this period, the cells induced multiple pathways to fight CoII intoxication such as efflux pumps for metal extrusion, enzymes for protein repair, and surface chemistry modifications that could mitigate CoII diffusion and promote cell-cell interactions leading to agglutination and biofilm formation. We also saw the downregulation of many genes encoding proteins that CoII could readily infiltrate and inactivate. The upregulation of genes encoding a cytochrome complex (CbcBA) for the reduction of electron acceptors with reduction potentials as low as the CoII/Co0 couple is a response only described in Geobacter, as of the publication of this thesis. I developed an assay to measure CoII removal by the cells (~25 µM) and used Transmission Electron Microscopy coupled with Energy Dispersive X- ray Spectroscopy (TEM-EDS) to demonstrate the extracellular precipitation of Co on discreet foci over the cell. Additional work to characterize the nanoparticles could be carried out. Techniques such as laser ablation with inductively coupled plasma and mass spectrometry (LA-ICP-MS) could characterize the chemical makeup of the nanoparticles. Furthermore, methods to measure the oxidation state, such as X-ray absorbance spectroscopy (XAS), should be applied to verify the reductive precipitation of CoII as Co0 127 and quantify the amount of metal reduced as opposed to the amount removed from the medium. In Chapter 3, I further characterized the role of CbcBA in CoII tolerance using a mutant strain that carried a deletion of the cbcBA genes and its genetically complemented strain. The CbcBA deficiency exacerbated some of the phenotypes associated with CoII intoxication. For example, the mutant cells agglutinated more than the wild type (WT) and genetically complemented strains when challenged with non-lethal concentrations of the metal during exponential growth. Yet, when the cells were allowed time to acclimate to the metal before initiating growth, they activated alternative pathways for detoxification and restart growth and cell division at WT rates. Additionally, resting cells of the ΔcbcBA mutant removed more CoII from solution and faster, becoming more intoxicated than the WT cells and delaying the recovery of the metal-stressed cells once transferred to metal- free medium. The hypervesiculation of the mutant cells suggested membrane permeability changes that facilitate CoII penetration into the cell envelope and exacerbated intoxication. Membrane permeability assays using dyes that typically cannot permeate the membrane, such as ethidium bromide, may prove useful for testing this proposal. These simple staining procedures use fluorescent stains that can move freely into the membrane, such as fluorescein diacetate, which can be coupled with flow cytometry to identify distinct populations of permeable cells. Notably, the CbcBA deficiency increased vesiculation even in the absence of metal stress, suggesting pleiotropic effects of the mutation on membrane stability. Given the pleiotropic effects of cytochrome mutations in G. sulfurreducens, it will be important to rule out compensatory expression of other cytochromes in the ΔcbcBA background by using a heme stain. Also 128 important is performing resting cell suspensions using cells previously grown on a medium that enhances their resistance to osmotic changes during the growth-resting state transitions. In Appendix 1 I describe how subtle changes in the salt content of the growth medium impact the viability of the cells in the resting state and the suitability of the NBAF medium formulation to prepare resting cells for CoII challenge assays for up to 12 h. In Chapter 4, another collaborative effort with Marcela Tabares, I demonstrated the growth advantage of piliated cells under CoII stress. These experiments used a temperature shift (from 30oC to 25oC) to induce piliation and also compared the CoII tolerance of a non-piliated WT strain to mutants unable to polymerize (ΔpilB) or depolymerize (ΔpilT3/4) pili. TEM images confirmed the monolateral mineralization of Co in the ΔpilT3/4 mutant, which is hyperpiliated, and the discreet deposition of the mineral particles on the outer surface in the pili-deficient mutant ΔpilB. Spectroscopic analyses of the Co minerals similar to those mentioned above are needed to evaluate the composition of the suspected Co0 nanoparticles. Future studies should also strive to further elucidate how the cells regulate the expression of these two reductive pathways (cytochrome and pili) and the adaptive roles that the reductive mineralization of CoII plays in the environment. The role of the cytochrome-loaded Xap exopolysaccharide (EPS) in CoII detoxification also needs to be considered as a contributor to the mineralization reaction. The role of pili dynamics (antagonistic cycles of pilus protrusion and retraction) may also be important for CoII mineralization. However, tools for the study of pilus retraction are limited to retraction- deficient mutants, which are also hyperpiliated. Nevertheless, it is possible to replace 129 tyrosines of the pilin (Tyr3 mutant) to reduce charge transport along the pili and produce piliated mutant strains unable to reduce metals via this pathway. Ultimately, experiments with mixed Co-Fe oxides will better characterize the role of Geobacter as keystone organisms in cobamide dependent ecosystems. Connecting the biomining of Co from these metal oxides to the secretion of cobamides will set the stage for future applications of Geobacter as biofertilizers and as drivers of green technologies for the reclamation of Co from spent Co products. 130 APPENDIX: EFFECT OF GROWTH MEDIA FORMULATIONS ON THE VIABILITY AND COII-DETOXIFICATION CAPACITY OF RESTING CELLS OF GEOBACTER SULFURREDUCENS ABSTRACT The many toxic effects of CoII trigger complex cellular responses to overcome intoxication, which may include the activation of repair mechanisms, extrusion pumps, and respiratory chains for reductive mineralization of the metal. Resting cell assays optimized for Geobacter sulfurreducens provide great tools to dissect the contributions of respiratory chains to other detoxification pathways. Here I present evidence that subtle changes in the salt content of the growth medium used to harvest cells for resting cell assays can greatly impact the ability of the cells to remain metabolically active while in the resting state. These findings emphasize the need to select growth media formulations with the osmotic balance needed to maintain the viability of the cells under non-growth conditions. INTRODUCTION Microbial metal respiration has been a central focus in the study of biogeochemistry, bioremediation, and biotechnology 1-4. Among metal respiring organisms, Geobacter bacteria have been investigated thoroughly for their ability to reduce a broad spectrum of metals such as FeIII and MnIV oxides 5-8, the uranyl cation (UO2 II) 9-11, and CuSO4 12 among many others 13. Techniques have been applied to study metals that support the growth of Geobacter bacteria in laboratory cultures with environmentally relevant electron acceptors. For example, the ferrozine assay is used to measure the release of FeII during the reductive dissolution of FeIII oxides 14. Geobacter bacteria can also use toxic metals and radionuclides as electron acceptors for growth 13. Energy yields from these reactions 131 are lower than expected thermodynamically 15, because some of the energy gained from the reduction of the electron acceptors must be diverted towards detoxification 9. For example, growth of the model representative Geobacter sulfurreducens with CoII induces a host of cell responses (repair, extrusion, surface chemistry modifications) that lead to removal of the metal from solution in a non-reductive manner 16. The poor energy yields predicted for the reduction of CoII to Co0 limit cell growth 16 but, nevertheless, provide a pathway for detoxification. Consistent with a reductive mechanism for detoxification, G. sulfurreducens cells precipitated Co as nanoparticles on their outer surface and upregulated cytochrome genes involved in extracellular electron transfer to low-potential electron acceptors 16. The contribution of respiratory chains to the reductive detoxification of CoII is often masked by other pathways used to overcome metal intoxication (chapter 3). Resting cell (RC) assays can help dissect this complex detoxification response. The resuspension of cells in buffers devoid of nutrients allows for the study of enzymatic reactions without the confounding effects of growth and cell division. Depending on the reaction formulation, cells can be maintained in a resting (non-growing) state for various periods of time, as needed to measure specific enzymatic reactions. The concentration of cells in the suspension can also be modified to increase reaction rates and measure reactions that would otherwise be difficult to measure in growing cultures. RC assays have been broadly applied in microbiology 17-19 and are particularly useful to study metal respiration 9,11,20. By limiting the production of new biomass, RC assays can allow for accurate measurements of metal respiration rates and assess the contribution of individual electron carriers to the measured reaction. Optimal conditions for the reduction of the uranyl cation 132 (UVI to UIV) by G. sulfurreducens resting cells highlighted the need to minimize osmotic stress to cells prepared for the RC assay 9,11. A stepwise adaptation of fumarate-grown cells to lower osmolarity than in the growth medium is essential for sustained cell viability in the resting state 11. In the optimized protocol, the cells are first harvested and resuspended in a wash buffer with half the salt (NaCl) content as in the growth medium before being transferred to a reaction buffer containing one-quarter of the growth medium salt content 11. In chapter 3 and 4, I described the use of the RC assays to investigate the contribution of the CbcBA cytochromes and electrically conductive pili to CoII detoxification. The buffer formulations used for these assays are those optimized for the uranium assays 11. Similar formulations to the FW medium such as NB (a nutrient broth used for genetic engineering studies 21 and DB (mineral medium used for electrode-reduction studies 22) are widely used for growth studies with metals. Indeed, we used the DB medium with acetate and fumarate (DBAF) to investigate the detoxification response of G. sulfurreducens to CoII (chapter 2). The NB and DB media differ slightly in the type and concentration of some Na+ and K+ salts and this, in turn, affects membrane fluidity and vesiculation in G. sulfurreducens (Morgen Clark, personal communication). Hence, in this Appendix, I investigated the impact of NB and DB growth media on the ability of resting cells to remain viable and metabolically active in the presence or absence of CoII. For these experiments, I grew G. sulfurreducens in either medium with acetate and fumarate (NBAF and DBAF, respectively) and harvested the cells for the RC assay. The results suggest that minor differences in the salt content of the media formulations can greatly impact the ability of G. sulfurreducens to overcome the osmotic and metal stress. 133 134 RESULTS Reduced ability of DBAF-grown cells to overcome osmotic and metal stress The DBAF medium is a mineral medium with acetate and fumarate that was optimized for growth of G. sulfurreducens cells used to inoculate microbial fuel cells and build electricity-producing biofilms on anode electrodes 22,23. This medium was also used to study the tolerance and detoxication response of G. sulfurreducens to CoII 16. For this reason, I harvested cells from mid-exponential phase DBAF cultures and prepared them for a RC assay in the presence or absence of 250 µM CoCl2. Addition of acetate (electron donor) to the cell suspension initiated the reaction (time 0). All of the samples, including untreated controls, required a variable amount of time (lag phase) to resume growth once transferred from the resting cell suspension into the metal-free growth medium (Fig. A.1), a sign of viability loss 9. Notably, the untreated resting cells taken at the start of the RC assay (0 h) had a delayed restart of growth and, for at least one replicate, only resumed growth after an extended lag phase (Fig. A.1A). This is indicative of an inability of the DBAF-grown cells to overcome osmotic shifts during the preparation of the resting cells. Growth delays were even more pronounced for the untreated cells after 6 h in the resting state (Fig. A.1B) and for all the samples taken from the CoII-treated resting cell reactions (Fig. A.1A-B). 135 Figure A.1 Growth recovery of DBAF-grown resting cells from CoII stress. Cells were grown and transferred in mid-exponential phase three times in DBAF medium before harvesting mid-exponential cells for the RC assays. Resting cells were challenged with 250 µM CoCl2 for 6 h (pink symbols) or left untreated (black symbols) and recovered in triplicate cultures with metal-free DBAF medium at the beginning (A) or end (B) of the RC assay (0 and 6 h, respectively). Enhanced ability of NBAF-grown cells to overcome osmotic and metal stress NBAF medium is routinely used to recover cells from stressed states, such as during the recovery of cells from frozen stocks and mutants after electroporation 21. Cells from NBAF cultures also have lower levels of vesiculation (Morgen Clark, personal communication), a phenotype associated with reduced membrane fluidity and permeability 24. Hence, I harvested mid-exponential cells from NBAF cultures and prepared them for the RC assay, following the same protocol used for DBAF-grown cells. As shown in Fig. A.2A, the resting cell samples resumed growth rapidly independently of the resting cell treatment (with or without CoII) and time in the resting state (0-12 h). The rapid growth restart of the resting cells shows that they remained viable throughout the duration of the assay, in this case for up to 12 h. This timeframe contrasts with the conventional RC assays using FWAF- grown cells, which maintain the viability of the cells in the resting state for approximately 136 6 h 11. The DBAF formulation was even worse, reducing the ability of even untreated cells to overcome the osmolarity shifts needed for the preparation of the resting cells. I also tested the ability of the cells to remain viable in the resting state at CoCl2 concentrations (500 µM) that can be lethal to growing cells 16. Increasing metal stress on the resting cells did not affect their ability to restart growth once transferred to the growth medium (Fig. A.1B). Hence, not only does the NBAF formulation maintain the viability of resting cells for prolonged periods of time (9-12 h), but it minimizes cell intoxication by CoII. The lower membrane permeability predicted for NBAF-grown cells is expected to reduce metal penetration, providing an effective mechanism to overcome metal stress. These same properties maintain the integrity of the outer membrane through osmotic shifts and during prolonged periods of time in the resting state. Figure A.2 Growth recovery of NBAF-grown resting cells from CoII stress. Cells were grown and transferred in mid-exponential phase three times in NBAF medium before harvesting mid-exponential cells for the RC assays. Resting cells were challenged with 250 µM (A) or 500 µM (B) CoCl2 for up to 12 h before being transferred to NBAF medium and incubated at 30oC to reinitiate growth (A660). Untreated controls (0 µM CoCl2) were also included. Shown are the average of duplicate (untreated) and triplicate (CoII-treated) cultures. Error bars are the standard deviation between replicates. 137 Comparison of NBAF and DBAF: a matter of salt? The apparent differences in the ability of NBAF- or DBAF-grown cells to remain viable and metabolically active during and after time in the resting state is surprising. The two media types tested are similar in their composition and there is no clear deviation in the key elements provided 22. And yet, DBAF-grown cells maintained in the resting state for 6 h needed a much longer lag phase to restart growth than NBAF-grown cells, a sign of media-induced stress (Fig. A.3A). Indeed, the NBAF-grown cells recovered from a 6-h resting state after 17.5 ± 0.1 h. By contrast, DBAF-grown cells maintained for 6 h in the resting state took 75 ± 47 h to restart growth. The presence of CoII did not significantly impact the recovery of the NBAF-grown (20.0 ± 2.5 h lag phase, p = 0.4 vs untreated NBAF cells) or DBAF-grown cells (98 ± 37 h lag phase, p = 0.5 vs untreated DBAF cells) (Fig. A.3A). This suggests that the DBAF cells are more sensitive to osmotic shifts than metal stress. 138 Figure A.3 Comparison of NBAF and DBAF effects on resting cell recovery . (A-B) Growth phenotypes of cells recovered after 6 h in the RC assay treated with 250 µM CoCl2 (pink) or untreated controls (black). The lag phase (A) and generation times (B) of cells prepared with NBAF or DBAF media are presented as individual replicates (circles) and averages (lines). (C) Differences in salt content between the two media as well as the wash (WB) and reaction buffer (RB) used int the RC assay. Despite the long periods needed for DBAF cells to recover from the resting state, the cells achieved generation times (4.5 ± 0.5 h for untreated cells and 4.2 ± 1.3 h for treated cells, p = 0.7) similar to the NBAF cultures (3.8 ± 0.1 h for untreated cells and 3.9 ± 0.3 h for treated cells, p = 0.9) (Fig. A.3B). However, these generation times were more variable in the DBAF than in the NBAF cultures (Fig. A.3B), possibly reflecting the activation of different pathways to overcome osmotic stress in each replicate DBAF culture. The dramatic effect that seemingly similar media formulations have on the viability of cells prepared for the RC assays points to salt differences as the major determinant of whether G. sulfurreducens overcomes the stress associated with osmotic shifts. Indeed, 139 there is a 10.8 mM difference in the Na+ and K+ ion content between NBAF (100.2 mM) and DBAF (89.4 mM) (Fig. A.3C). These ions can have broad effects on cell physiology such as alteration of the ionic motive force within the membrane 25,26. They can also affect membrane fluidity and vesiculation in G. sulfurreducens (Morgen Clark, personal communication). These data therefore highlight the importance that growth media formulations have in producing cells suitable for RC assays and the impact that seemingly minor differences in salt content have on cell viability and/or metabolic activity. DISCUSSION Suspension in a resting state is a stressful experience for cells and can drastically affect viability 11. While a lack of nutrients and the washing process can inhibit recovery, the osmolarity of the buffers used is pivotal to survival 11. Our data show that osmolarity of the medium used to grow the cells for the RC assays also matters. As a result, cells previously grown on NBAF or DBAF media have different sensitivities to the osmotic shifts optimized to prepare resting cells of G. sulfurreducens (Fig. A.3). CoII challenge to the resting cells also impacts recovery from the resting state, albeit not as significantly as the osmotic transitions required for the preparation of resting cells. Thus, while NBAF-grown cells readily transitioned between the growth and resting states, DBAF-grown cells experienced significant losses of viability during the transition. This, in turn, reduced the concentration of resting cells that were able to resume growth and produced extended lag phases even in cells that had not experienced metal stress (Fig. A.3). The key difference between these two media formulations seems to be a 10.8 mM higher Na+/K+ salt content in the NBAF media compared to DBAF. This difference, though seemingly insignificant, can translate into osmotic pressure differences that may alter the membrane 140 stability of G. sulfurreducens 27. In support of this, cells grown in the medium with lower salt concentration (DBAF) have a nearly 2-fold increase in OMV production compared to NBAF (Morgen Clark, personal communication). Furthermore, amending the DBAF medium with Na+ and/or K+ salts to match the Na+/K+ content of NBAF ameliorates OMV levels (Morgen Clark, personal communication). The biogenesis and functions of bacterial OMVs are not fully understood 24 and are just beginning to be explored in G. sulfurreducens 28. It is however well-accepted that some cations can bind and neutralize the negative charge of the LPS that coat the outer membrane of Gram-negative bacteria, relieving the electromagnetic repulsion between the LPS molecules and stabilizing the membrane 29,30. Hence, the higher salt content in NBAF may stabilize the LPS and provide permeability control and osmoprotection. This is also expected to mitigate CoII permeation and the toxic effects derived from the accumulation of the metal in the periplasm 16. MATERIALS AND METHODS Bacterial strains and culture conditions Cultures of G. sulfurreducens strain PCA were obtained from our laboratory culture collection and grown anaerobically in either DB or NB minimal medium as previously described 22. Media were supplemented with 20 mM of acetate as the electron donor and 40 mM of fumarate as the electron acceptor. Growth was monitored spectrophotometrically as the absorbance at 600 nm (A600) and cultures were routinely grown to late exponential phase (A600, 0.5-0.6) prior to transfer to fresh medium. CoII resting cell assays Resting cell assays were performed as previously described 9 with some modifications. Unless otherwise indicated, all procedures were performed inside an anaerobic chamber 141 (COY Labs). Cultures of G. sulfurreducens were grown in either DB or NB media at 30oC to mid-exponential phase (A600 ~0.4) and dispensed into 50-ml conical tubes. 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