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DATE DUE DATE DUE DATE DUE NITRATE REDUCTION PATHWAY IN SHE WANELLA ONEIDENSIS MR-l by Claribel Cruz-Garcia A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Crop and Soil Sciences and Environmental Toxicology Programs Department of Crop and Soil Sciences 2005 ABSTRACT NITRATE REDUCTION PATHWAY IN SHEWANELLA ONEIDENSIS MR-l by Claribel Cruz-Garcia Shewanella oneidensis MIR-1 is a gram-negative bacterium with an extraordinary metabolic versatility in anion reduction, including the reduction of NO3’, Fe(III), U(VI), Mn(IV), Se(V I), Cr(VI). While reduction of nitrate and nitrite has been described for this microorganism, it is not known whether the reduction is by denitrification or dissimilatory nitrate reduction into ammonium (DNRA). By both physiological and genetic evidence, I proved that DNRA is the nitrate reduction pathway in this organism. Using the complete genome sequence of S. oneidensis MR-l , I identified a gene encoding a periplasmic nitrate reductase based on its 72% sequence identity with the napA gene in E. coli. Anaerobic growth of MR-l on nitrate was abolished in a site directed napA mutant I constructed, indicating that NapA is the only nitrate reductase present. The anaerobic expression of the napA and nrfA, a homolog of the cytochrome b552 nitrite reductase in E. coli, increased with increasing nitrate concentration until a plateau was reached at 3 mM KNO3. This indicates that these genes are not repressed by increasing concentrations of nitrate. The reduction of nitrate generates intermediates that can be toxic to the microorganism. To determine the genetic response of MR-l to high concentrations of nitrate, DNA microarrays were used to obtain a complete gene expression profile of MR- 1 at low (1 mM) versus high (40 mM) nitrate concentrations. Genes encoding transporters and efflux pumps were up-regulated, perhaps as a mechanism to export toxic compounds. In addition, the gene expression profile of MR-l, grown anaerobically with nitrate as the only electron acceptor, suggested that this dissimilatory pathway contributes to N assimilation. Hence the nitrate reduction pathway could serve a dual purpose. The role of EtrA, a homolog of Fnr (global anaerobic regulator in E. coir“) was examined using an etrA deletion mutant I constructed, S. oneidensis Etra7-1. The global transcriptome suggested a starvation response for anaerobic cultures of EtrA7-l when nitrate was the electron acceptor. Genes involved in the activation and synthesis of the LambdaSo, MuSol and MuSoZ prophages of MR—l were up-regulated, suggesting a phage infection. This could be responsible for the low growth yields observed for EtrA7- 1 when compared to the wild type. Starvation is a stress condition that is known to induce viral infections. Even though starvation was not directly targeted for examination, the results in this study suggest that EtrA might play an important role in the survival of MR- 1 under starvation. Moreover, the low biomass suggests a greater sensitivity of MR-l to starvation than the toxicity associated with high nitrate concentrations. Down-regulation of genes involved in the nitrate reduction pathway was also observed for EtrA7-1 relative to the wild type, which suggests a positive regulatory role for this protein in the nitrate reduction pathway of S. oner’densis MR-l. Copyright by Claribel Cruz-Garcia 2005 DEDICATION T o my family for all the support, love and encouragement. ACKNOWLEDGEMENTS I would like to thank the College of Agriculture at Michigan State University especially Dr. James E. Jay for all the support he gave me when I most needed it. Thanks to him, Dr. Melvin Yokoyama and Dr. Tony Nufiez, I was able to continue and finish my PhD. I need to give special thanks to my advisor, Dr. James M. Tiedje, for accepting me in his laboratory even when my background was unrelated to his work. Dr. Tiedje always demonstrated trust and faith in his students and for him there is nothing impossible. This great quality inspired me and motivated me to continue and finish my goal. I was very lucky of having a second but not less great advisor, Dr. Alison E. Murray. She introduced me to this research area with a passion and such enthusiasm that was impossible to walk away from it. Her encouragement and positive enforcement motivated me to explore and get involved in all this new research area. Even in the distance, she was able to help me and always managed to have time for long conversations and great scientific discussions. I also need to thank all the people at the Tiedje Lab, especially Héctor L. Ayala, Veronica Grilntzig, Xiaoyun Qiu, Carlos Rodriguez Minguela, Claudia Etchebehere, Mary Beth Leigh, Elica Moss, and Debora Rodriguez for all the laughs and the good times and for always being there for me to help with good scientific and personal advices. I am very grateful of Lisa Pline, Pat Englehart and Joyce Wildenthal for all the help, great attentions and beautiful friendship. Many people at Michigan State University help me with their expertise in some of the analyses presented in this thesis. Thanks to Jonathan Dahl at Soil Testing Lab for the N-species analyses and Annette Thelen and Jeff Landgraph at the Genomic Technical Support Facility for helping with the Q-RT-PCR and DNA microarray analyses. I would also like vi to thank Liyou Wu and J izhong Zhou at Oakridge National Laboratory for providing the Shewanella oneidensis MR-l complete DNA microarray slides. I would like to thank the members of my graduate committee, Dr. Clayton Rugh and Dr. Syed Hashsham, for all the help and the good professional and personal advices. Thanks to Dr. Joel Klappenbach for revising my writing and for the help in the mutagenesis process. My friends in Michigan made of this experience a very sweet one, being away from home was not easy. The Figueroa family, the Sullivan-Figueroa family, the Jimenez family, the Caulkett family, the Estrada-Feliu family, Jaime Graulau, Juliana Pérez, Simone Charles, Tomeka Prioleau, Pedro Torres and Michelle Martinez became the family and friends that we could always count on. Thank you all. I do not have words to thank my family for all their love, support and patience. I need to thank my dad, Carlos Cruz, for being my greatest inspiration and my model. Thanks for being the greatest dad ever, for all the love and endless words of encouragement. Thanks to my mom for all the love and her faith in me. Thanks for all the encouragement and the guidance throughout my school years. This title is yours. I need to thank God for my wonderful sisters Brenda Cruz and Waleska Cruz, for always being there for me with words of love and hope. I have to thank all my grandparents, aunts, uncles and cousins for all the prayers and good wishes. Gustavo and Carlos G. Sepulveda, the light in my life, thank you for the patience, help, love and support. Gustavo, thanks for all the nights spent in the lab helping me with experiments and data analyses. I am very grateful of the scientific discussions and the challenging questions. Thanks for the love and understanding during the bad times and vii moreover, thanks for Carlos and for all the happiness you have brought into my life. I have to thank the Sepulveda Family for taking me into their family and for all the prayers. Thank you God for given me this Opportunity and for always being with me. viii TABLE OF CONTENTS LIST OF TABLES ................................................................................. xi LIST OF FIGURES ................................................................................ xii LIST OF SUPPLEMENTAL TABLES ........................................................ xiv CHAPTER I. INTRODUCTION AND RATIONALE ................................................ 1 OBJECTIVES AND EXPERIMENTAL APPROACHES .......................... 15 REFERENCES ............................................................................ 20 CHAPTER II. REDUCTION OF NITRATE IN SHE WANELLA ONEIDENSIS MR-l ................... 26 ABSTRACT ............................................................................... 27 INTRODUCTION ......................................................................... 28 MATERIALS AND METHODS ....................................................... 30 RESULTS .................................................................................. 32 DISCUSSION .............................................................................. 37 REFERENCES ............................................................................ 41 CHAPTER III. NapA IS THE ENZYME RESPONSIBLE FOR THE REDUCTION OF NITRATE IN SHE WANELLA ONEIDENSIS MR-I ............................................................ 44 ABSTRACT ............................................................................... 45 INTRODUCTION ......................................................................... 46 MATERIALS AND METHODS ....................................................... 49 RESULTS .................................................................................. 58 DISCUSSION .............................................................................. 78 REFERENCES ............................................................................ 84 CHAPTER IV. ROLE OF EtrA IN THE REGULATION OF THE NITRATE REDUCTION PATHWAY IN SHE WANELLA ONEIDENSIS MR-l ........................................ 88 ABSTRACT ................................................................................ 89 INTRODUCTION ........................................................................ 90 MATERIALS AND METHODS ....................................................... 92 RESULTS ................................................................................. 101 DISCUSSION ............................................................................ 120 REFERENCES ........................................................................... 129 CHAPTER V. SUMMARY AND FUTURE RESEARCH ................................................... 134 SUMMARY .............................................................................. 135 FUTURE RESEARCH .................................................................. 137 ix REFERENCES ........................................................................... 141 APPENDIX A. SHE WANELLA ONEIDENSIS MR-l DELETION MUTANTS. . . . . . 142 APPENDIX B. SUPPLEMENTAL TABLES ................................................. 146 TABLE 1.1 TABLE 2.1 TABLE 3.1 TABLE 3.2 TABLE 3.3 TABLE 4.1 TABLE 4.2 TABLE 4.3 TABLE 4.4 TABLE 4.5 LIST OF TABLES Genes involved in nitrate reduction in E. coli and their homologues in Shewanella oneidensis MR-l ................................................ 5 Stoichiometry of nitrogen ions measured in Shewanella oneidensr's MR-l anaerobic cultures with 2 mM KNO3 as the electron acceptor and lactate as the electron donor ............................................... 36 Bacteria, plasmids, primers and oligonucleotides used in this study. .....51 Genes induced in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO3 ............................................................. 73 Genes repressed in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO3 ............................................................ 76 Bacteria, plasmids, and primers used in this study ........................... 93 Genes induced in anaerobic cultures of EtrA7-1 relative to the wild type (reference strain) ........................................................... 112 Genes repressed in anaerobic cultures with nitrate of EtrA7-1 relative to the wild type (reference strain) ............................................. 115 Genes induced in anaerobic cultures of EtrA7-1 at 1 mM (reference) versus 40 mM KNO3 ............................................................ 118 Genes repressed in anaerobic cultures of EtrA7-l at 1 mM (reference) versus 40 mM KNO; ............................................................ 119 xi FIG. 1.1 FIG. 2.1 FIG. 2.2 FIG. 2.3 FIG. 2.4 FIG. 3.1 FIG. 3.2 FIG. 3.3 FIG. 3.4 FIG. 3.5 FIG. 3.6 FIG. 3.7 FIG. 3.8 LIST OF FIGURES Model proposed for the regulation of the expression of the mf and nap genes in Escherichia coli ................................................... 12 Growth of Shewanella oniedensis MR-l on different concentrations of nitrate .................................................................................................... 33 Growth of Shewanella oneidensis MR-l on different concentrations of nitrite ............................................................................ 33 Growth rate of Shewanella oneidensis MR-l in anaerobic cultures at different nitrate and nitrite .................................................... 34 Nitrate, nitrite and ammonium concentrations in Shewanella oneidensis MR-l in anaerobic cultures grown in the presence of nitrate as the electron acceptor ........................................................... 36 Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensr’s MR-l used for Q-RT-PCR analyses at the time the cells were harvested for RNA extraction .................................. 59 Expression of napA in cultures of Shewanella oneidensis MR-l grown at different nitrate concentrations under anaerobic conditions (0), aerobically with no nitrate (I) and aerobically with 3 mM nitrate ( ). . .60 Expression of nrfA in cultures of Shewanella oneidensis MR-l grown at different nitrate concentrations under anoxic conditions (9), aerobically with no nitrate (I) and aerobically with 3 mM nitrate ( ). . .62 napA gene deletion confirmation by PCR ..................................... 64 DNA sequence of napA deletion in MR-l .................................... 65 Growth curves for Shewanella oneidensr’s MR-l wild type grown in M1 medium aerobically (O), and anaerobically with 3 mM nitrate ( ) and Shewanella oneidensis MR-l AnapA grown aerobically (I) and anaerobically with 3 mM nitrate ( -') ...................................... 67 Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensr's MR-l wild type and Shewanella oneidensr's MR-l AnapA for growth curve after 24 h incubation period ............... 68 Distribution of differentially expressed genes (> 2-fold change) grouped in 20 functional categories after cultivation on 1 mM (reference) versus 40 mM nitrate concentration .................................................... 72 xii FIG. 4.1 FIG. 4.2 FIG. 4.3 FIG. 4.4 FIG. 4.5 FIG. 4.6 etrA gene deletion confirmation by PCR ..................................... 102 DNA sequence of the etrA deletion in MR-l ................................. 103 Growth of Shewanella oner’densis MR-l wild type (4), EtrA7-l (I), EtrA7-l complement ( ),and EtrA7-1 harboring pCM62 (a) under anaerobic conditions with 3 mM KNO3 .............................. 105 Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensis MR-l wild type , EtrA 7-1, Etra7-1 complement (harboring pCCG02c) and EtrA7-1 harboring pCM62 during growth curve after 10 h incubation period .......................................................... 105 Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensis MR-l wild type, EtrA 7-1, Etra7-1 complement (harboring pCCG02c) and EtrA7-1 harboring pCM62 during growth curve after 23 h incubation period .......................................................... 106 Distribution of differentially expressed genes (> 2-fold change) grouped in 20 functional categories in anaerobic cultures of EtrA7-1 with 2 mM KN03 as the sole electron acceptor relative to the wild type (reference strain) .............................................................................. 108 xiii LIST OF SUPPLEMENTAL TABLES SUPPLEMENTAL TABLE A. 1. Shewanella oner'densis MR-l deletion mutants and primer sequences ............................................................................................ 145 SUPPLEMENTAL TABLE B.l. Genes induced in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO3 ........................................................ 147 SUPPLEMENTAL TABLE B.2. Genes repressed in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO3 ..................................................... 167 SUPPLEMENTAL TABLE 33. Genes induced in anaerobic cultures of EtrA7-1 relative to the wild type (reference strain) ...................................................... 184 SUPPLEMENTAL TABLE B.4. Genes repressed in anaerobic cultures with nitrate of EtrA7-1 relative to the wild type (reference strain) ...................................... 194 SUPPLEMENTAL TABLE 35. Genes induced in anaerobic cultures of EtrA7-1 at 1 mM (reference) versus 40 mM KNO3 ..................................................... 206 SUPPLEMENTAL TABLE B.6. Genes repressed in anaerobic cultures of EtrA7-l at 1 mM (reference) versus 40 mM KNO3 ...................................................... 212 xiv CHAPTER I INTRODUCTION AND RATIONALE INTRODUCTION Nitrogen is one of the crucial elements in the building blocks of life: nucleic acids and proteins. Nitrogen exists in nature in various oxidative states that range from +5 for nitrate (the most oxidized specie) to -3 for ammonium (the most reduced compound). Nitrogen compounds found in the biosphere undergo several transformations, which constitute the biogeochemical nitrogen cycle. These transformations that are predominantly carried out by bacteria and archaea regulate the local and global concentration of each nitrogen form. Thus, these processes are important in maintaining the various nitrogen species in balanced or non-harmful levels. Nitrate is one of the most important nitrogen species in agriculture and in the environment. Due to excessive use of fertilizers, nitrate has become a contaminant in groundwater. Nitrate is also of clinical concern since elevated levels in drinking water have been associated with some forms of cancer and methaemoglobinemia (11, 18, 49). Nitrate is a reactant in three pathways in bacteria. (i) Assimilatorv nitrafi reduction is one of the most important biological transformations since it provides a nitrogen source for organism growth. This pathway reduces nitrate to nitrite, and finally to ammonium, which is used for the synthesis of amino acids. (ii) Nitrate respiration uses nitrate as the electron acceptor for the generation of energy from a proton motive force. It is a component in denitrification, which is a respiring process where nitrate is converted sequentially into dinitrogen (NO3'—>N02'—)NO—>N20—+N2). The electron accepting capacity of nitrate in denitrification is 5 electrons per nitrogen. Nitrogen oxide gases produced as intermediates in this pathway are associated with the green house effect and ozone depletion. In agriculture, this process represents an area of great concern because nitrogen is the most limiting nutrient in crop cultivation and significant loss of nitrogen fertilizer is attributed to denitrification. On the other hand, bacterial denitrification is used for nitrate removal as part of a wastewater treatment prior to the release in the environment. (iii) Dissimilatorv nitrate reduction into ammonium NRA is an important process in some anaerobic environments. Three possible functions have been proposed for DNRA: nitrite detoxification, redox balancing, and energy generation. DNRA is characterized by the reduction of nitrate to nitrite but, instead of being reduced to N2 as in denitrification, nitrite is reduced directly to ammonium. In contrast to denitrification, DNRA conserves nitrogen, which makes it advantageous. This process is most favorable in anaerobic environments due to the high capacity of nitrate to accept electrons when reduced into ammonium (8 electrons per nitrogen). Due to its importance, nitrate reduction has become the subject of many research studies. However, there are crucial aspects that are yet to be elucidated. Shewanella oneidensr’s MR-l Shewanella oneidensis MR-l is a Gram negative, facultatively anaerobic, polarly flagellated y proteobacterium. Formerly known as Shewanella putrefaciens, MR-l was first isolated from the Oneida Lake, NY, USA from which its name was acquired. This bacterium has been found in soil, sediments, the water column and clinical environments (19, 51). Bacteria from this genus have been studied for decades, but due to recent findings, which identify Shewanella oneidensis MR-l’s potential metal bioremediation, there has been increasing interest to better understand the metabolic capabilities of this organism (20, 47, 51). Unlike most isolated bacteria, MR-l is a metal-ion reducer and has the advantage of utilizing a wide array of compounds as electron acceptors including oxygen, nitrate, nitrite, fumarate, thiosulfate, elemental sulfur, dimethyl sulfoxide, trimethylamine N- oxide, iron oxide, manganese oxide, chromium oxide and uranium oxide (19). The Institute for Genomic Research (TIGR) released the genome sequence of Shewanella oneidensis MR—l (NCOO4347 whole genome, NC004349 megaplasmid) in 2002. The genome sequence revealed the presence of numerous genes that encode for an array of electron transport systems including up to 39 c-type cytochromes, the greatest number identified in a bacterial genome thus far. Although the genome does not show an extremely high number of metal ion transporters, the large number of cytochrome genes might explain the metabolic versatility of this microorganism. Studies have indicated that MR-l can utilize nitrate and nitrite as electron acceptors in a two-step reaction coupled to growth where nitrate is reduced to nitrite and nitrite into ammonium (21). In addition, studies have indicated that MR-l can reduce Cr(IV) by co-metabolism with nitrate reduction, for energy acquisition (25). MR-l reduces U(V I) and Cr(IV) into the insoluble forms U(IV) and Cr(VI), which are immobile, thus less toxic and bioavailable. Chromium reduction by co-metabolism with nitrate has been observed in S. oneidensis MR-l while reduction of uranium has been associated with nitrite reduction in other members of the genus (52). Even though ammonium has been detected as a product of nitrate reduction in MR-l , there is a controversy about whether denitrification is taking place in the cell (14, 21). According to the genome sequence of Shewanella oner'densr’s MR-l, the genes necessary to carry out this process are absent; however, those required for DNRA are present (TABLE 1.1). TABLE 1.1. Genes involved in nitrate reduction in E. coli and their homologues in Shewanella oneidensr's MR-l. narGHJI narZYWX narL, narX narP, narQ nrfABCDEFG ner fnr napFDA GHBC napDAGHB, napF, cyruA (napC homologue) narP, narQ nrfA, nrflr‘, nrflJCG etrA Periplasmic nitrate reductase Membrane-bound nitrate reductase Membrane-bound nitrate reductase Two-component regulatory system nitrate sensitive Two-component regulatory system nitrate sensitive Periplasmic nitrite reductase Membrane-bound nitrite reductase Oxygen sensing regulator Dashed lines represent no homologues found in MRI-1. Genes and proteins involved in bacterial nitrate reduction Most of the genetic and biochemical studies on nitrate respiration and DNRA have been done in E. coli (26, 39, 45, 46). Two types of bacterial nitrate reductases have been described: assimilatory nitrate reductases (Nas) and dissimilatory nitrate reductases (Nap and Nar). Assimilatory nitrate reductases have been found in phototrophic and heterotrophic bacteria. They are comprised of two types, ferredoxin- or flavodoxin-dependent. These enzymes reduce nitrate to nitrite, which is then fiuther reduced into ammonium and incorporated into cell material. Genes involved in assimilatory nitrate reduction are found in the same operon as those involved in assimilatory nitrite reduction. Different nomenclature has been given to homologous genes of these enzymes in different bacteria but the nas term is more commonly used and some authors believe it is more appropriate (26, 38). In contrast to the assimilatory nitrate reductases, many types of dissimilatory nitrate reductases have been observed in bacteria and archaea. A single organism may possess more than one type (e.g. E. coli expresses three dissimilatory nitrate reductases) (5, 10, 32, 45). These enzymes can be distinguished not only by their structure and gene sequence but also by their biochemical properties (5, 26, 39, 46). There are two types of dissimilatory nitrate reductases: the membrane-bound respiratory (Nar) and the periplasmic (Nap) nitrate reductases. In E. coli the membrane-bound nitrate reductases can be further divided into NarZ and NarA. NarZ, encoded by narZYWV genes, is constitutively expressed in the cell at low levels under aerobic or anaerobic conditions (5, 26). NarA, encoded by narGHIJ genes, and in contrast to NarZ, is only expressed under anaerobic conditions. The activity of NarA represents 90% of the total activity of the membrane-bound nitrate reductases in E. coli K12 (26). The bacterial membrane-bound nitrate reductase has three subunits: or (112- I4OKDa), B (52-64KDa) and y (19-25KDa). Nar uses NADH as an electron donor, can reduce chlorate and is inhibited by low concentrations of azide (3, 21, 45). This enzyme has been found in microorganisms capable of denitrification and anaerobic respiration (26). This protein is expressed under anoxic conditions. Since it is membrane-bound, it has the ability to generate energy by a proton motive force. In contrast to Nar, the periplasmic nitrate reductase (Nap) is expressed under aerobic and/or anaerobic conditions and its activity is not affected by low concentrations of azide (3, 45). Several biochemical properties have been used to identify NapA from NarG but the two most common are inability to use NADH as an electron donor and to reduce chlorate. Nap has been purified as a two-subunit enzyme consisting of a molybdopterin-containing catalytic subunit A (~90 KDa) and a [4Fe4S] cluster and a diheme cytochrome 0552 subunit B (~16KDa). Since Nap cannot generate a proton motive force for energy conservation due to its location (periplasm), different roles have been attributed to this enzyme in different bacteria. Experiments on the Nap systems of Thiosphaera panthotropha, Rhodobacter capsulatus and Rhodobacter sphaeroides DSM 158 suggest a redox-balancing role when there is an excess of reducing agents (37, 38, 41). Due to the presence of Nap in most aerobic denitrifiers, a second role has been suggested for Nap and that is as the enzyme responsible for the first step in aerobic denitrification (3, 33). These studies suggest that since Nap is expressed under aerobic and anaerobic conditions, it plays a role in the transition from aerobic to anaerobic respiration. Once the conditions become more anaerobic, Nar is the primary enzyme and Nap plays a secondary role. However, a study on Pseudomonas sp. strain G-179 showed that while this is true for P. denitrificans it is not for Pseudomonas sp. strain G-l79 where Nap is the primary enzyme required for the reduction of nitrate into nitrite in denitrification (3). This investigation suggested that the periplasmic nitrate reduction in this organism generates energy by showing the inability of Nap' mutants to grow on nitrate. The mechanism of energy generation using Nap in this organism is not clear. Pseudomonas sp. strain G-179 was originally classified as a Pseudomonas but a phylogenetic analysis using the 16S rDNA sequence of various nitrate reducers revealed a 97% similarity to Rhyzobium galegae (3). In E. coli, Nap is encoded by the napFDAGHBC operon. Studies in this organism have demonstrated that NapABCD, but not NapFGH, are essential for periplasmic nitrate reduction when glycerol, forrnate and glucose are used as substrate (32). In an attempt to elucidate the role of NapFGH, mutants defective in either ubiquinol or menaquinol biosynthesis, revealed that NapG and H, but not F, are essential for electron transfer from ubiquinol to NapAB (5). This investigation also proved that NapC has an essential role in electron transfer from both ubiquinol and menaquinol to NapAB. In further investigations, the deletion of either napG or napH abolished the activity of NapA in cultures of naphtoquinone defective strains of E. coli K12 (6). This study indicates that napG and napH encode an electron transfer complex. The function of this complex seems to be important to maintain a redox-balancing growth. Several studies in the Nap systems of enteric bacteria have shown expression only under anaerobic conditions (32, 33, 45, 53). This contrast to the biochemical properties observed for the Nap in some denitrifiers, however this difference has been attributed to the variation in the organization of its operon (45). This group of bacteria, including E. coli, is capable of DNRA, but not of denitrification, which suggests a different but unclear role for this system. The genome sequence of various microorganisms reveals a pattern in the genetic organization of the nap operon that might determine its physiological role (32, 33). In all organisms, for which the genome sequence has been published, the nap operon consists of napDABC genes as a common template. In addition to this template, different permutations of other genes have been documented in different types of bacteria. For example, the nap operon in denitrifiers consists of five genes napEDABC, whilst E. coli, which is a non-denitrifier and other ammonifying bacteria do not carry the napE gene but contain napGHF genes in addition to the napDABC. Pseudomonas sp. G-179, which utilizes a different denitrification pathway, as described above, possesses a nap operon that even when it contains the napE gene, is different from that of the denitrifiers (napEFDABC). The nap operon of Shewanella oneidensis MR-l is different from any nap operon described; this strain does not possess napC or napF (napDAGHB) (19). The organization of the nap operon of MR-l is very interesting since the internal genes are arranged as those in the nap operon of E. coli, however homologues of napF and napC (cymA) are found in different loci (30). This genetic organization may explain similarities and uniqueness of the nitrate reduction pathway of MR-l compared to those in E. coli and in other bacteria Recent experiments on E. coli have demonstrated that the nap operon is preferentially expressed under low concentrations of nitrate and/or nitrite, whilst the nar Operon is expressed when the concentrations of nitrate and/or nitrite are high (33, 53). In an effort to demonstrate whether the Nap system was an energy generating process in E. coli, an experiment was performed using NarA'/NarZ' mutants, thus only Nap was carrying out any nitrate reduction in the cell. Since the mutant grew poorly, a second mutation was performed abolishing NarL (response regulator) activity. The NarL' mutation resulted in improved growth, which confirms that the poor growth was partly due to repression of nap expression by NarL. Another experiment using NarL' mutants expressing only nap as the sole nitrate reductase, demonstrated that Nap activity could be high enough to support anaerobic respiration but that it does not constitute a site to generate a proton motive force (45). This experiment confirmed the observations for the regulation of the expression of nar and nap operons. The results demonstrated that NarL and NarP induce the nap and nor genes in response to NarX and NarQ (12, 13). E. coli possesses a nitrate and nitrite two-component regulatory system consisting of NarX, NarQ, NarP and NarL. NarQ and NarX are the two sensor transmitter proteins while NarP and NarL are the two response regulators. It has been shown that NarP and NarL compete for the same DNA-binding site. They both induce nar and nap but it has been shown that NarL, but not NarP, represses nap at high concentrations of nitrate. This has also been observed and studied in depth for the nitrite reductase systems reported in E. coli (54). The reduction of nitrate is followed by the action of a nitrite reductase. E. 0011' has two nitrite reductases encoded by the nrfABCDEFG and the nirB operons, which reduce nitrite into ammonium (7, 9, 54). Even though these enzymes catalyze the same reaction they have different roles and biochemical properties. NirB is a cytoplasmic enzyme while NrfA is located in the periplasm. NarL, NarP, NarQ and NarX regulatory systems along 10 with the regulatory protein Fnr regulate the expression of these two enzymes (31, 48, 54, 57). In a study of E. coli, NrfA was highly expressed under low concentrations of nitrate and/or nitrite while NirB was induced only under high concentrations of nitrate (54). Hence, NirB was proposed to have a role in detoxifying the cell from high levels of nitrite produced by the action of NarG, which is also induced under high concentrations of nitrate. The authors also suggested that NirB might recycle NADH by oxidizing it when there is an excess of reducing equivalents. On the other hand, since NrfA is expressed under low concentrations of nitrate and inhibited at high concentrations, it may work together with Nap using the nitrite that it generates. As previously mentioned, recent investigations suggest a redox-balancing role for NapA in E. coli conferred by a NapG/NapH complex (6). It is still not clear whether E. coli can generate energy from this process but it has been suggested that Nap and Nrf form a complex where some of their components are membrane-associated enabling the conservation of energy (34). A model for the regulation of these genes in E. 6011' establishes that at low concentrations of nitrate, NarP and NarL are autophosphorylated, which will allow them to bind the activation sites at positions —79 and —70 of the mfA operon (FIG. 1.1). Fnr will bind to its recognition site in the operon and the expression of mf will be induced As nitrate reaches a higher concentration, NarL will also bind to —50 and —22 sites, for which the affinity is lower. At site -50 NarL will interfere with Fnr binding and at site —22 it will interfere with RNA polymerase binding to the promoter. These will cause suppression in the expression of nrf(45, 53, 54). ll Expression Sggntratlons level of nap and MA OmM no, flan Ol’ <1mM N03. ”if NO! NO? . . __._L‘Lm "8P“ High >101" N03. 1 _ NI! f f rm f f fix. it: w 'ItL’Ef‘l Him L“ FIG. 1.1. Model proposed for the regulation of the expression of the nrf and nap genes in Escherichia coli. When the concentrations of nitrate are low, the expression level of these genes is induced by the response regulator NarP and the transcription factor Fnr. Once the nitrate concentrations increase, NarL gets activated and blocks the sites for Pm and NarP, therefore the expression of napA and mfA is repressed (Wang and Gunsalus, 2000; Potter et al., 2001). 12 The benefits of periplasmic nitrate reduction in terms of energy acquisition are not completely clear. Many experiments to elucidate the role and mechanism of this pathway have revealed intriguing results on the physiology of various organisms that keep challenging researchers around the world. The nitrate reduction process in S. oneidensis MR-l seems to be even more interesting, since it is very likely that periplasmic nitrate reduction into ammonium is the primary nitrate reduction pathway used by this organism when nitrate is the only electron acceptor. S. oneidensis MR-l possesses homologues for the genes that have been reported as necessary to carry out only this pathway (TABLE 1.1). Previous studies have suggested that S. oneidensis MR-l is capable of denitrification, however this has been a subject of controversy (14, 21). An experiment with different strains of Shewanella putrefaciens (including MR-l) using nitrate and nitrite as sole electron acceptors has primarily detected production of N20, and ammonium in low concentrations in the cultures of MR-l (21). This study also describes the partial purification of a membrane-bound nitrate reductase from MR-l, which the authors claim corresponds to Nar, and a membrane-bound nitrite reductase. These results do not coincide with the genome sequence of S. oneidensis MR-l since only the nap and nrfoperons are present, which encode periplasmic proteins. The study of the nitrate reduction pathway in Shewanella oneidensis MR-l has been limited to physiological investigations. There has been a lack of detailed genetic studies. Understanding expression of the genes involved in this process in MR-l is needed to clarify some of the questions regarding the role of the Nap system in this organism. Also, genetic expression analyses can help understand the metabolism of nitrate and its potential to conserve energy for cell survival in other organisms, some of 13 them pathogens like Haemophilus influenzae (34). Since MR-l is genetically similar to E. coli, the models discussed above can be used to help explain what is occurring in MR-l (27). The evolutionary implications of the absence of the napF and napC homologues from the nap operon of MR-l might help answer more questions about the pathway. Several studies mentioned above have been performed using mutants defective in alternative nitrate reduction pathways, which have given rise to important models proposed for this system. Based on these approaches and on the fact that the genome sequence of MR-l does not show the genes for the NarL/NarX two-component regulatory system, and Nap is the only nitrate reductase encoded, this is an excellent organism to study and to confirm what has been proposed for this system without the need of extensive DNA manipulation. This research focuses on the study of the nitrate reductase(s) involved in the reduction of nitrate and the elucidation of this mechanism in Shewanella oneidensis MR-l. This investigation also clarifies some of the contributions of the periplasmic nitrate reduction to the anaerobic metabolism, which is an important process in the global nitrogen cycle. 14 OBJECTIVES AND EXPERIMENTAL APPROACHES Objective 1. Determine the growth rate of Shewanella oneidensis MR-l when either nitrate, nitrite or nitrous oxide was used as the only electron acceptor. This objective will not only provide the optimal concentrations of nitrate and nitrite necessary for growth but will also provide information about the tolerance of this microorganism to the levels of nitrate and nitrite supplied. The growth of Shewanella oneidensis MR-l was examined and compared to that of the positive control, Pseudomonas stutzeri, for the reduction of nitrous oxide when the later was the only electron acceptor. Nitrous oxide is one of the intermediates in denitrification and this experiment is positive evidence for this pathway. Objective 2. Determine the intermediates in the nitrate reduction pathway in S. oneidensis MR—l by their sequential production and consumption. The growth rate, and the concentration of nitrate, nitrite and ammonium were analyzed over time in anaerobic cultures of Shewanella oneidensis MR-l grown in minimal medium with 2 mM KNO3 as the only electron acceptor. This experiment also establishes the balance of N-compounds. Objective 3: Determine the gene expression pattern of S. oneidensis MR-l when grown anaerobically under different concentrations of nitrate. To quantify napA, nrfA, narQ and narP mRNA copy numbers, Quantitative-Real Time- PCR was used (8). The principle of Q-RT-PCR resides in a constant monitoring of the 15 PCR amplification of the targeted template allowing the quantification of the initial gene copy number. The cautious design of a set of primers, and sometimes of a probe, that anneals between the primers specific for the gene of interest is important for the success of the Q-RT-PCR. The TaqMan technology from Applied Biosystems was used for the Q-RT-PCR analysis. This technology requires the addition of the probe that contains a reporter dye at the 5’end and a quencher at the 3’end (www.appliedbiosystems.com). The amplification of the template starts with the annealing of the primers and the probe to the template following the elongation step by the DNA polymerase. Once the polymerase encounters the probe, it degrades the probe by its 5’ nuclease activity. At this point the dye is released and separated from the quencher, which was keeping it from expressing all the fluorescence. Fluorescence increases with the increase in amplification of the template. This fluorescence is then detected by a RT PCR machine through an optic fiber. These data are translated into a value known as the CT or threshold cycle which is calculated from the curve generated from the fluorescent measurements recorded every few seconds. In order to ensure accuracy of the technique, many safety measures should be taken into consideration. For example, it is important to include a standard and an internal control in the experiment if an absolute quantification is desired. The standard could be a PCR product of the gene of interest in a known concentration. This will allow the extrapolation of the CT to the actual concentration or copy number of the targeted transcript. The internal control should be a gene that does not change between conditions or treatments, so it can be utilized to account for mechanical errors and for reaction inhibitors. Primers and a probe specific for the gene chosen as control are used to 16 quantify its transcripts as it is done for the genes of interest. Quality of the samples and the use of quality reagents are also critical for obtaining accurate results. Objective 4: Analyze the global gene expression profile of S. oneidensis MR-l when grown anaerobically under high and low concentrations of nitrate. In order to globally analyze the difference in gene expression under a low and a high concentration of nitrate, a Shewanella oneidensis MR-l complete genome DNA microarray was used (17). The spotted DNA microarray contains 4648 unique fragments representing individual open reading frames from Shewanella oneidensis MR-l. Total RNA extractions fi'om MR-l anaerobic cultures grown on 1 mM and 40 mM KNO3 were labeled with two different fluorescent dyes and hybridized in triplicate on these microarray slides. This experiment examines the genetic or molecular behavior of the cell when the concentrations of nitrate are increased dramatically. Objective 5: Determine existence of more than one nitrate reductase in MR—l. The construction of a nap' mutant was performed using the Cre-lox mutagenesis protocol for Gram-negative bacteria (24). Mutagenesis in Shewanella oneidensis MR-l as well as in many other bacteria has been a great challenge (23). This protocol had to be modified in order to overcome some of the obstacles presented when it was applied to MR-l. This protocol consists of four major steps, which will be discussed briefly. The first step is the insertion of the PCR products of the regions flanking the nap gene by ligation. This procedure requires the design of two pairs of primers (amplifying a region of 400-600 bp 17 in length for better efficiency of recombination) for the amplification of the regions flanking the nap gene. Each primer is designed so their respective 5’ termini posses complementary tags to permit insertion into the suicide vector. This can be accomplished if the tags include an enzyme recognition site (two different tags were used per primer set to allow directional insertion of the fragments). The napA flanking regions are then cloned into the suicide vector, which in this case was pCM184. This suicide vector possesses a kanamycin resistance (Kan) cassette flanked by two loxP sites and two multiple cloning sites. The flanking regions of the napA gene were ligated, flanking the two loxP sites. Once the vector is characterized by sequencing, the second step is its electroporation into E. coli [32155 competent cells for subsequent transference of the vector into MR-l. This E. coli strain is an auxotroph for di-amino-pimelic acid (DAP), which in its absence disables the growth of the bacterium. This is a great advantage because, after the mating E. coli B2155 harboring the vector with the flanking regions of the gene of interest and Shewanella oneidensis MR-l, they are spread on LB plates with kanamycin but no DAP. This media will allow only the growth of the MR-l cells that were successfully transformed with the vector. Since this is a suicide vector it is expect to be eliminated as the bacteria duplicates, because it should not replicate in MR-l. Unfortunately, that was not the case and the vector remained in the cell. Hence, another vector, pKNOCK-Gm, known to be a suicide vector in MR-l was then selected and the fi‘agment including the flanking regions of napA, loxP sites and the kanamycin cassette from pCM184 were excised by enzymatic digestion and moved into the multiple cloning site of pKNOCK-Gm (as the name implies, it is gentarnycin resistant) (1). Then, this vector (pCCGOI) was electroporated into E. coli 02155 competent cells, from which it 18 was subsequently transferred by conjugation into MR-l. The MR-l kanamycin resistant, gentamycin susceptible colonies were then diagnosed for the deletion by PCR. The kanamycin cassette replaces the gene by homologous recombination between the flanking regions in the suicide vector with those in the chromosome. The third step involves the introduction by conjugation of a plasmid encoding the Cre recombinase (pCM157) into the mutated colonies. The Cre recombinase recognizes the loxP sites flanking the kanamycin cassette and recombines them, excising the kanamycin cassette and leaving a residual loxP sequence. The fourth and last step is the curing of the plasmid. This was achieved by inoculating the positive transforrnants in liquid media without tetracycline, the antibiotic for which this plasmid confers resistance, and it is transferred until the resistance is lost. Characterization of the mutant was performed by diagnostic PCR and DNA sequencing. Growth on nitrate as the only electron acceptor was tested for the mutant. Objective 6: Determine the role of EtrA in the nitrate reduction pathway of Shewanella oneidensis MR-l. A S. oneidensis MR-l etrA deletion mutant was generated as described above. The growth, nitrate reduction capabilities and global gene expression profile when cultivated with nitrate as the only electron acceptor Were analyzed in comparison with those of the wild type. DNA microarray analyses were done as described above. The results obtain from this work will resolve how S. oneidensis MR-l reduces nitrate and provides insight into the global regulatory control of this process in response to nitrate concentrations. 19 10. REFERENCES . Alexeyev, M.F. 1999. The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. 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Isolation of U(VI) reduction-deficient mutants of Shewanella putrefaciens. FEMS. Microbiol. Lett. 184:143-8. 53. Wang, H., C.-P. Tseng, and R.P. Gunsalus. 1999. The napF and napG nitrate reductase operons in Escherichia coli are differently expressed in response to submicromolar concentrations of nitrate but not nitrite. J. Bacteriol. 18125303- 5308. 54. Wang, H., and R.P. Gonsalus. 2000. The mfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. J. Bacteriol. 182:5813-5822. 55. Ye, R.W., W. Tao, L. Bedzyk, T. Young, M. Chen, and L. Li. 2000. Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions. J. Bacteriol. 182:4458-4465. 56. Yuen, T., E. Wurmbach, R.L. Pfeffer, B.J. Ebersole, and SC. Sealfon. 2002. Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res. 30:e48. 57. Zumft, W.G. 2002. Nitric oxide signaling and NO dependent transcriptional control in bacterial denitrification by members of the FNR-CRP regulator family. J. Mol. Microbiol. Biotechnol. 4:277-286. 25 CHAPTER II Reduction of Nitrate in Shewanella oneidensis MR-l 26 ABSTRACT Nitrate is an important environmental resource and can also be a contaminant. Bioremediation of nitrate can be achieved by nitrate reduction to dinitrogen gas, which in nature is mostly performed by denitrifying bacteria. The enzymes and the mechanisms behind nitrate reduction have been the subject of many studies in E. coli as well as in denitrifying bacteria Shewanella oneidensis MR-l is a y-proteobacterium, which has the ability to utilize a wide variety of electron acceptors including nitrate and nitrite. It has been a subject of controversy as to whether this gram-negative bacterium reduces nitrate by denitrification or by dissimilatory nitrate reduction into ammonium (DNRA). In this study, Shewanella oneidensis MR-l was grown anaerobically using KNO3 or NaNO2 as the sole electron acceptor, showing a maximum growth yield at 4 mM and 2 mM, respectively. Anaerobic cultures of MR-l in minimal medium using 20 mM lactate as the electron donor and 2 mM KNO3 as the electron acceptor showed a sequential reduction of nitrate to nitrite and then to ammonium. Nitrate reduction into ammonium was coupled to growth. When MR-l was inoculated under anaerobic conditions using N20 as the only electron acceptor, no growth or gas production was observed. These results establish that Shewanella oneidensis MR-l is not capable of denitrification but instead carries out DNRA. 27 INTRODUCTION Shewanella oneidensis MR-l is a metal ion reducer that has extraordinary versatility in the variety of compounds it can reduce under oxygen-limited conditions. Among the compounds it can utilize, there is oxygen, nitrate, nitrite, fumarate, Mn(III), Fe(III), elemental sulfur, sulfide, thiosulfate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), Cr(VI), and U(VI) (9). S. oneidensis MR-l reduces nitrate, generating energy for growth, as it reduces Cr(VI) into its insoluble form, a less harmful form (12). Nitrate, chromium and uranium, all reduced by S. oneidensis MR-l, are regulated pollutants of great concern due to their human health implications. All of them have been linked to various forms of cancer (5, 7, 8, 12, 16, 21). In S. oneidensis MR-l two different pathways have been described for the reduction of nitrate: the dissimilatory nitrate reduction into ammonium (DNRA) and denitrification (6, 10). In denitrification nitrate is sequentially reduced into dinitrogen (NO3'—-)NO2'—)NO->N2O—>N2), whilst in DNRA, nitrate is reduced into nitrite and then into ammonium. No bacterium has been reported to carry out both complete pathways (18). In order to determine which of these pathways takes place in the organism, the nitrogen intermediates and products are determined (20). To measure denitrification, acetylene is used since it inhibits the enzyme that catalyzes the reduction of nitrous oxide into dinitrogen (nitrous oxide reductase) causing the accumulation of nitrous oxide which is easily quantified. Dinitrogen production can also be measured but due to its high abundance in the atmosphere it is harder to quantify its production. Accumulation of ammonium is measured in cultures to determine whether DNRA is the nitrate reducing 28 process. In the studies mentioned above where the reduction of nitrate was measured in cultures of MR-l , ammonium was measured only in one of them (10) and the concentration detected was very low. Each of these previous reports detected nitrous oxide but in very low amounts compared to the concentration of nitrate supplied. Although these studies concluded that denitrification is occurring in MR-l, the data from its genome sequence does not support this conclusion. The complete genome sequence of MR-l revealed the presence of all the genes necessary to carry out DNRA but key genes for denitrification, such as those for denitrifying nitrite, nitric oxide and nitrous oxide reductases, are not present (9). Preliminary studies in our laboratory have shown that insignificant amounts of N20 and no N2 have been detected when S. oneidensis MR-l was cultivated anaerobically with nitrate as the only electron acceptor suggesting that the nitrate reduction pathway in this organism is yet to be resolved. Nitrate reduction in bacteria has been studied for decades (20). Despite all of the knowledge acquired on this subject, there are still important genetic and biochemical aspects that need clarification. This study focuses on nitrate metabolism of Shewanella oneidensis MR-l. MR-l was cultivated under different growth conditions to determine the optimal concentrations of nitrate and nitrite for growth as well as the intermediates produced from the reduction of nitrate when the later is used as the sole electron acceptor. The inability of Shewanella oneidensis MR-l to use nitrous oxide and the lack of gas production from the reduction of nitrate confirm that denitrification is not taking place in this microorganism and the production of ammonium shows that DNRA is the operative nitrate reduction pathway. 29 MATERIALS AND METHODS Growth conditions and bacterial strains. Shewanella oneidensis MR-l (ATCC 700550) was the strain used in this study. Pseudomonas stutzeri (provided by Veronica Griintzig at the Center for Microbial Ecology at Michigan State University) was the bacterium used as a positive control in the nitrous oxide utilization assay. Growth curves were performed in anaerobic modified LML liquid medium (2). This media was supplied with 20 mM lactate as the electron donor, 0.01% of vitamin-free Casamino Acids, and 0.01% of a trace metals solution (11). The medium also contained either 0.5, l, 2 and 4 mM NaNO2 or 0.5, 1, 3, 4, 10, 40 and 100 mM KNO3 as electron acceptors. The anaerobic cultivation was performed in 30 ml Balch tubes with 15 ml of medium, which was previously degassed by boiling and purged with helium. The tubes were closed with butyl rubber stoppers to prevent aeration. A vitamin solution (23) was added by injection after autoclaving. These cultures were inoculated by injection of a 1% Shewanella oneidensis MR-l culture that had been grown aerobically for 12 h in LML. This inoculum originated from an overnight starter culture grown in LB medium. Incubation was performed at 30°C without shaking. Cultures used to determine consumption and production of nitrate, nitrite and ammonium by Shewanella oneidensis MR-l were cultivated in Modified M1 minimal medium (14) without NH4CI. The M1 medium was supplemented with 20 mM sodium lactate as the electron donor. HEPES (pH7.2) was added to buffer the medium at a 50 mM final concentration. KNO3 was added as the electron acceptor at 2 mM final 30 concentration. Other medium components were prepared as specified above. Negative controls of each growth condition were no inoculation and media without the electron acceptors but inoculated. Nitrate, nitrite and ammonium analyses. To determine the consumption and production of intermediates during the bacterial reduction of nitrate, three 30 ml Balch tubes containing 15 ml of M1 medium with 2 mM KN03 were inoculated as indicated above. These cultures were incubated at 30°C and an OD measurement at 600 nm was taken every 3 to 4 h after an initial 8 h incubation period using a Varian Cary 50 B10 UV-Vis spectrophotometer (V arian, Zug, Switzerland). After the OD measurements were taken, a 2 ml sample was sterile filtered (0.22 um syringe filter). These samples were analyzed for nitrate and nitrite using a Lachat QuickChem Automated Flow Injection Ion Analyzer following the Copperized Cadmium Reduction Method as in QuickChem Method No. 10-107-04-1-A (Lachat Instruments, 1988) at the Soil Testing Lab at Michigan State University. The ammonium analysis was performed by the salicylate colorimetric method (15). Cell growth on nitrous oxide as sole electron acceptor. To determine whether or not Shewanella oneidensis MR-l is capable of utilizing nitrous oxide as an electron acceptor, a 1% inoculum from an aerobic culture grown for 12 h was used to inoculate 20 Balch tubes with 10 ml of LML medium prepared anaerobically. Nitrous oxide gas was added to 10 tubes. The remaining tubes with their helium headspace were used as negative controls for anaerobicity, without nitrous oxide or any other electron acceptor. These cultures were incubated at 30°C without shaking for 3 weeks. Each tube had a Durham tube (inverted smaller tube) at the bottom to observe gas production. The same procedure 31 was performed as a positive control for nitrous oxide utilization, using a Pseudomonas stutzeri culture grown for 12 h as an inoculum. Ten tubes without inoculum served as negative controls for gas production. This same experiment was also performed using 4 mM KNO3 as the only electron acceptor, instead of nitrous oxide, with S. oneidensis MR- 1 and P. stutzeri as inocula. RESULTS Effects of increasing concentrations of nitrate or nitrite in the growth of Shewanella oneidensis MR-l. When Shewanella oneidensis MR-l was inoculated in anaerobic LML medium in the presence of KNO3 as the only electron acceptor, the biomass increased with increasing concentrations of nitrate until 4 mM KNO3, where maximum yield occurred (FIG. 2.1). When the nitrate concentrations were higher than 4 mM, the biomass was dramatically reduced and the maximum yield reached was similar at all concentrations tested up to 100 mM, which was the highest. However, the growth rate in cultures with NO3' showed an increase until 2 mM (1 hi), and decreased at higher concentrations (FIG. 2.3). Similar growth rates were calculated for MR-l cultures on N03" concentrations higher than 2 mM (0.5 b"). When nitrite was supplied as the only electron acceptor, biomass of MR-l increased with increasing concentrations of NaNO2 until 2 mM, where the maximum cell yield was observed (FIG. 2.2). Higher concentrations of NaNO2 resulted in lower grth yields. The growth rate of MR-l cultures on N02' decreased with increasing NO2' concentrations (FIG. 2.3). 32 0.25 0 2 + 0.5mM Nitrate . —I— 1mM Nitrate E 015 ——A— 2mM Nitrate § . ”,4.- 3mM Nitrate © 0 1 ‘ —>K— 4mM Nitrate 8 . /;»; —l— ‘10an Nitrate 0 05 [1,6 + —-— 40mM Nitrate ‘. // {r— 100mM Nitrate o 2'93!’ _ , . o 10 20 30 Incubation Time (hr) FIG. 2.1. Growth of Shewanella oniedensis MR-l on different concentrations of nitrate. The data are mean and standard deviation of three biological replicates monitored by optical density measurements. + 0.5mM Nitrite —I— 1mM Nitn'te —A— 2mM Nitrite —9(— 4mM Nitn’te Incubation Time (hr) FIG. 2.2. Growth of Shewanella oneidensis MR-l on different concentrations of nitrite. The data are mean and standard deviation of three biological replicates monitored by optical density measurements. 33 1.2 0.8 0.6 0.4 0.2 w o I . . . . 0 20 40 60 80 100 120 Concentration (mM) —c—— NOz‘ —-I— N031 Growth rate (1Ih) fl— P2,... In I FIG. 2.3. Growth rate of Shewanella oneidensis MR-l in anaerobic cultures at different nitrate and nitrite. The growth rate was calculated from the results of the growth curves shown in FIG 1 and 2. These values were calculated using the average of the optical densities obtained in three biological replicates. Consumption and production of intermediates in nitrate reduction of Shewanella oneidensis MR-l. After 8 h incubation, the culture was in early log phase and nitrate was almost completely depleted and nitrite was produced to a stoichiometric maximum (2 mM)(FIG. 2.4). Ammonium remained at its initial concentration. After 12 h, growth was approximately at mid-log phase and nitrite consumption was commenced along with ammonium accumulation. Nitrate and nitrite were completely consumed and growth reached its maximum at 15 h. At this point ammonium concentration reached 2 mM. After this time the cultures continued to accumulate ammonium to approximately 3 mM 34 presumably as a result of cell death. The maximum consumption rate of NO3' was 0.2 mM/h, whilst for nitrite it was 0.5 mM/h. Growth of Shewanella oneidensis MR-l when N20 is the sole electron acceptor. When S. oneidensis MR-l was incubated with N20 or NO3' as its sole electron acceptor, no N2 production was observed. In the contrary, those tubes inoculated with P. stutzeri showed N2 production, and growth from both electron acceptors, as expected for the positive control. Growth of MR-l did occur with N03' but not in N20 cultures. This study indicates the inability of Shewanella oneidensis MR-l to grow or produce any N2 gas from the reduction of nitrate or nitrous oxide. 35 4 . " 0.12 ‘~ 0.1 E +N03' O 2 '~ 0.08 ‘v + + c 5 NH‘ O O E o 06 § C N02- 0 § 0 ‘r 0.04 0 "‘HGrowth CUTVO * 0.02 0 30 Time (hr) FIG 2.4. Nitrate, nitrite and ammonium concentrations in Shewanella oneidensis MR-l in anaerobic cultures grown in the presence of nitrate as the electron acceptor. OD measurements were obtained at each sampling. These measurements (including the N- species concentrations) are the mean and standard deviation of three biological replicates. TABLE 2.1. Stoichiometry of nitrogen ions measured in Shewanella oneidensis MR-l anaerobic cultures with 2 mM KN03 as the electron acceptor and lactate as the electron donor. The concentrations of each of the N-compounds were an average from three biological replicates and the averages were added for the inorganic nitrogen budget of the cultures. Incubation Balance N Time (h) N03'(mM) NO2'(mM) NH4*(mM) (mM) 0 1.84 0.00 0.33 2.17 8 0.10 1.98 0.37 2.45 12 0.07 0.65 1.31 2.03 15 0 0.00 2.03 2.03 21 0.00 0.00 3.04 3.04 __ 24 0.00 0.00 2.29 2.29 36 DISCUSSION This study clarifies the dissimilatory nitrate reduction pathway of Shewanella oneidensis MR-l . Stoichiometric results of inorganic nitrogen species confirmed that nitrate is reduced to ammonium. These experiments showed a sequential reduction of nitrate into nitrite and subsequently into ammonium. The production of the N02' in amounts equivalent to the nitrate supplied indicates that the entire nitrate concentration was converted to first nitrite before been converted to ammonium. This indicates that DNRA is the pathway occurring in Shewanella oneidensis MR-l. Also, the inability of MR-l to reduce N20 supports DNRA as the nitrate reduction pathway, instead of denitrification. In this study, neither nitrous oxide nor dinitrogen were measured, but previous experiments performed in our laboratory have demonstrated that when MR-l was grown using nitrate as the only electron acceptor insignificant amounts of N20 and no N2 were detected. Studies on MR-l that led to the claim of MR-l as a denitrifier were based on nitrous oxide production but without establishing the stoichiometry of the products (6, 10). Detection and production of nitrous oxide from nitrate has been reported for a number of non-denitrifiers but in all cases this production is less than 30% of the nitrate reduced (3, 4, 19). An investigation to examine the different sources of nitrous oxide in the environment confirmed that all the nitrate-respiring bacteria analyzed, including the Escherichia and the Enterobacter genera, produced N20 (3). Moreover, studies in the non-denitrifier Escherichia coli, detected N20 as a product of the reduction of NO2' by the nitrate reductase. In this study, reduction of nitrite and production of nitrous oxide decreased significantly in an E. coli nitrate reductase mutant. Reduction of nitrite into NH; and N20 was observed for the wild type, however no energy was generated when 37 N2O was produced. Nitrous oxide production has also been suggested to be the result of an abiotic process, where hydroxylamine (an intermediate in the nitrite reduction into ammonium by NrfA) is converted to nitric oxide and N20 under specific conditions (4, 24). Denitrification is defined as the complete reduction of nitrate into dinitrogen (20). In this study, cultures of MR-l failed to produce gas when grown with N03' or N20 as electron acceptors. In contrast to cultures with N20, growth on nitrate was detected, which indicates that even when the cell had enough energy to grow, the metabolic pathway used to generate the energy was not producing gas intermediates as would occur in denitrification. Moreover, cultures of P. stutzeri, a well-characterized denitrifier, which was used as a positive control, produced gas. This indicates that contrary to standard denitrifiers, MR-l is incapable of carrying out the last step of denitrification. This coincides with the lack of the nosZ gene in the genome, which encodes the nitrous oxide reductase described in other bacteria (18). No organism has been described that can execute the complete pathway of both DNRA and denitrification. Wolinella succinogenes, a s-proteobacterium, has been hypothesized to reduce nitrous oxide to dinitrogen with nitrate as the electron acceptor when either H2 or formate is supplied as electron donor (18). The genome sequence of W. succinogenes, a non-denitrifier that, like MR-l, can reduce nitrite into ammonium, has been recently reported as encoding a unique nos cluster lacking some of the genes found in other bacteria but having a novel N20 reductase gene. This nitrous oxide reductase has been identified as a cytochrome c and the authors hypothesized that it can be part of a novel electron transport when either H2 or formate is used as the electron donor. Thus, 38 even when the microorganism cannot carry out denitrification some of its products can still be detected in the cultures. Some of the nos genes (nosLDF YA) have been described in the genome sequence of MR-l but there is no homolog for a nitrous oxide reductase. In a study comparing the gene expression profile of MR-l under aerobic versus anaerobic growth with nitrate as the electron acceptor, the nos genes were induced (1). Even though MR-l does not posses a homolog of this particular nos gene, it is possible that MR-l might be capable of a novel nitrous oxide reduction like W. succinogenes, but its conversion to N2 was not seen under the grth conditions used in this study. However, the studies mentioned previously in which nitrous oxide was detected as the result of nitrate reduction in cultures of MR-l , added H2 to the medium (6, 10). Even though the genome of MR-l codes for some of the proteins involved in denitrification, it does not seem to possess all of the genes required for this process. The genome sequence does not show homologues of nir (genes for the reduction of nitrite into nitric oxide), nor (genes involved in the reduction of NO into N20), and nosZ (enzyme required for the reduction of N20 into N2). In experiments were the gene expression profile of MR-l was studied using DNA microarray in cultures grown under anaerobic conditions with nitrate as the sole electron acceptor, significant induction of napBGHA, nrfA, narQ, and cymA (napC homolog, ref. 13) genes was reported (1). All of these genes are required for the DNRA pathway in E. coli when the reaction takes place in the periplasm (17, 22). Although this study presents physiological evidence to support that MR-l is not capable of denitrification, mutational and gene expression analyses is needed to better characterize the nitrate reduction process in this bacterium. 39 This study confirms the ability of Shewanella oneidensis MR-l to use the DNRA pathway for growth. However, the reduction in biomass and growth rate with increasing concentrations of nitrate and nitrite indicates cytotoxicity at higher concentrations. Growth rate on nitrate decreased at concentrations higher than 2 mM, while the growth rate on nitrite decreased at concentrations higher than 0.5 mM. This indicates a higher cytotoxicity caused by nitrite than nitrate. Also the consumption rate of nitrite was faster than that of nitrate, which might indicate a need of the organism for a faster disposal of the more toxic compound. Increasing concentrations of nitrate become toxic as well but the cell seems to tolerate it better showing a higher and constant growth yield independent of the nitrate concentration up to 100 mM. These results suggests that nitrite causes toxic effects in MR-l and that MR-l probably has a mechanism to protect the cell against the nitrite toxicity, perhaps by sensing the concentrations of nitrate and controlling the amount that gets reduced, which will result in lower growth yields that could be kept constant independent of the nitrate concentration. In conclusion, even when the cell is sensitive to low concentrations of nitrite, it can still grow under considerably high concentrations of nitrate and use it for growth. 40 REFERENCES . Beliaev, A.S., D.K. Thompson, T. Khare, H. Lim, C. C. Brandt, G. Li, A.E. Murray, J.F. Heidelberg, C.S. Giometti, J. YatesIII, K.H. Nealson, J.M. Tiedje, and J. Zhou. 2002. 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The unprecedented nos gene cluster of Wolinella succinogenes encodes a novel respiratory electron transfer pathway to cytochrome c nitrous oxide reductase. FEBS. Lett. 569:7-12. Smith, M.S. 1983. Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity. Appl. Environ. Microbiol. 45: 1 545-1547. Tiedje, J.M. 1994. Denitrifiers. Methods of soil analysis, Part 2. Chapter 14:245- 267. . Van Leeuwen, J.A., D. Watner-Toews, T. Abernathy, B. Smit, M. Shoukri. 1999. Associations between stomach cancer incidence and drinking water 42 contamination with atrazine and nitrate in Ontario (Canada) agroecosystems, 1987-1991. Int. J. Epidemiol. 28:836-840. 22. Wang, H., and R.P. Gunsalus. 2000. The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. J. Bacteriol. 182:5813-5822. 23. Wolin, E.A., M.J. Wolin, and R.S. Wolfe. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-2886. 24. Zumft, W.G. 1993. The biological role of nitric oxide in bacteria. Arch. Microbiol. 160:253-264. 43 CHAPTER III NapA is the Enzyme Responsible for the Reduction of Nitrate in Shewanella oneidensis MR-l 44 ABSTRACT NapA is a microbial periplasmic nitrate reductase that is of interest due to a possible relationship between the organization of its operon and the physiological role it plays in the organism. S. oneidensis MR-l has one ORF ($00848) similar to known nitrate reductases, and it has 72% sequence identity to the napA gene of the well-studied Escherichia coli K12. A napA deletion mutant was unable to grow on nitrate, confirming that NapA is the only functional nitrate reductase in MR-l. MR-l also possesses a gene that codes for nitrite reductase ($03980), NrfA. The expression of napA was up- regulated at high (40 mM KNO3) concentrations of nitrate, which indicates that there is not a repressor regulation system for this operon at high nitrate concentrations as there is in E. coli, and is consistent with the absence of an alternative pathway in MR-l. napA and nrfA, quantified by Q-RT-PCR, were both expressed under aerobic conditions, although reduction of nitrate, was highly inhibited, indicating that the lack of activity under this condition was likely due to oxygen inhibition of the nitrate reductase and not to its transcription. 45 INTRODUCTION Nitrate reduction has been extensively studied in many bacteria. In Gram-negative bacteria nitrate reduction is characterized by the location and biochemical properties of their nitrate reductase. This enzyme catalyzes the reduction of nitrate into nitrite, the first step in the nitrate reduction pathway. Two types of enzymes have been described, an assimilatory and a dissimilatory nitrate reductase. The assimilatory nitrate reductase is a membrane-bound protein that catalyzes the conversion of nitrate into ammonium. The ammonium is then incorporated into amino acids and then into cell material. There are two dissimilatory nitrate reductases, classified by their location in the cell: a membrane- bound (Nat) and a periplasmic (Nap) nitrate reductase. Nar is associated with the cytoplasmic membrane and Nap is solubilized in the periplasmic space (23). In most non- denitrifying bacteria these enzymes have been associated with a nitrite reductase. Nar has been associated with the NirB nitrite reductase, which is also located in the cytoplasmic membrane. On the other hand, Nap has been found in association with NrfA, which is a periplasmic nitrite reductase (6, 23). The proximity of these proteins within the cell has been described as energetically advantageous for cell growth. Moreover, a multi-enzyme complex between the Nap and Nrf proteins has been proposed for E. coli and other bacteria. This complex might help conserve energy in the cell by avoiding losses of nitrite ensuring high efficiency in its reduction to ammonium (23). NapA has been identified in different kinds of bacteria including denitrifiers, non- denitrifiers, pathogens and even phototrophic bacteria. Studies in Shewanella oneidensis MR-l have described the capabilities of MR-l to reduce nitrate (7, 13, 38). A nitrate 46 reductase was partially purified from membrane extracts of MR-l suggesting that it is a membrane-associated protein (13). In this same study, a nitrite reductase was also partially purified and it also seemed to be membrane-bound. However, the biochemical and structural properties of the nitrate and nitrite reductases described in that study do not correspond to those previously described for the membrane-bound nitrate and nitrite reductases in other organisms. One of the criteria used to classify the nitrate reductase of MR-l as membrane-bound was its activity in the membrane fraction of the cell but, in a study of Pseudomonas spp. G-179, Nap was found in both the periplasmic and the membrane fractions of the cell (2). This study suggested that Nap might undergo a maturation process where Nap is cleaved from the membrane. Another possibility might be the formation of a protein complex between the soluble subunits and a membrane- bound protein such as NapC, which is essential for the Nap system. Although these authors found three subunits of the nitrate reductase in MR-l , their molecular weights are not in the range of those previously isolated: or, B and 7 were 90, 70 and 55 KDa, respectively for MR-l. In addition, the nitrite reductase detected, contrary to the Nir isolated from denitrifiers, was not periplasmic but membrane-bound. The other biochemical and structural properties observed for the nitrate reductase detected in MR-l are similar to those observed for NapA in other bacteria (2, 25, 32, 34). Some of these properties include insensitivity towards low concentrations of azide and activity in the presence of oxygen (significantly lower than that under anoxic conditions). According to the complete genome sequence of MR-l (11), this microorganism possesses a likely periplasmic nitrate reductase, which shares 72% sequence identity to the NapA of E. coli. The nap operon has been sequenced in different bacteria and a 47 relationship between its organization and its physiological role has been proposed (21, 22, 23). In all organisms, for which the genomic sequence has been published, the nap operon consists, among others, of napDABC genes in this same order. S. oneidensis MR- I possess napDAGHB, and is the only organism so far which does not have the napC gene in the same gene cluster, although it has a homolog (cymA) located in a different loci. There is also a homolog of the napF but it is also located in a different loci. MR-l also possesses the nrfABCDEF genes, and the narP and narQ genes, which code for one of the two-component regulatory systems involved in the regulation of the nrf and nap operons in E. coli. The cymA gene encodes a cytochrome c proven to be required for nitrate reduction in MR-l as well as for its homolog in E. coli (18, 19). Studies in a S. oneidensis MR-l cymA deletion mutant indicate its requirement not only for nitrate respiration but also for the reduction of nitrite via NrfA, Fe(III), and fumarate and for grth with dimethyl sulfoxide (DMSO) (28, 29). That investigation proved the role of CymA as a common electron supplier for at least five different anaerobic energy- generating processes. DNA microarray data from MR-l cultures grown under anaerobic conditions with nitrate as the sole electron acceptor demonstrated significant induction of nrfA, napBGHA, narQ, and cymA genes (3). In this study the expression of the napA and nrfA genes was monitored in aerobic and anaerobic cultures of MR-I under different nitrate concentrations. An increase in the expression of these genes was observed with increasing concentrations of nitrate until it reached a plateau. Also, a global gene expression profile was examined for MR-l anaerobic cultures grown at a low versus a high concentration of nitrate. A Shewanella 48 oneidensis MR-l napA deletion mutant was generated to test whether this was the enzyme responsible for the nitrate reduction. MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains, plasmids, primers and probes used in this study are described in TABLE 3.1. Cultures of Shewanella oneidensis MR-l for DNA microarray and Q-RT-PCR experiments were incubated at 30°C after inoculation in Modified M1 minimal medium (17) with no NH4C1 to avoid interference with chemical analyses. HEPES (pH 7.2) was added to buffer the medium at a 50 mM final concentration. The medium was supplemented with 20 mM lactate and KN03 was added as the electron acceptor at 1 mM and 40 mM final concentration for the DNA microarray experiments. The Q-RT-PCR experiments also included expression analyses of cultures under 0.1 mM, 0.25 mM, 0.5 mM, 3 mM, 10 mM, and 15 mM KN03. Aerobic cultures of MR-I with 3 mM KN03 and without it were analyzed using Q-RT-PCR. In order to make the media anoxic, the media were degassed by boiling and purged with helium. The medium (100 ml) was transferred to 250 ml serum bottles and closed with a butyl black stopper. The medium was inoculated by injection with a 1% inoculum from a 12 h aerobic culture in M1 media, which originated from an overnight starting culture in aerobic M1 media as well. The medium was autoclaved and 0.1 ml of Wolfe’s vitamin solution (41) was added by injection with a sterile syringe. Incubation was performed at 30°C without shaking. Negative controls for each grth condition were no inoculation and medium without the electron acceptors but inoculated. 49 Cultures of Escherichia coli [321 5 5 (auxotroph of diaminopirnelic acid) were grown in Luria-Bertani (LB) medium supplemented with 100 ug/ml of diaminopirnelic acid (DAP). These cultures were incubated at 37°C. Shewanella oneidensis MR-l was cultivated in LB media and incubated at 30°C during the mutagenesis process. Antibiotics for E. coli were prepared and added as described elsewhere (26). The antibiotics used for the selection of MR-I positive transforrnants were added in the following concentrations: 25 11ng of kanamycin, 7.5 ug/ml of gentamycin, and 10 ug/ml tetracycline. Total RNA preparations. To determine gene expression profiles total RNA was extracted from cultures of S. oneidensis MR-l grown in triplicate as described above. Cells were collected at mid-log phase and concentrated by centrifuging at 4°C for 30 min at 7,500 rpm. The pellets were washed with 1 ml of an ice-cold 1X DEPC-treated PBS solution (26). The RNA was extracted with The RNAwiz Solution following the instructions of the manufacturer (Ambion, Inc.). The RNA extraction was followed by an isopropanol precipitation (26) and its resuspention in the RNA storage solution (Ambion, Inc.). The RNA samples used for DNA microarray analyses were treated with RNase-free DNaseI (Roche) to eliminate residual DNA. The samples were purified by phenol, phenolzchloroform (1:1) and chloroform extractions, and stored in ethanol at -80°C until ready for use. The RNA samples used for Q-RT-PCR were DNase treated using the DNA-free Kit (Ambion, Inc.) and purified using the RNeasy Mini Kit (Qiagen). Quality of the RNA was observed using the RNA 6000 Pico LabChip kit and the 2100 Bioanalyzer (Agilent Technologies). The RNA concentration was determined with 0D measurements at 260 nm using a Varian Cary 50 BIO UV-Vis spectrophotometer (V arian, Zug, Switzerland). 50 TABLE 3.]. Bacteria, plasmids, primers and oligonucleotides used in this study. Strain, plasmid, Description or nucleotide sequence“ Source, reference primer or probe or relative position of primer or probe Bacterial Strains E. coli [32155 Diaminopimelic acid auxothroph used for cloning and conjugation 8 S. oneidensis Lake Oneida, N.Y., sediment 16 MR—l S. oneidensis MR- napA gene deletion derived from MR-l This study 1 Amp! Plasmids pCM157 Broad-host-range cre expression vector 14 pCMl84 Broad-host-range allelic exchange vector 14 pCCG185 pCMl 84 with napA upstream flank This study pCCGl86 pCCGl85 with napA downstream flank This study pKNOCK-Gm Broad-host-range allelic exchange vector 1 pCCGOl pKNOCK-Gm with napA flanking regions separated by two loxP This study sites flanking a kanamycin resistance gene Primersc napAN Fwd napAN Rev GCATATGGGCGGCTAATGCTCATAGTGTT CAT Start codon CGAATTCTCT‘TGCCCCATTCCTCCCT 504m upstreamd napAC Fwd the start codon napAC Rev CGAGCTCCAGACATCGCAGCGTAATCCTI‘ 2452 GCCGCGGAGTGCCCCGTAAAAGTGATGAA 517nt downstream napAScreen Fwd stop codon AGACATCGCAGCGTAATCTC 499m downstream napAScreen Rev stop codon TCCCTCTCCAAAGGGATAGC 484nt upstream napAScreenout start codon Fwd GTGTCATGCT CT GCGGAT'I‘ 728m downstream napAScreenout stop codon Rev AATGCGCCTGGGATTGAA 591nt upstream 23 SRT Fwd start codon 23 SRT Rev TAGCGAAATTCC’I'I‘GTCGGG 1920 23 Stemp Rev GAGACAGCGTGGCCATCAT‘T 1985 napART Fwd GTATCAGTTAGCT CAACGCCT C 2847 napART Rev AGAAAGCCCTGTTAACCGTGG 210 napAtemp Rev TCATCCGCAGCAATGGTGT 101 nrfART Fwd GATCGAAGCTACGGTTCTCG 751 nrfART Rev GCCACATGTATGCCGTGACT 257 nrfAtemp Rev TTTACAGCTCCAGCAAGCCA 357 ACGTTTCATACTCGGGATGC 775 Probes 23SRTProbe napARTProbe AGTTCCGACCTGCACGAATGGCG 1942 nrfARTProbe CTGTATTAAAGGT‘TACTI‘CCTGTCGAAAATCAT‘GTACGG 237 CGTAATACCTTGCGTACTGGCGCGC 283 ‘ The sequence for the primers is written from the 5’end to the 3’end. b Primers were designed using putative gene sequences of S. oneidensis MR-l. ° For primer sequences, the restriction sites incorporated are underlined. CATATG, Ndel; GAATTC, EcoRI; GAGCT C, SacI; CCGCGG, SacIl. d Even though the napA gene is in the opposite direction in the genome, the sequence at the right end of the start codon will be denominated as upstream and the one at the left side of the stop codon as downstream. 51 Quantitative-Real-Time-PCR (Q-RT-PCR). Total RNA (1 ug) was reverse-transcribed using the Superscript II Kit (Invitrogen) and hexamer primers. cDNA was purified using the Qiagen Purification Kit (Qiagen). The cDNA concentration was determined by spectrophotometry at 260 nm and 700 pg was used as the template for the Q-RT-PCR. Gene specific primers and probes were designed using Primer Express® 1.0 software (Applied Biosystems). BLAST of the sequences against the MR-l genome was performed to test for specificity. The reaction was carried out with 1X TaqMan® Universal PCR Master Mix (Applied Biosystems) and 500 nM each of, primers and probes. The quantification of napA transcripts was done by Q-RT-PCR using the name Fwd and napART Rev primers, and the napART probe (TABLE 1). In the same manner, nrfA transcripts were quantified using the nrfART Fwd and nrfART Rev primers, and the nrfART probe. The reaction was performed using the ABI PRISM® 79OOHT Sequence Detection System (Applied Biosystems) at the Genomics Technical Support Facility at Michigan State University. A standard calibration curve was prepared with a serial dilution of a PCR product of the putative genes in MR-l as templates. The PCR product for the napA calibration curve was amplified with the napART Fwd and napAtemp Rev. The PCR product for the nrfA calibration curve was amplified with nrfART Fwd and nrfAtemp Rev. Primers and a probe specific for the 23S rDNA (23SRTFwd, 23SRTRev and 23SRT probe) were used to quantify the 23S rRNA transcripts. This gene was used as an internal control to normalize the difference in the efficiency of the amplification and technical manipulations (4). A calibration curve was also prepared for the 23S rRNA gene using a PCR product amplified with the 23SRT Fwd and the 23 Stemp Rev. The template concentration for this reaction was optimized to 52 7 pg but the concentration of primers and probe was the same as for the other genes. Triplicates of each sample were run for all the reactions. A negative control (no cDNA added) was run for each of the reactions. Absolute quantification was calculated by interpolating each sample with their corresponding standard curve. napA deletion mutagenesis. Molecular procedures such as genomic and plasmid purifications, restriction digestions, sticky ends repair, ligations and electroporations were performed as previously described (26). Primers for PCR reactions (TABLE 3.1) were designed using the Vector NTI® software (InforMax, Inc.) and synthesized at Integrated DNA Technologies (www.idtdna.com). a) naQA allelic exchange vector generation. The MR-l napA flanking regions were cloned into the broad-host-range vector pCMl 84 for further replacement of the napA gene in MR-l by a kanamycin cassette. This replacement occurred by homologous recombination of the flanking regions in the vector with those in the genome. The kanamycin cassette in pCMl 84 is flanked by two loxP sites, which are in turn flanked by two multiple cloning sites (MCS) (14). The primers napAN Fwd (NdeI) and napAN Rev (EcoRI) were used to amplify the region upstream of the napA gene start codon while the napAC Fwd (SacI) and napAC Rev (SacII) amplified the region downstream the stop codon. The product of these reactions was approximately 500 bp each, to ensure high efficiency of recombination (14). Each primer had a restriction site linker at the 5’end for an enzyme that was chosen using the pCMl 84 MCS as a reference for directional cloning of the fragments. Afier amplification, the N fragment (upstream) was double digested 53 with NdeI and EcoRI and the C fragment (downstream) with SacI and SacII. These two fragments were cloned one at a time into the vector (pCM184) that was previously digested with the corresponding restriction enzymes depending on the fragment to be cloned. After the fragment and the vector were digested, these were gel purified using the QIAquick® Gel Extraction Kit (Qiagen). A vectorzinsert ratio of 1:3 was used for the ligation reaction and 1 ul of this overnight reaction was used to electroporate E. coli 132155 electrocompetent cells. The positive transforrnants were selected by plating on LB agar supplemented with kanamycin and DAP. The colonies were screened with the primers used to amplify each fragment. Once both fragments were inserted a final PCR screen was performed to check for the correct incorporation of the fragments into the vector generating pCCGl 86. A set of primers including napAScreen Fwd, which anneals to the inside region of the C fragment and napAScreen Rev that anneals to the inside of the N fragment were used for the screen. Due to replication of the suicide vector (pCCG186) in MR-l, pCCGl 86 was digested with EcoRI and SacI to excise the kanamycin cassette with the loxP sites and the N and C fi'agments (a fragment of approximately 2.5 kbp), which were transferred into pKNOCK-Gm (1) for the generation of pCCGOI. The pKNOCK-Gm broad-host-range vector has a gentamycin resistance gene and a R6K origin, which needs a it protein for propagation. Since this protein is not present in MR-l this plasmid made a good suicide vector. To clone the fragment excised from pCCGl 86 into pKNOCK-Gm, this vector was digested with SmaI, which is a blunt end cutter and the plasmid was dephosphorylated with calf intestinal phosphatase to avoid self-ligation. The sticky ends product of the digestion of the insert with EcoRI and SacI were repaired using T4 DNA 54 polymerase. The reaction products were gel purified as described above and a vectorzinsert ratio of 1:16 was used for the ligation reaction. Electroporation and screening of the transformants was performed as described above. b) napA allelic exchange vector nflfer into Shewanella oneidensis MR-l. pCCG01 was introduced into MR-l by conjugation (protocol from Margie Romine, Pacific National Laboratory). The E. coli 02155 harboring pCCG01 was inoculated in LB liquid media supplemented with 100 ug/ml of DAP and 50 ug/ml of kanamycin. Overnight cultures of MR-l and E. coli 02155 harboring pCCGOl were mixed in microtubes, the mixtures consisted of 0.5 ml of MR-l with either 0.5 ml or 1 ml of E. coli cultures. Individual controls for each culture were also prepared simultaneously. These cultures were mixed by vortex and concentrated by centrifuging at 4,000 rpm for 2 min at room temperature. The supernatant was discarded and the pellet was gently swirled with the pipette tip in the remaining of the medium (approximately 100 ul). Then, this solution was spotted on an LB plate with DAP and incubated at room temperature for 12 to 16 h. Controls were spotted on individual plates. After incubation, the spotted cultures were scrapped and resuspended in 1 ml of IX Phosphate-Saline Buffer (PBS) (26). Two 10- fold serial dilutions were prepared and plated on LB agar with 25 ug/ml of kanamycin and no DAP to avoid growth of the E. coli 02155, thus only the MR-l cells that were successfully transformed with pCCG01 were able to grow. To check for the loss of the vector and replacement of the napA gene by homologous recombination the colonies obtained were transferred to LB plates with kanamycin and LB plates with kanamycin and gentamycin. Positive MR-l napA deletion mutants were expected to be resistant to kanamycin and susceptible to gentamycin. Those colonies were screened by PCR using 55 napAScreenout Fwd and napAScreenout Rev which anneal approximately 210 bp downstream and 90 bp upstream from the napA flanking regions included in pCCGOl , respectively. This screening ensures that the recombination occurred in the targeted area. c) Removarloffikagamycin cassette. The two loxP sites flanking the kanamycin cassette are recognition sites for Cre recombinase. This recombinase excises the region inside the loxP sites by recombination leaving one of the loxP sites. pCM157 is a cre gene expression vector (14) which was electroporated into E. coli 02155. This vector was transformed into Shewanella oneidensis MR-l AnapA Kanr by conjugation as described above. Colonies susceptible to kanamycin and resistant to tetracycline were selected as positive transformants. These were screened by PCR using napAScreenout primers to confirm the loss of the kanamycin cassette. d) pCM157 curation from S. oneidensis MR-l AM. Cultures of MR-l AnapA (Tet' Kans) were transferred three times on LB liquid media with no antibiotics and then screened for tetracycline susceptibility on LB agar. This phenotype indicates the loss of the pCM157 plasmid. Colonies were transferred and screened until the correct phenotype was obtained. Colonies susceptible to kanamycin and tetracycline were diagnosed with PCR the napAScreen primers and with primers targeting the inside region of the MR-l napA gene (napART Fwd and napAtemp Rev). DNA sequencing performed at the Genomics Technical Support Facility at Michigan State University confirmed the deletion. The napAScreen Fwd was used to sequence the upstream region from the loxP site and the napAScreen Rev for the sequence downstream. These two sequences were assembled into one sequence using Vector NTI Suite 8.0 software. 56 Growth comparisons of Shewanella oneidensis MR-l wild type and MR-l AnapA. The cultures of the the wild type and three MR-l AnapA independent mutants were cultivated aerobically and anaerobically with 3 mM KNO3 in M1 minimal medium as described above. Growth was monitored constantly by 0D measurements at 600nm. Samples from each culture were collected throughout the incubation period for determination of nitrate, nitrite and ammonium concentrations by a Lachat QuickChem Automated Flow Injection Ion Analyzer following the Copperized Cadmium Reduction Method as in QuickChem Method No. 10-107-04-1-A (Lachat Instruments, 1988) at the Soil Testing Lab at Michigan State University. The ammonium analysis was performed by the salicylate colorimetric method (20). Gene expression profiles of MR-l growth anaerobically with 1 mM and 40 mM KNO3. Global gene expression profiles of anaerobic cultures of MR-l at a low and a high nitrate concentration were compared using DNA microarray technology. A Shewanella oneidensis MR-l complete genome microarray containing a total of 4197 PCR amplicons and 451 oligonucleotides representing individual open reading frames (9) was provided by Liyou Wu and J izhong Zhou at Oakridge National Laboratory, Oakridge, TN, USA. cDNA preparation and labeling were performed as previously described (27) using a 2:3 ratio of 5-(3-aminoallyl)-dUTP and dTTP. Hybridization and post-hybridization washes were done as described elsewhere (10). Three biological replicates per treatment were used for the hybridization of six microarray slides including technical duplicates (dye- swap). The slides were scanned using an Axon 4000B scanner (Axon Instruments, Inc.). The data analysis was performed using the GeneSpring 6.0 software (Silicon Genetics). The data was normalized per chip and per gene (Lowess Normalization) and the spots 57 with less than 55% of pixels greater than background plus two standard deviations were eliminated from the analyses (15). The data was filtered using the Benjamini and Hochberg false discovery rate with 95% confidence and only those genes with a >2-fold change in magnitude were considered significant. RESULTS Expression analyses of napA and nrfA in cultures of Shewanella oneidensis MR-l by Q-RT-PCR. Liquid samples from the cultures used in the Q-RT-PCR analyses were analyzed to quantify nitrate, nitrite and ammonium concentrations in the medium. Insignificant amounts of nitrite and ammonium were produced in aerobic cultures supplemented with 3 mM KN 03, and not much nitrate consumption was seen (FIG. 3.1). The anaerobic cultures with 0.1 mM to 1 mM KNO3 showed disappearance of nitrate and nitrite, although in cultures with the higher initial concentrations, nitrate, nitrite and ammonium remained. The expression of napA in anaerobic cultures of MR-l increased with increasing concentrations of KN03 until 3 mM after which the expression reached a plateau (FIG. 3.2). The expression of napA in the MR-l aerobic cultures with 3 mM KNO3 and without nitrate did not show a significant difference between each other or when compared with the anaerobic cultures grown on 0.5 mM, 1 mM, 3 mM KN03 (Fa=o_05 9, 20). The expression of napA in anaerobic cultures with 3 mM, 10 mM, 15 mM and 40 mM KN03 was not significantly different between each other. However, the difference in its expression in anaerobic cultures on 40 mM KN03 when compared to that of aerobic and 58 45 40 g 35 30 25 2° .. 15 10 Concentration at collection time (mM) OmM 0.1mM 0.25mM 0.5mM 1mM 3mM 10mM 15mM 40mMAerobic Aearotslc m initial nitrate concentration (mM) I N03. . N02- U NH; FIG. 3.1. Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensis MR-l used for Q-RT-PCR analyses at the time the cells were harvested for RNA extraction. Three biological replicates were used for each condition to calculate the mean and the standard deviation. 59 8 __ 2 f 3’ 0.9 - e - .L a 0.8 aw— > ab 33 <5 3: 0.7 c>~ L on. -8 5’ o 0.6 '5 a “a 0 a» 0.5 __ a 2 < 0.4 I T T 1 I I I I I I o 4 81216 20 24 28 32 36 4o Nitrate Concentrations (mM) FIG. 3.2. Expression of napA in cultures of Shewanella oneidensis MR-l grown at different nitrate concentrations under anaerobic conditions (0), aerobically with no nitrate (I) and aerobically with 3 mM nitrate ( ). Three biological replicates each done with three analytical replicates were used to calculate the mean and the standard deviation for each growth condition. 60 anaerobic cultures on 0.1 mM, 0.25 mM, 0.5 mM and 1 mM KN03 was statistically significant. The maximum expression of nrfA was reached at 1 mM KNO3 and higher concentrations showed a constant expression of the gene (FIG. 3.3). The expression of nrfA was not significantly different (F u=0.05 9, 20) between anaerobic cultures grown on nitrate at concentrations higher than 0.1 mM KN03. The difference in the expression of nrfA in the aerobic cultures without nitrate when compared to that of aerobic cultures with 3 mM KN03 was statistically significant. The nrfA expression in aerobic cultures with and without nitrate was not significantly different when compared to the other anaerobic cultures (except for those at 0.1 mM KN 03, which was statistically different from all the other conditions tested). 61 .0 co 1—1 .0 co copy number 0 \l Average of ratio of Log10 nrfA copy number/Log10 23 0.6 0.5 0.4 I I I I I f I I T I 0 4 8 12 16 20 24 28 32 36 40 Nitrate Concentration (mM) FIG. 3.3. Expression of nrfA in cultures of Shewanella oneidensis MR-l grown at different nitrate concentrations under anoxic conditions (0), aerobically with no nitrate (I) and aerobically with 3 mM nitrate ( ). Three biological replicates each done with three analytical replicates were used to calculate the mean and the standard deviation for each growth condition. 62 napA deletion mutagenesis and complementation analysis. The napA deletion mutants obtained were confirmed by two diagnostic PCR reactions. The first reaction generated a fragment of approximately 1300 bp, which corresponds to the size of the flanking regions and the loxP residual left after the recombination by the Cre recombinase (FIG. 3.4). This PCR product was sequenced. A control using the MR-l wild type genomic DNA as template was used for the same reaction. Since in this case the napA gene is present the fragment size is approximately 3800 bp long. This PCR reaction does not only confirm the absence of the gene but also demonstrates the correct location for the incorporation of the construct at the time of the homologous recombination. This is a critical problem for mutagenesis in MR—l, where after the transformation with the allelic exchange vector, the construct often gets integrated somewhere else in the genome. The second diagnostic PCR reaction amplified a region inside the napA gene. No amplification was observed in the case of the AnapA mutants as expected if the gene was deleted. A positive control for this reactions was performed using the MR-l wild type genomic DNA as the template. The fragment for this reaction was approximately 541 bp long. The PCR products of the mutated region in the AnapA (FIG. 3.4; lanes 1, 2 and 3) were used for DNA sequencing (FIG. 3.5). This sequence includes approximately 500 bp to each side of the deletion and the loxP residual (in boldface). 63 4 Kb 3 Kb 2 Kb 1.65 Kb 1 Kb 0.85 Kb 0.65 Kb 0.50 Kb FIG. 3.4. napA gene deletion confirmation by PCR. This is a 1% agarose gel 1X TAB which has the PCR reactions to confirm the MR-l AnapA mutants. Lanes labeled as M are for 1 Kb plus DNA ladder (Invitrogen Life Technologies). Lanes 1-5 correspond to PCR screening with napAScreenout primers. Lanes 6-10 correspond to PCR screening with napART Fwd and napAtemp Rev primers. Sample order: lanes 1 and 6, MR-l AnapA 22; lanes 2 and 7, MR-l AnapA 66; lanes 3 and 8, MR-l AnapA 68; lanes 4 and 8, MR-l wild type; and lanes 5 and 10 negative control (no DNA). 64 l agcatttttc tcatcgatca gtctccaatc cccattttag catcgctaat gtgctcaagt 61 tgaggatcaa gcgcacccga tgggcaggct tttatgcagg gaatatcctc acacatttcg 121 cagggaatgt gtcttgcggt aaagaatggc gtgccagtgg cggcgccatc gaaccaacgt 181 gccagtgtta gcgtgtcgta agggcaagcc tccacacaca aaccgcagcg cacgcaggcc 241 gagagaaaat cgctctcctc aagggcgccc ggtggtctgc aagcttgagg agcgagctgg 301 ccttggcttt ttgccgtagc cgttaagcct aatcccacga gtcccatcac acagccagcc 361 tttgccgtcg tggccaaaaa ctggcgacg'g ttgacttgct tggctgtgaa tgcactctta 421 acttgctgac tcaccttaga ctccttaatt gctattaatt gacgccatcg ctatcaaatg 481 aaggcttagg ccttcatcac ttttacgggg cactccgcgg tatcgataag ctggatccat 541 aacttcgtat aatgtatgct atacgaagtt atgcggccgc catatgggcg gctaatgctc 601 We ctcactcatt ttttctaaca gttcttgttc taggggctcg acttggtggt 661 aaatcaaact ggcggataac acgccggaca gggcattgat ggcttcaaca ttatcgagaa 721 tggccttttg gctatctcct tcgagggtaa taaccaattt accttcgggg gaaatggcgt 781 ggatatcgca gccctttaag gcggttatat cggcctctac ctgttgtaag gcattgggcg 841 cggcatgtac cacgaggctg gtaacatggt attcctgact catagcggtg atccttatct 901 ggagatgcat tcagttgcgt ttgataaatt ttagtacaac taaatgtgga tacctgcgag 961 tctagacctg attaaaaatg tgggtatacc tcacaagagg tattgaaggg gatagtcgat 1021 cgggatcaaa gtttta FIG. 3.5. DNA sequence of napA deletion in MR-l. This sequence was assembled using vector NTI Suite 8.0 software. The assembly included the sequence upstream the napA gene and the one downstream using primers napAScreen Fwd and napAScreen Rev, respectively. The sequences underlined represent the sequence of the napAC Rev and napAN Fwd primers. The sequence in boldface corresponds to the loxP residual. 65 Growth of MR-l wild type versus MR-l AnapA mutant. Growth under aerobic and anaerobic conditions with nitrate as the electron acceptor was compared in cultures of the wild type versus the AnapA mutant (FIG. 3.6). Based on 0D measurements, the aerobic growth was similar in the wild type and in the mutant. However, for anaerobic cultivation with 3 mM nitrate, no growth was detected for the AnapA mutant, contrary to the wild type for which growth was observed. No growth was detected in the anaerobic controls without nitrate. Analyses of nitrate, nitrite and ammonium were performed in the medium to determine whether the mutant could reduce nitrate (FIG. 3.7). No nitrite was detected in the medium. Low concentrations of ammonium were detected in cultures lacking nitrate as well as in the mutant cultures supplied with nitrate probably generated from cell material. As expected, in the wild type cultures reduction of nitrate and production of nitrite and ammonium was detected (FIG. 3.7). Due to the length of the napA gene (2,484 bp), the construct of an expression vector for the complementation of the AnapA mutant was not possible. However, the inability to grow and reduce nitrate when nitrate was the only electron acceptor was observed for three independent MR-l AnapA deletion mutants. This ensures that no other spontaneous mutation was causing the phenotype. These results are not shown for simplicity of the graphs. 66 0.14 0.12 0 5 10 15 20 25 30 Incubation Time (hr) FIG. 3.6. Growth curves for Shewanella oneidensis MR-l wild type grown in M1 medium aerobically (O), and anaerobically with 3 mM nitrate ( ) and Shewanella oneidensis MR-l AnapA grown aerobically (I) and anaerobically with 3 mM nitrate (‘- ). Cultures without nitrate inoculated with the wild type ( * ) and the AnapA mutant (C) were included as negative controls. 67 OD N hamburgers: A Concentration (mM) —L ‘ I N03- I N02- Cl NI‘14+ .° or O I WT No nitrate napA' No WT 3mM napA' 3mM FIG. 3. 7. Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensis MR- 1 wild type and Shewanella oneidensis MR- 1 AnapA for growth curve after 24 h incubation period. 68 Gene expression profiles of MR-l anaerobic growth on 1 mM and 40 mM KN03, The total number of genes differentially expressed, greater than 2-fold in anaerobic cultures of MR-l on 1 mM KN03 compared to those on 40 mM, was 1082 genes. Of these, 517 were up-regulated and 565 down-regulated at 40 mM relative to 1 mM KNO3 cultures. The genes up-regulated and down-regulated has been grouped in 20 functional “TIGR Role” categories (FIG. 3.8).The categories with higher percentage of up-regulated as well as the down-regulated genes were the “conserved hypothetical” (22% of the down-regulated and 15.2% of the up-regulated genes) and the “hypothetical proteins” (19.7% of the down-regulated and 12.4% of the up-regulated genes). The other categories with high percentage of up-regulated genes were “Protein synthesis” (11.5%), “Transport binding proteins” (8.85%) and “Energy metabolism” (8.85%)(FIG. 3.8). The up-regulated genes are arranged per category and only those showing a five-fold change or higher were reported in this chapter, except for the genes involved in regulatory functions for which all the genes with a fold change higher than two were included (TABLE 3.2). A complete list of all the genes induced two-fold or higher is provided (SUPPLEMENTAL TABLE B.1). The genes induced in the protein synthesis category include a variety of ribosomal protein genes, different tRNA synthetase genes and other genes involved in translation. Among the transport binding proteins there are a large number of genes encoding ABC transporters such as sulfate, copper and molybdenum ABC transporters. Also, three genes included in this category and annotated as proton/glutamate symporters ($00157, $00922 and $03562) were highly induced. In the energy metabolism category, genes associated with nitrate metabolism such as hcp, napADG and H were significantly induced. In this category a large number of up- 69 regulated genes encode cytochromes such as cydAB, ccoNOPQ, ccmF -1 and $04047, $04048 and $04643. Genes encoding ATP synthases were also induced (athBE and F). The “purines, pyrimidines, nucleosides and nucleotides” category also comprise a significant percentage of up-regulated genes (4.42%). This group includes carAB, nrdDG, guaB, apt, udp, upp, among others. There are also genes involved in amino acid biosynthesis that were significantly induced. Some up-regulated genes included in the “other categories” and in “conserved hypothetical” and “hypothetical” are associated with the LambdaSo phage, which is one of the three phages reported in MR-l (11). Induction of genes associated with redox response and oxidative stress was also observed (katG-2, dsbB, uvrA, uer and some co-chaperone genes) (TABLE 3.2, SUPPLEMENTAL TABLE 3.1). The categories with higher percentages of down-regulated genes following the “conserved hypothetical” and the “hypothetical proteins” are the “unknown firnction” (11.8%), the “regulatory functions” (7.9%) and the “energy metabolism” (6.4%)(FIG.3.8). The down-regulated genes have been grouped per functional “TIGR Role” category and only those down-regulated 5-folds or higher were reported in this chapter, except for those involved in regulatory functions for which all the genes with a fold change higher than two were included (TABLE 3.3). A complete list of all the genes repressed two-fold or higher is provided (SUPPLEMENTAL TABLE B.2). Under the “unknown function” category there are genes that encode hydrolases (e. g. $00177, $01585, $01670, $02333, $04039, $04092), oxydoreductases ($00900, $03382, $02813), domain proteins (e.g. $00033, $00296, $00805, $00815, $01208, $02495, $02862, $03489) and AMP binding proteins ($00075, $00355, $01971). Among the 70 genes that belong to the "regulatory function” there is an array of transcriptional regulators for genes involved in the synthesis of amino acids (glnB-Z, gInD, metJ, among others) and of other proteins such as flagellin (flgM), prophage LambdaSo Cro/CI family ($02990) and AraC/XylS family ($01762, $03488). Down-regulated genes involved in energy metabolism include genes of the TCA cycle, DMSO anaerobic reduction (dmaA- I, dmaB-I) and genes involved in redox response such as thioredoxin (trxA C), glutaredoxin ($02745) and NADH dehydrogenase I (nqu). Other genes involved in redox response and oxidative stress in MR-l and in other bacteria (24) that were down- regulated include katB, hemH-Z, phrB, ahpCF, sodB, ohr and some genes that encode heat shock proteins (hsl U, grpE, ipr)(TABLE 3.3, SUPPLEMENTAL TABLE B2). 71 100% Percentage of genes differentially expressed based on functionality L 90% *——"-‘-l ’1] Unknown function In Transport and binding proteins 1 [1 Transcription 80% 0% I i l l Down-regulated genes Up-regulated genes 4 I Signal Transduction I Regulatory Functions l Purinas pyrimidines. nucleosides and nucleotides I Protein synthesis I Protein fate Other categories C1 Hypothetical I Fatty Acid and Phospholipid Metabolism I Energy metabolism [:1 DNA metabolism I Disrupted Reading Frame I Consened Hypothetical Proteins l I Central Intermediary Metabolism [:1 Cellular Processes [:1 Cell Envelope I Biosynthesis of Co-Iactors. Prosthetic Groups and Carriers Amino Acid Biosynthesis 72 FIG. 3.8. Distribution of differentially expressed genes (> 2-fold change) grouped in 20 functional categories after cultivation on 1 mM (reference) versus 40 mM nitrate concentration. The total of genes down-regulated is 519 and the up-regulated is 571. TABLE 3.2. Genes induced in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KN03. Gene Relative Gene ID name expression' COG Annotation 1) Transport and Binding Proteins $00827 lldP 22.48 (21:1 1.00) L-iactate perrnease $04652 sbp 18.15 (:1: 8.56) sulfate ABC transporter, periplasmic sulfate-binding protein $04654 cysW-2 10.07 (i 2.89) sulfate ABC transporter, perrnease protein $03553 9.973 (:t 2.32) sulfate perrnease family protein $04150 9.901 (d: 2.23) transporter, putative $03599 cysP 9.506 (t 1.97) sulfate ABC transporter, periplasmic sulfate-binding protein $04653 cysT-2 8.15 (i 1.79) sulfate ABC transporter, perrnease protein $04077 6.736 (t 1.30) TonB-dependent receptor, putative $02857 6.33] (i 1.71) sodium/solute symporter family protein 2) Energy Metabolism $00849 napD 18.8 (i 9.08) napD protein $01363 hcp 15.57 (:t 4.04) prismane protein $01926 gltA 14.24 (3: 5.58) citrate synthase NADqubiquinone oxidoreductase, Na translocating, alpha $00902 nqrA-l 13.64 (A: 5.33) subunit $02136 adhE 12.51 (i 3.13) aldehyde-alcohol dehydrogenase $01364 8.712 (:1: 2.22) iron-sulfur cluster-binding protein $00848 napA 8.617 (d: 4.86) periplasmic nitrate reductase NADH:ubiquinone oxidoreductase, Na translocating, $00903 nqu-l 6.49 (at: 1.93) hydrophobic membrane protein quB $02743 acs 6.439 (d: 1.54) acetyl-coenzyme A synthetase $03286 cydA 5.869 (i 1.3 7) cytochrome d ubiquinol oxidase, subunit 1 $04509 5.607 (d: 2.30) formate dehydrogenase, alpha subunit '3) Amino Acid Biosynthesis $00277 argF 8.366 (i 1.39) omithine carbamoyltransferase $02903 cysK 5.94 (i 2.23) cysteine synthase A 4) Purines, Pyrimidines, nucleosides and nucleotides $01218 deoA 6.847 (i 1.45) thymidinc phosphorylase $02791 cdd 5.448 (:t 1.39) cytidine deaminase $02403 cmk 5.285 (:h 1.39) cytidylate kinase $01301 pyrB 5.165 (t 1.73) aspartate carbamoyltransferase 5) Regulatory Functions $01415 11.18 (:I: 3.81) transcriptional regulator, TetR family $03901 icc 7.949 (i 3.12) lacZ expression regulator $03627 6.185 (i 2.77) transcriptional regulator, TetR family $03059 5.778 (t 0.90) formate hydrogenlyase transcriptional activator, putative $02305 lrp 5.163 (d: 1.63) leucine-responsive regulatory protein $00843 5.153 (t 1.40) transcriptional regulator, LysR family $01806 pspF 4.98 (i 1.18) psp operon transcriptional activator $01916 2.802 (i 0.34) transcriptional regulator, LysR family $02490 2.714 (:1: 0.62) transcriptional regulator, RpiR family $03538 hlyU 2.457 (:t 0.42) transcriptional regulator HlyU $00393 fis 2.422 (:t 0.34) DNA-binding protein Fis $01328 2.3 (:t 0.46) transcriptional regulator, LysR family $03874 2.228 (:h 0.61) transcriptional regulator, LysR family $01687 2.17 (:t 0.57) transcriptional regulator, MerR family (Continued) 73 TABLE 3.2. (Cont’d) Genes induced in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO, Gene Relative Gene ID name expression' COG Annotation $02652 2.154 (:h 0.30)b prophage Mu$02, transcriptional regulator, Cro/CI family $03460 2.135 (:h 0.33) transcriptional regulator, LysR family 6) Protein Synthesis $00230 rsz 7.618 (t 2.33) ribosomal protein $10 $01855 rmf 6.964 (:1: 3.00) ribosome modulation factor $00007 rpmH 6.416 (t 2.58) ribosomal protein L34 $01288 rpsU 6.221 (t 1.08) ribosomal protein $21 $02261 6.027 (:1: 1.03) RNA methyltransferase, TrmH family, group 1 $03940 rle 5.857 (:h 1.86) ribosomal protein L13 $01357 rpsP 5.855 (:I: 1.81) ribosomal protein $16 $00241 rplN 5.674 (at: 1.32) ribosomal protein L14 $01359 trmD 5.544 (:1: 1.39) tRNA (guanine-N1)-methyltransferase $00231 rplC 5.379 (:1: 1.44) ribosomal protein L3 $02402 rpsA 5.299 (:t 2.07) ribosomal protein S1 $03939 rpsI 5.279 (:t 1.91) ribosomal protein S9 $00242 rplX 5.148 (d: 0.66) ribosomal protein L24 7) Protein Fate $02196 8.50 (:t 1.45) LPXTG-site transpeptidase family protein $02267 hscB 7.891 (t 3.31) co-chaperone Hsc20 $01252 5.58 (a: 1.02) peptidase, U32 family $00218 secE 5.48 (i 1.27) preprotein translocase, SecE subunit 8) Cellular Processes $03245 flgF 9.09 (:l: 2.30) flagellar basal-body rod protein FlgF $03229 fliE 7.49 (i 1.13) flagellar hook-basal body complex protein FliE $00837 6.70 (a: 1.82) beta-lactamase, putative $03250 flgB 5.46 (d: 6.74) flagellar basal-body rod protein FlgB 9) Conserved Hypothetical Proteins $04302 21.56 ($12.00) conserved hypothetical protein $00944 13.57 (:I: 4.92) conserved hypothetical protein $02821 13.16 (s: 6.13) conserved hypothetical protein $03542 12.73 (:1: 3.19) conserved hypothetical protein $04504 9.54 (:t 2.98) conserved hypothetical protein $01287 9.45 (i 3.25) conserved hypothetical protein $04505 8.70 (i 2.79) conserved hypothetical protein $00449 7.53 (:t 1.11) conserved hypothetical protein $03891 7.14 (d: 4.93) conserved hypothetical protein $03507 6.88 (d: 1.14) conserved hypothetical protein $01657 6.68 (i: 1.88) conserved hypothetical protein $00324 6.51 (i 1.24) conserved hypothetical protein $04651 6.12 (:i: 5.86) conserved hypothetical protein $03720 5.29 (i 1.09) conserved hypothetical protein $03085 5.15 (at 2.76) conserved domain protein $04131 5.08 (:I: 0.95) conserved hypothetical protein 10) Hypothetical Proteins $00941 8.25 (22.07) hypothetical protein (Continued) 74 TABLE 3. 2. (Cont’d) Genes induced in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO, Gene Relative Gene ID name expression' COG Annotation $04656 7.59 ($2.84) hypothetical protein $00581 7.55 (at 2.36) hypothetical protein ORF03631 7.05 (:I: 2.75) hypothetical protein $0A0157 6.87 (i 2.38) hypothetical protein $01947 5.91 (:t 1.01) hypothetical protein $04701 5.50 (i 1.27) hypothetical protein $01516 5.35 (i 2.44) hypothetical protein SOA0158 5.13 (i 1.51) hypothetical protein 11) Central Intermediary Metabolism $03738 cst 13.93(i3.94) sulfite reductase (N ADPH) flavoprotein alpha-component $01871 11.13(i6.92) S-adenosylmethionine decarboxylase proenzyme, putative $03727 cysD 8.33 ($3.00) sulfate adenylyltransferase, subunit 2 $03726 cysN 7.3 (i 1.87) sulfate adenylyltransferase, subunit 1 $03737 cysI 7.01 (3:1.53) sulfite reductase (NADPH) hemoprotein beta-component ‘ The relative expression is presented as the ratio of the dye intensity of the anaerobic cultures of MR-l own at 40 mM KNO, to that of the anaerobically grown at 1 mM KNO; (reference). e standard deviation was calculated fi'om six data points, which included three independent biological samples and two technical samples for each biological sample. 75 TABLE 3.3. Genes repressed in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KN03. Gene Relative Gene ID name expression' COG Annotation 1) Energy Metabolism $00274 ppc 0.17(:EO.04)b phosphoenolpyruvate carboxylase $00452 ter 0.11 (i 0.05) thioredoxin 2 $00406 trxA 0.09 (:i: 0.05) thioredoxin 1 $03683 0.07 (i 0.01) coniferyl aldehyde dehydrogenase 2) Amino Acid Biosynthesis $03019 trpE 0.19 (i 0.05) anthranilate synthase component 1 $01268 0.17 (t 0.03) glutamine synthetase $04349 iva 0.05 (:1: 0.02) ketol-acid reductoisomerase 3) Protein Synthesis $01473 smpB 0.20 (:t 0.08) SsrA-binding protein $03403 yfiA-l 0.18 (i 0.05) ribosomal subunit interface protein $01786 glnS 0.13 (:1: 0.04) glutaminyl-tRNA synthetase 4) Regulatory Functions $04057 metJ 0.50 (t 0.17) met repressor $02990 0.50 (i 0.14) prophage LambdaSo, transcriptional regulator, Cro/CI family $01393 0.50 (d: 0.08) transcriptional regulator, TetR family $03519 glnB-2 0.50 (i 0.12) nitrogen regulatory protein P-11 1 $00529 trpl 0.46 (:1: 0.14) trpba operon transcriptional activator $01259 0.44 (d: 0.12) transcriptional regulator, LysR family $00346 0.44 (i 0.17) transcriptional regulator. GntR family $03419 trpR 0.43 (:1: 0.09) trp operon repressor $01762 0.42 (:t 0.07) transcriptional regulator, AraC/XylS family $02493 0.42 (:t 0.07) transcriptional regulator, TetR family $03082 sixA 0.41 (d: 0.09) phosphohistidine phosphatase SixA $04567 0.41 (i 0.11) transcriptional regulator, Aan family $0A0165 0.40 (i 0.06) transcriptional regulator, LysR family $02455 0.40 (:1: 0.07) transcriptional regulator, LysR family $00295 0.38 (i 0.07) transcriptional regulator, LysR family $03494 0.38 (i 0.06) transcriptional regulator, TetR family $0A0041 0.38 (d: 0.03) transcriptional regulator, PemK family $03254 flgM 0.37 (:1: 0.10) negative regulator of flagellin synthesis FlgM $00402 0.33 (:1: 0.07) transcriptional regulator, LysR family $00989 0.33(i: 0.11) transcriptional regulator, LysR family $01626 glnD 0.29 (:t 0.07) protein-P-II uridylyltransferase $00433 rsd 0.28 (i 0.10) regulator of sigma D $01265 0.27 (:I: 0.09) transcriptional regulator, putative $01603 0.25 (i 0.10) transcriptional regulator, putative $04326 0.24 (:1: 0.06) transcriptional regulator, TetR family $01343 rseA 0.23 (at 0.05) sigma-E factor negative regulatory protein $02847 0.23 (at 0.11) transcriptional regulator, LysR family $03799 aan 0.22 (i 0.09) regulatory protein Aan $02282 0.22 (:t 0.03) transcriptional regulator, GntR family $01898 0.20 (:t 0.08) transcriptional regulator, putative $01669 tyrR 0.17 (i 0.04) transcriptional regulatory protein TyrR (Continued) 76 TABLE 3.3. (Cont’d) Genes repressed in anaerobic cultures of MR-l at 1 mM (reference) versus 40 mM KNO3. Gene Relative Gene ID name expression' COG Annotation $01607 0.16 (i 0.04) transcriptional regulator, LysR family $02046 0.15 (i 0.04) transcriptional regulator, MarR family $03684 0.15 (:1: 0.02) transcriptional regulator, TetR family $03488 0.14 (i: 0.02) transcriptional regulator, AraC/XylS family $03660 0.13 (i 0.02) sigma-54 dependent transcriptional regulator/sensory box protein $04312 0.09 (i 0.06) adenylate cyclase CyaA, putative $00443 0.06 (:1: 0.03) transcriptional regulator, MerR family 5) Transport and Binding Proteins $03 802 0.20 (:t 0.06) ABC transporter, ATP-binding protein $00139 ftn 0.19 (i 0.07) ferritin $04598 0.17 (:1: 0.07) heavy metal efflux pump, Cch family $02045 0.11 (:t 0.04) cation efflux family protein $00857 0.08 (i 0.03) ABC transporter, ATP-binding protein 6) Unknown Function $02849 0.20 (d: 0.03) acetyltransferase, GNAT family $03715 0.18 (at: 0.12) oxygen-insensitive NAD(P)H nitroreductase $00911 0.15 (i 0.14) ParA family protein, degenerate $02228 0.14 (dc 0.07) CBS domain protein $01609 syd 0.14 (d: 0.16) syd protein $03382 0.12 (i 0.31) oxidoreductase, short-chain dehydrogenase/reductase family $00698 fsxA 0.12 (:1: 0.19) fst protein $03586 0.12 (i 0.04) glyoxalase family protein $02850 0.11 (i 0.05) acetyltransferase, GNAT family 7) Protein Fate $04699 prlC 0.20 (:I: 0.05) oligopeptidase A $02447 0.17 (:t 0.04) channel protein, hemolysin 111 family subfamily $03577 clpB 0.12 (:1: 0.02) clpB protein $01126 dnaK 0.07 (:t 0.02) chaperone protein DnaK $02277 ipr 0.03 (d: 0.03) 16 kDa heat shock protein A 8) Cellular Processes $04170 0.18 (i 0.04) C-factor, putative $03585 0.17 (:1: 0.06) azoreductase, putative $02754 motY 0.17 (i 0.07) sodium-type flagellar protein MotY, authentic frameshift $03349 0.13 (i 0.79) glutathione peroxidase, putative $00956 ahpF 0.09 (d: 0.05) alkyl hydroperoxide reductase, F subunit $00958 ahpC 0.08 (:1: 0.05) alkyl hydroperoxide reductase, C subunit $01158 0.07 (:t 0.75) Dps family protein $01070 katB 0.05 (i 0.05) catalase 9) DNA Metabolism $03866 0.20 (:h 0.04) site-specific recombinase, phage integrase family $03384 phrB 0.07 (i 0.05) deoxyribodipyrimidine photolyase 11) Biosynthesis of co-factors, prothetic groups and carriers hemH-2 0.05 (i 0.06) ferrochelatase ‘ The relative expression is presented as the ratio of the dye intensity of the anaerobic cultures of MR-l own at 40 mM KN03 to that of the anaerobically grown at 1 mM KNO, (reference). e standard deviation was calculated from six data points, which included three independent biological samples and two technical samples for each biological sample. $03348 77 DISCUSSION This study demonstrates that NapA is the sole nitrate reductase in Shewanella oneidensis MR-l. The gene content derived from the complete genome sequence of MR- 1, the gene expression analyses, and the deletion of the napA gene are all consistent in establishing that this periplasmic nitrate reductase is in fact responsible for the first step in the nitrate reduction pathway of Shewanella oneidensis MR-l. The inability of the MR-l AnapA mutant to grow when nitrate was the only electron acceptor demonstrated its role as the sole enzyme responsible for the reduction of nitrate into nitrite in this microorganism. We observed an increase in the expression of napA and nrfA with increasing concentrations of nitrate until a plateau was reached. This was also confirmed in the DNA microarray analyses where napADGH expression was induced at high concentrations of nitrate (40 mM KN03). Expression of nrfA did not change under these two conditions as was previously observed in the Q-RT-PCR analysis where its expression reached a maximum at 1 mM nitrate and its level remained constant at higher concentrations. These results indicate that MR-l does not posses an alternative nitrate/nitrite reductase system for high concentrations of nitrate as is observed in E. coli (39). Gene expression studies of napA and narG in E. coli have demonstrated an increase in the expression of napA but not narG when nitrate is present in concentrations below 1 mM and a repression of napA and induction of narG when nitrate concentrations are higher than 1 mM nitrate (39). This trend was also observed for the nitrite reductases NrfA and NirB (40). In this case nrfA is expressed at low concentrations and repressed at high concentrations of nitrate whilst nirB is expressed at high concentrations of nitrate. 78 Moreover, in E. coli there are two different two-component regulatory systems for the regulation of the expression of nap and nor genes, NarP/NarQ and NarL/NarX. NarP and NarQ activate the expression of the nap and mf genes when the concentrations of nitrate are below 1 mM (33, 39, 40). When the concentration of nitrate exceeds 1 mM, NarL and NarX repress the nap and nrfoperons and induce the nar and nir genes. In contrast to E. coli, Shewanella oneidensis MR-l possesses only homologs for narP and narQ, which explains the expression pattern observed for the nap and nrfA genes in this organism. This is also true for the human pathogen Haemophilus influenzae, which colonizes many human body fluids (35). Compared to H. influenzae, Shewanella oneidensis MR-l has been found in more diverse environments such as sediments, water columns as well as an opportunistic pathogen in humans and aquatic animals (11, 37, 38). These environments, including bodily fluids, are scarce in nitrate, therefore it is advantageous for MR-l and for any other organism that lives in such habitats to posses a nitrate scavenging system to generate energy for growth. This is a good survival strategy for MR-l since this microorganism seems to prefer microaerophilic conditions. In aerobic conditions MR-l cultures form clumps of cells that keep the cultures in a microaerophilic space (38). Since NapA has a very high affinity for nitrate and is much more active in anaerobic conditions, its presence in MR-l is vital. The gene expression profile of MR-l growth at 1 mM nitrate when compared to that at 40 mM nitrate changed dramatically. A high percentage of up-regulated genes belong to the “protein synthesis” functional category, while the number of down- regulated genes in this category is considerably low. This might indicate that there is an increase in the expression of genes necessary for the synthesis of the proteins when the 79 concentrations of nitrate increase. It might also indicate that at higher concentrations the synthesis of more proteins is necessary. “Transport and binding proteins” and “energy metabolism” are also categories with a high percentage of induced genes. These three categories contain genes that are important for growth and energy generation. Induction of these genes represents an increase in metabolic activity in the cell. Many of the genes in the “transport and binding proteins” category belong to the ABC transporter system which has been associated with the transport of nitrate, sulfate, copper and molybdenum that are important molecules involved in cell growth and energy generation. Changes in the expression of these genes in response to nitrate reduction have been reported previously in bacteria, including MR-l (3, 5). Genes encoding sulfate transporters and enzymes involved in sulfate metabolism were highly induced. This indicates a need for sulfate due to the increase in metabolic activity, especially in protein synthesis. Genes that belong to the “energy metabolism” category that were up-regulated at 40 mM KN03 include genes involved in glycolysis, nitrate reduction, electron transport and synthesis of ATP. This up-regulation suggests that an increase in nitrate reduction by NapA promotes growth and energy generation in MR-l. Induction of genes involved in purine, pyrimidine, nucleoside, nucleotide and amino acid biosynthesis was also observed. This not only indicates an increase in metabolic activity but also might suggest nitrate assimilation in MR-l. Nitrate assimilation has been reported in organisms which possess an assimilatory nitrate reductase, which reduces nitrate into ammonium in the cytoplasm. This ammonium is a precursor for amino acid and nucleotide biosynthesis. Enzymes involved in the conversion of ammonium into organic material present in MR-l include carbamoyl-phosphate and glutamine synthases. Induction of the genes that encode the 80 small and the large subunit of the carbamoyl-phosphate synthase were observed at 40 mM nitrate. Repression of the gene encoding glutamine synthase and of other genes involved in its transcription activation gInD, glnB-Z, ntrB and ntrC was observed at high concentrations of nitrate. This repression may be caused by the accumulation of some of the metabolites in the synthesis of amino acids. The transcription of the genes encoding the glutamine synthase is halted at high concentrations of glutamine by feedback inhibition. Glutamine is the product of the ATP-dependent arnidation reaction of glutamate, which is catalyzed by glutamine synthase. High concentrations of ammonium also inhibit the activation of this enzyme. Nitrate assimilation is an ATP consuming process, which is carefully regulated to avoid unnecessary energy expenditure. In cultures of MR-l examine in this study, nitrite gets reduced into ammonium, which accumulates in the medium. Similar to Rhodobacter capsulatus, which is capable of assimilating nitrate (5), growth curves of MR-l in which nitrate, nitrite and ammonium were monitored over time indicated a start of ammonium consumption late in the growth curve. In Rhodobacter capsulatus, when hydroxylamine was supplied to the medium, ammonium assimilation did not occur until hydroxylamine was completely reduced and ammonium accumulated in the medium. The reduction of nitrate produces intermediates that are highly toxic to the cell. Nitrite, nitric oxide, hydroxylamine and even ammonium cause cytotoxic effects in many organisms (5, 30). One of the important queries in MR-l is, how does this microorganism grow on such high concentrations of nitrate? Nitrite, an oxidative-stress causing agent, is produced following the reduction of nitrate. Nitric oxide and hydroxylamine are intermediates that are generated in the reduction of nitrite into ammonium. These 81 intermediates are generated within the nitrite reductase and remain trapped during the reaction. Studies in bacteria suggest that when high concentrations of these intermediates are generated they are release from the enzyme (30, 31). However, some bacteria posses an enzyme called prismane that reduces hydroxylamine into ammonium detoxifying and reducing oxidative stress in the cell. This enzyme is encoded by the hcp gene ($01363) in MR-l , which is significantly induced at high concentrations of nitrate. There are also a number of up-regulated genes that have been associated with oxidative stress response in MR-l as well as in other microorganisms (24). In our study there is a shift in the oxidative stress responses when there is an increase in the concentrations of nitrate. Some genes are down-regulated(dmaA-1, dmaB-I, trxAC, katB, hemH-Z, phrB, ahpCF, sodB, ohr) and others up-regulated (katG-Z, dsbB, uvrA, uer) in response to a higher nitrate concentration. The products of these genes protect the cell from DNA damage or convert some of the toxic intermediates in less toxic forms. There is also induction of 15 genes that are involved in the activation of prophage LambdaSo, which is one of the three prophages described in MR-l. Activation of LambdaSo has been previously described in MR-l when subjected to oxidative stress conditions (24). This prophage can cause lysis, affecting cell growth. In cultures of MR-l where the concentrations of nitrate exceeded 5 mM, growth rate decreased and reached a plateau with increasing nitrate concentrations. The oxidative stress and the activation of this prophage might be causing an inhibitory effect on cell growth. This study provides a better understanding of the nitrate reduction pathway of MR-l and also gives a global profile of its genetic expression in response to an increase in nitrate concentration. We conclude that NapA is the sole enzyme responsible for 82 nitrate reduction in MR-l. Even though NapA is a periplasmic protein, our study suggests that there is energy generation in this process. Since CymA is associated with the membrane and it is required for nitrate and nitrite reduction, it is possible that it can be generating a membrane potential that provides energy for the cell (28, 29). Also, MR-l has the largest number of cytochromes among bacteria, which play a key role in electron transport in various anaerobic processes. This might also be a very important mechanism in MR-l to generate energy from many other processes since many of the reductases (including the metal reductases) present are periplasmic. We also suggest that nitrate might be assimilated, however more studies on this subject need to be performed. There are also striking similarities in the genetic machinery and nitrate reduction pathway of MR-l when compared to some pathogenic bacteria. These make MR-l a very good model to study and understand the mechanisms of these microorganisms to survive in their natural environments. 83 REFERENCES . Alexeyev, M.F. 1999. The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. Biotechniques. 26:824-6, 828. . Bedzyk, L., T. Wang, and R. W. Ye. 1999. 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Sundin, L. Wu, J. Zhou and J.M. Tiedje. 2005. Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-l following exposure to UVC, UVB and UVA radiation. J. Bacetriol. 187:3556- 3564. 25. Roldan, M.D., F. Reyes, C. Moreno-Vivian, and F. Castillo. 1994. Chlorate and nitrate reduction in the phototrophic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides. Curr. Microbiol. 29: 241-245. 26. Sambrook, J., and D.W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY. 27. Schroeder, R.G., L.M. Peterson, and R.D. Fleischmann. 2002. Improved quantitation and reproducibility in Mycobacterium tuberculosis DNA microarrays. J. Mol. Microbiol. Biotechnol. 4:123-126. 28. Schwalb, C., $.K. Chapman, and G.A. Reid. 2002. The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella. Biochem, Soc. Trans. 30:658-662. 29. Schwalb, C., $.K. Chapman, and G.A. Reid. 2003. The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis. Biochemistry. 42:9491-9497. 30. Simon, J. 2002. Enzymology and bioenergetics of respiratory nitrite ammonification. FEMS. Microbiol. Rev. 26:285-309. 31. Simon, J., O. Einsle, P.M. Kroneck, and W.G. Zumft. 2004. The unprecedented nos gene cluster of Wolinella succinogenes encodes a novel respiratory electron transfer pathway to cytochrome c nitrous oxide reductase. FEBS. Lett. 569:7-12. 86 32. Stewart, V., Y. Lu, and A.J. Darwin. 2002. Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12. J. Bacteriol. 184:1314-1323. 33. Stewart, V., P.J. Bledsoe, and SB. Williams. 2003. Dual overlapping promoters control napF (periplasmic nitrate reductase) operon expression in Escherichia coli K-12. J. Bacteriol. 185:5862-70. 34. Stolz, J.F., and P. Basu. 2002. Evolution of nitrate reductase: molecular and structural variations on a common function. Chembiochem. 3: 198-206. 35. Tatusov, R.L., A.R. Mushegian, P. Bork, N.P. Brown, W.S. Hayes, M. Borodovsky, K.E. Rudd, and E.V. Koonin. 1996. Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli. Curr. Biol. 6:279-91. 36. Thormann, K.M., R.M. Saville, S. Shukla, and A.M. Spormann. 2005. Induction of rapid detachment in Shewanella oneidensis MR-l biofihns. J. Bacteriol. 187:1014-21. 37. Tiedje, J.M. 2002. Shewanella-the environmentally versatile genome. Nat. Biotechnol. 20: 1093-4. 38. Venkateswaran, K., D.P. Moser, M.E. Dollhopf, D.P. Lies, D.A. Saffarini, B.J. MacGregor, D.B. Ringelberg, D.C. White, M. Nishijima, H. Sano, J. Burghardt, E. Stackebrandt, and K.H. Nealson. 1999. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. Nov. Intl. J. Syst. Bacteriol. 49:705-724. 39. Wang, H., C.-P. Tseng, and R.P. Gunsalus. 1999. The napF and napG nitrate reductase operons in Escherichia coli are differently expressed in response to submicromolar concentrations of nitrate but not nitrite. J. Bacteriol. 181:5303- 5308. 40. Wang, H., and R.P. Gonsalus. 2000. The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. J. Bacteriol. 182:5813-5822. 41. Wolin, E.A., M.J. Wolin, and R.S. Wolfe. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-6. 87 CHAPTER IV Role of EtrA in the Regulation of the Nitrate Reduction Pathway in Shewanella oneidensis MR-l 88 ABSTRACT EtrA is an Escherichia coli Fnr homolog, which has been identified as a possible global regulator of the anaerobic metabolism of MR-l. EtrA shares 50.8% and 73.6% amino acid sequence identity with the oxygen-sensing regulator in E. coli, Pm, and with the Anr (anaerobic regulator of arginine deaminase and nitrate reductase regulator) protein of Pseudomonas aeruginosa, respectively. This similarity suggests an oxygen sensing regulatory role for EtrA in MR-l. Physiological and genetic expression analyses of a S. oneidensis MR-l etrA knockout strain (EtrA7-1) indicates a regulatory role of EtrA in the expression of genes likely involved in nitrate reduction, specifically the nap genes, nrfA, cymA and hcp, and in other anaerobic metabolism processes. This was concluded after detecting a significant decrease in the expression of these genes in EtrA7- 1 relative to the wild type. However, the nitrate reduction activity was not shut down in the mutant, suggesting the existence of other regulator(s) involved in the regulation of this process. Evidence for negative regulation of putative genes related to aerobic metabolism was also obtained. A starvation genetic response was observed for this mutant and the effects on its growth were examined. A significant decrease in the growth of the mutant was observed when compared to that of the wild type. During this period, the cells entered a state of prophage activation possibly in response to stressful growth conditions (starvation and oxidative stress) as a result of absence of EtrA. 89 INTRODUCTION The regulatory mechanisms that control the bacterial anaerobic metabolism have been of interest in Shewanella oneidensis MR-l and other microorganisms (3, 26, 37, 38, 45, 47). In Escherichia coli, for example, the transition from aerobic to anaerobic conditions is mainly regulated by Fnr (firmarate-nitrate reduction regulatory protein) and by the two-component regulatory system ArcAB (aerobic respiratory control) (39, 44). This regulation occurs at the transcriptional level. Recently a genetic expression study in E. coli K12 indicated that one-third of its 4,290 genes were differentially expressed during aerobic versus anaerobic growth. Among the differentially expressed genes, 712 (49%) genes were directly or indirectly affected by Fnr. Fnr possesses a [4Fe-4S]2+ cluster that acts as a sensory domain for oxygen (8). When oxygen levels increase, a two- step reaction transformation occurs where 02 reacts with the cluster and transforms it into a [3Fe-4S]1+ cluster. The second step is a non-redox reaction in which the [3Fe-4S]1+ is converted into a [2Fe-2S]2+ cluster. This transformation apparently changes the conformation of the protein impeding its binding to DNA, which in turns affects the transcription of the genes it regulates. The regulation of the nap and nrf genes has been studied in detailed in Escherichia coli (7, 9, 10, 25, 39, 45). Mutational studies have revealed the direct participation of F nr and of two additional two-component regulatory systems NarL/NarX and NarP/NarQ in the regulation of the expression of the nap and nrf genes. In S. oneidensis MR-l, there are homologues for narQ and narP but not for narX or narL. In E. coli, NarP enhances the expression of the nap and nrf genes, while NarL acts as a negative regulator at high concentrations of nitrate (45). 90 In contrast to E. coli, the regulation of the nitrate reduction pathway of MR-l has not been studied in depth. Mutational studies in MR-l have identified two possible regulators for the nitrate reduction pathway, EtrA (electron transport regulator protein) and CRP (cyclic AMP receptor protein) (37, 38). EtrA is an Fnr homolog that shares 50.8% and 73.6% of amino acid sequence identity with Fnr (fumarate-nitrate reduction regulatory protein) in Escherichia coli and Anr (anaerobic regulator of arginine deaminase and nitrate reductase) in Pseudomonas aeruginosa, respectively. This high degree of similarity suggests a potential for EtrA to regulate metabolic activities by sensing oxygen-limiting conditions. Despite the lack of physiological evidence to support a regulatory role of EtrA in the anaerobic metabolism of MR-l (26), a genetic expression study using a partial S. oneidensis MR-l DNA microarray suggested an involvement of EtrA in the regulation of proteins associated in aerobic and anaerobic metabolism (3). This study compared the growth of Shewanella oneidensis MR-l D$P10 strain (a rifampicin spontaneous mutant) with a DSPlO etrA deletion mutant. The results suggested a negative regulation in the expression of genes related to aerobic metabolism and a positive regulation in the expression of genes associated with anaerobic metabolism by EtrA, as it has been observed for Fnr in E. coli (39). The study of Shewanella oneidensis MR-l has recently increased due to its potential as a bioremediator (19, 20, 46), which also motivated support for a genomic sequencing effort. Bioremediation is a challenging but cost-effective procedure. A variety of strategies have been developed to enhance the effectiveness of the inocula in the environment and starvation is one of these. It has been observed that cells that have been exposed to long periods of nutrient and energy limitation express proteins that protect 91 them against stressful growth conditions. Thus, these cells are pre-adapted to survive the harsh conditions in the environment (16). Starvation has been studied in a few species of the Shewanella genus (2, 6, 16), however to our knowledge there is not much information regarding the cultivation of S. oneidensis MR-l in starvation conditions. To demonstrate the fimction of EtrA, we generated a gene-deletion mutant in a wild type background. Previous experimentation in etrA mutant strains are complicated by rifampicin resistance in the host strain which modifies the electron transport function of the cell membrane. In this study, genetic expression of an etrA knockout strain (EtrA7- 1) under anaerobic conditions with nitrate as the sole electron acceptor was compared with that of the wild type using a complete S. oneidensis MR-l genome array. The genetic expression pattern of the anaerobic grth of the EtrA7-1 was also examined at a high and a low concentration of nitrate. The genetic expression profile of EtrA7-1 indicates a dramatic starvation response at the transcriptional level. The physiological and genetic analyses of EtrA7-l suggest an involvement of EtrA in the regulation of the expression of the nap operon, the cymA and the nrfA genes. Regulation of other genes associated with energy metabolism is suggested and these results were compared with the previous findings of Beliaev et al., 2002 done in the DSPIO host. MATERIAL AND METHODS Bacterial strains and growth conditions. The bacterial strains, plasmids and primers used in this study are presented in TABLE 4.1. Cultures of Shewanella oneidensis MR-l , Shewanella oneidensis MR-l AetrA, Shewanella oneidensis MR-l AetrA complement 92 TABLE 4.1. Bacteria, plasmids, and primers used in this study. Strain, plasmid, primer or probe Bacterial Strains E. coli 02155 S. oneidensis MR-l EtrA7-1 EtrA7-1 complement EtrA7-1 with pCM62 Description or nucleotide sequence“ Diaminopimelic acid auxothroph used for cloning and conjugation Lake Oneida, N.Y., sediment etrA gene deletion derived fi'om MR-l etrA deletion mutant complemented with the etrA gene cloned into pCM62 etrA deletion mutant transformed with the pCM62 as a negative control for complementation Plasmids pCM62 Cloning vector pCM157 Broad-host-range cre expression vector pCMl 84 Broad-host-range allelic exchange vector pCCGl95 pCMl84 with etrA upstream flank pCCGl96 pCCGl95 with etrA downstream flank pKNOCK-Gm Broad-host-range allelic exchange vector pCCG02 pKNOCK-Gm with etrA flanking regions separated by two loxP sites flanking a kanamycin resistance gene pCCGOZc pCM62 with MR-l putative etrA gene Primersc etrAN Fwd GCCGCGGTCATGTCGGTTCTCAAGT etrAN Rev CGAGCTCCGACAGCTATCTGTTAGTCT etrAC Fwd CGAATTCAAATCACCGC I'I'I'I'AACTT G etrAC Rev GCATATGCCAGATAAATCACACCTTTI‘ etrAScreenout AAT'I‘CT'I‘CAGGCA’ITI‘GACT CG Fwd etrAScreenout GGCCGTATCTTGAGTTATACCC Rev etrAcomp Fwd GGATCCAGGTGTGATTI‘ATCTGGCG etrAcomp Rev GAATTCCCGACATGACAATAGAGCAGA Source, reference or relative position of primer ogrobe 12 33 This study This study This study 28 27 27 This study This study 1 This study This study CAT Start codon 503 nt upstreamd the start codon 493 nt downstream the stop codon TAA Stop codon 1188 nt downstream the stop codon 559 nt upstream start codon TTA Stop codon ATG Start codon ° The sequence for the primers is written from the 5’end to the 3’end. ° Primers were designed using putative gene sequences of S. oneidensis MR-l. ° For primer sequences, the restriction sites incorporated are underlined. CATATG, Ndel; GAATTC, EcoRI; GAGCT C, $ac1;CCGCGG, SacII; GGATCC, BamHI. ° Even though the etrA gene is in the opposite direction in the genome, the sequence at the right end of the start codon will be denominated as upstream and the one at the left side of the stop codon as downstream. 93 and Shewanella oneidensis MR-l harboring pCM62 (as the negative control for complementation) were incubated at 30°C after inoculation in Modified M1 minimal medium (32) with no NI~I4C1 to avoid interference with chemical analyses. HEPES (pH 7.2) was added to buffer the medium at a 50 mM final concentration. The medium was supplemented with 20 mM lactate. KN03 was added as the electron acceptor in concentrations specified below. The medium prepared for the RNA extractions was degassed by boiling, purged with helium and transferred to 250 ml serum bottles. The cultures used for growth curve determinations were performed in 30 ml Balch tubes. The serum bottles and tubes were closed with butyl black stoppers to avoid oxygenation of the medium. The medium was autoclaved and 0.1 ml of Wolfe’s vitamin solution (52) was added by injection with a sterile syringe. The medium was inoculated by injection with a 1% inoculum from a 12 h aerobic culture in M1 medium, which originated from an overnight starting culture in aerobic M1 medium, as well. Incubation was performed at 30°C without shaking. Negative controls (i) without inoculation and (ii) medium without the electron acceptors but inoculated, were run in parallel with all grth and gene expression experiments. Cultures of Escherichia coli 02155 (auxotroph of diaminopimelic acid) were grown in Luria-Bertani (LB) medium supplemented with 100 ug/ml of diaminopimelic acid (DAP). These cultures were incubated at 37°C. Shewanella oneidensis MR-l was cultivated in LB medium and incubated at 30°C during the mutagenesis process. Antibiotics for E. coli were prepared and added as described elsewhere (40). The antibiotics used for the selection of MR-l positive transformants were added in the 94 following concentrations: 25 ug/ml of kanamycin, 7.5 ug/ml of gentamycin, and 10 ug/ml tetracycline. RNA extractions. To compare the gene expression profile of the wild type with that of the EtrA7-1, total RNA was extracted from cultures of S. oneidensis MR-l (OD600 um 0.03-0.05) and EtrA7-1 (OD600 um 0.012-0.015) grown in triplicate as described above. The wild type was grown at 2 mM KN03, while EtrA7-1 was inoculated in 1, 2 and 40 mM KN03 media. Cells were collected at mid-log phase and concentrated by centrifuging at 4°C for 30 min at 7,500 rpm. The pellets were washed with 1 ml of an ice- cold 1X DEPC-treated PBS solution (40). The RNA was extracted with The RNAwiz Solution following the instructions of the manufacturer (Ambion, Inc.). The RNA extraction was followed by an isopropanol precipitation (40) and its resuspension in the RNA storage solution (Ambion, Inc.). These samples were treated with RNase-free DNaseI (Roche) to eliminate residual DNA. The samples were purified by phenol, phenolzchloroform (1:1) and chloroform extractions, and stored in ethanol at -80°C until ready for use. Quality of the RNA was observed using the RNA 6000 Pico LabChip kit and the 2100 Bioanalyzer (Agilent Technologies). The RNA concentration was determined with OD measurements at 260 nm using a Varian Cary 50 BIO UV-Vis spectrophotometer (V arian, Zug, Switzerland). etrA deletion mutagenesis. Molecular procedures such as genomic and plasmid purifications, restriction digestions, sticky ends repair, ligations and electroporations were performed as previously described (40). Primers for PCR reactions (TABLE 4.1) were 95 designed using the Vector NTI® software (InforMax, Inc.) and synthesized at Integrated DNA Technologies (www.idtdna.com). a) etrA allelic exchange vector genergtion. The MR-l etrA flanking regions were cloned into the broad-host-range vector pCMl 84. This vector was used to replace the etrA gene in MR-l with a kanamycin cassette by homologous recombination. The kanamycin cassette in pCMl 84 is flanked by two loxP sites, which are in turn were flanked by two multiple cloning sites (MCS) (27). The primers etrAN Fwd (SacII) and etrAN Rev (SacI) were used to amplify the region upstream of the etrA gene start codon while the etrAC Fwd (EcoRI) and napAC Rev (NdeI) amplified the region downstream of the stop codon. The product of these reactions was approximately 500 bp each, to ensure good efficiency of recombination (27). Each primer had a restriction site linker at the S’end for an enzyme that was chosen using the pCMl 84 MCS as a reference for directional cloning of the fragments. These two fragments were cloned individually into pCMl 84 and transformed into E. coli 132155 electrocompetent cells by electroporation. The positive transformants were selected by inoculating on LB agar supplemented with kanamycin and DAP. The colonies were screened using the primers used to amplify each fragment. The pCCGl96 vector (pCMl 84 with the etrA flanking regions) was replicating independently in MR-l due to the presence of a ColEl origin present in pCMl 84, and therefore in pCCGl96. To generate a suicide vector for MR-l , pCCGl96 was digested with EcoRI and SacI. This reaction excises the kanamycin cassette with the loxP sites and the N and C fragments (a fragment of approximately 2.5 kbp) to further clone it into pKNOCK-Gm (1) for the generation of pCCG02. The pKNOCK-Gm broad-host-range 96 vector has a gentamycin resistance gene and a R6K origin, which needs a it protein in order to be propagated. Since this protein is not present in MR-l, this plasmid made a good suicide vector. To clone the fragment excised from pCCGl86 into pKNOCK-Gm, this vector was digested with SmaI, which is a blunt end cutter and the plasmid was dephosphorylated with calf intestinal phosphatase to avoid self-ligation. The sticky ends product of the digestion of the insert with EcoRI and SacI were repaired using T4 DNA polymerase. These reactions were gel purified and a vectorzinsert ratio of l :16 was used for the ligation reaction. Electroporation and screening of the transformants was performed as described above. b) etrA flelic exchgge vector transfer into Shewanella oneidensis MR-l. pCCG02 was transformed into MR-l by conjugation (protocol from Margie Romine, Pacific Northwest National Laboratory). The E. coli 02155 harboring pCCG02 was inoculated in LB liquid media supplemented with 100 11ng of DAP and 50 ug/ml of kanamycin. Overnight cultures of MR-l and E. coli 02155 harboring pCCG02 were mixed in ratios of 1:2. This mixture was then concentrated by centrifugation and the supernatant was discarded. The conjugation mixtures were spotted on an LB plate with DAP and incubated at room temperature for 12 to 16 h. Controls were spotted on individual plates. After incubation, the spotted cultures were scrapped and resuspended in 1 mL of IX Phosphate-Saline Buffer (PBS) (40). Two 10-fold serial dilutions were prepared and plated on LB plates with 25 ug/mL of kanamycin and no DAP to avoid grth of the E. coli 02155. Selection of positive transformants was performed by screening on LB plates with kanamycin and LB plates with kanamycin and gentamycin. Positive MR-l etrA deletion mutants were expected to be resistant to kanamycin and 97 susceptible to gentamycin. Colonies with this phenotype were screened by PCR using etrAScreenout Fwd and etrAScreenout Rev which anneal approximately 700 bp downstream and 56 bp upstream from the etrA flanking regions included in pCCG02, respectively. This screening ensures that the recombination occurred in the targeted area. c) RemovaLOf kflamvcinflsgtte. The two loxP sites flanking the kanamycin cassette are recognition sites for Cre recombinase. This recombinase excises the region inside the loxP sites by recombination leaving one of the loxP sites. pCM157 is a cre gene expression vector (27) which was electroporated into E. coli 02155. This vector was transformed into Shewanella oneidensis MR-l AetrA Kanr by conjugation as described above. Colonies susceptible to kanamycin and resistant to tetracycline were selected as positive transformants. These were screened by PCR using etrAScreenout primers to confirm the loss of the kanamycin cassette. d) pCM157 curation from S. oneidensis MR-l AetrA. Cultures of MR-l AetrA (Tetr Kans) were transferred three times on LB liquid media with no antibiotics and then screened for tetracycline susceptibility on LB agar. This phenotype indicates the loss of the pCM157 plasmid. Colonies were transferred and screened until the correct phenotype was obtained. Colonies susceptible to kanamycin and tetracycline were diagnosed by PCR with the etrAScreenout primers and using primers targeting the inside region of the MR-l etrA gene (etrAcomp Fwd and etrAcomp Rev). DNA sequencing performed at the Genomics Technical Support Facility at Michigan State University confirmed the deletion. The etrAC Fwd was used to sequence the upstream region from the loxP site and the etrAN Rev for the sequence downstream. These two sequences were assembled into one sequence using Vector NTI Suite 8.0 software. 98 AetrA complement construct. Plasmid pCM62 was used as the vector for the expression of the etrA gene in one of the etrA knockout strains obtained called EtrA7-1. The etrA expression vector was called pCCG02c. The etrA gene was amplified from S. oneidensis MR-l genomic DNA using the etrAcomp Fwd and etrAcomp Rev. The amplicon was double digested with BamHI and EcoRI, which were the restriction sites linked to the 5’end of etrAcomp Fwd and etrAcomp Rev, respectively. The pCM62 plasmid was also double digested with BamHI and EcoRI. The products of these digestion reactions were gel purified and the vectorzinsert ratio was 1:3. Ligation, electroporation into E. coli 02155 and conjugation into MR-l was performed as described above. The vector pCM62 was transferred to MR-l by conjugation. This strain was used as a control for the complementation analyses to check for any effects caused due to its presence. Antibiotic selection of positive transformants was performed by streaking on LB plates with tetracycline. Tetracycline-resistant colonies were diagnosed by PCR using the etrAcomp primers and subsequently sequenced to verify the deletion. Growth comparisons of Shewanella oneidensis MR-l and EtrA7-l. Cultures of the wild type, EtrA7-1, EtrA7-1 complement and EtrA7-1 harboring pCM62 were grown anaerobically with 3 mM KN03 in M1 minimal medium as described above. Growth was monitored constantly by OD measurements at 600 nm. Samples from each culture were collected throughout the incubation period .for determination of nitrate, nitrite and ammonium concentrations by a Lachat QuickChem Automated F low Injection Ion Analyzer following the Copperized Cadmium Reduction Method as in QuickChem Method No. 10-107-04-1-A (Lachat Instruments, 1988) at the Soil Testing Lab at 99 Michigan State University. The ammonium analysis was performed by the salicylate colorimetric method (34). Gene expression analyses of EtrA7-l. A Shewanella oneidensis MR-l complete genome microarray containing a total of 4197 PCR amplicons and 451 oligonucleotides representing individual open reading frames (15) was used to examine the global genetic expression of EtrA7-1 under different growth conditions. Gene expression profiles of anaerobic cultures of the wild type S. oneidensis MR-l and EtrA7-1 grown at 2 mM nitrate were compared. Also, the gene expression pattern of MR-l AetrA grown at a low (1 mM KNO3) concentration of nitrate was compared to that at a high (40 mM KNO3) concentration. cDNA preparation and labeling were performed as previously described (41) using a 2:3 ratio of 5-(3-aminoallyl)-dUTP and dTTP. Hybridization and post- hybridization washes were done as described elsewhere (18). Three biological replicates per treatment were used for the hybridization of six microarray slides including technical duplicates (dye-swap) per experiment. The slides were scanned using an Axon 4000B scanner (Axon Instruments, Inc.). The data analysis was performed using the GeneSpring 6.0 software (Silicon Genetics). The data was normalized per chip and per gene (Lowess Normalization) and the spots with less than 55% of pixels greater than background plus two standard deviations were eliminated from the analyses (30). The data was filtered using the Benjamini and Hochberg false discovery rate with 95% confidence and only those genes with a >2-fold change in magnitude were considered significant. 100 RESULTS etrA deletion mutagenesis and physiology of the MR-l AetrA mutant. Six MR-l AetrA deletion mutants were diagnosed by two different PCR reactions (FIG. 4.1). The primers used for the first reaction targeted the inside region of the gene and as expected, no amplification was observed when using the DNA from the MR-l etrA deletion mutants as a template (FIG. 4.1; lanes 1-6). In lane 8, where the DNA of the wild type was the template, there was amplification of a band of approximately 750bp (expected size for the PCR product using the etrAcomp primers). The second diagnostic reaction included the etrAScreenout primers that should generate PCR products of approximately 1.75 kb and 2.5 kb when the DNA of the MR-l AetrA and the wild type are used as the templates, respectively. This is shown in lanes 9-14 for the MR-l AetrA mutants and lane 16 for the wild type. The product of this last reaction was used as the template for sequencing using the etrAC Fwd and the etrAN Rev primers (FIG. 4.2). This sequence shows the replacement of the gene with the loxP site residual and confirms the complete deletion of the etrA gene from S. oneidensis MR-l genome. 101 M12345678910111213141516 M 3kb 2 kb 1.6 kb 1 kb 0.85 kb 0.75 kb FIG. 4.1. etrA gene deletion confirmation by PCR. This is a 1% agarose gel 1X TAE which has the PCR reactions to confirm the MR-l AetrA mutants. Lanes labeled as M are for 1 Kb plus DNA ladder (Invitrogen Life Technologies). Lanes 1-8 correspond to PCR screening with the etrAcomp primers. Lanes 9-16 correspond to PCR screening with the etrAScreenout primers. Sample order: lanes 1 and 9, EtrA7-l; lanes 2 and 10, EtrA14-1; lanes 3 and 11, EtrA15-l; lanes 4 and 12, EtrA15-2; lanes 5 and 13, EtrA55-3; lanes 6 and 14, EtrA55-7; lanes 7 and 15, negative controls (no DNA); lanes 8 and 16, MR-l wild type. 102 1 caatcgcatg gttaacaatg ctttcaaacg gacgattatg ccaaatgact tgagtatcaa 61 tgttgagtcc taggtgattg tagggggcga gtagatcttg gatccacgcc acacgctgat 121 caatcacacc ttggcgcatc gcttcgcgct cttggctcga taaaattgaa gtcatttcat 181 aggagaagtc aaatattgag agaaacacag tcacatgggc gttactttta ctggccaagg 241 taacagctcg ggcgagagca acctgatttt ctgtcgtggg atcgacaact accagtattt 301 tttgataatc cttcatagca tgttccttta gtcgtaggct catgtttatc atgagccttt 361 ggcaattagc tgtattgttc tagatcaaaa ctcttttcaa aaccgatgcc tagcgtaaag 421 cataaacgct aaaaggtgtg afltctgg catatggcgg ccgcataact tcgtatagca 481 tacattatac gaagttatgg atccagctta tcgataccgc ggtc_atgtcg gttctcaagt 541 taatccactg cagccatgtt aaaccaattc attcgcttgg gctagtttag ctgcgacagg 601 gcgatatata aagtttggcc accaaagacg attaatagta agcccactca ataatctaac 661 ggttttttgt tgtacccaat tggctaaacg ctttgctgcc acacgccagc actaagtagc 721 gccgggagag tccctaaacc aaaggcgagc ataatcaagg cgccttggct ggcagaacct 781 gccgccacag accaagttaa ggtgctatat accagtccac agggcagcca tccccatatc 841 aatccagcgg tgatggcttg cattggcgtg gtgatcggca caagacgctg ggctatgggt 901 tttaaataac gccacaacac ttggccgagg cgttcaattt gtactattcc gacccaaatt 961 ttagcaatgt ataaacctgt cgcgatcatc atgat FIG. 4.2. DNA sequence of the etrA deletion in MR-l. This sequence was assembled using vector NTI Suite 8.0 software. The assembly included the sequence upstream the etrA gene and the one downstream using primers etrAScreenout Fwd and etrAScreenout Rev, respectively. The sequences underlined represent the stop (left side) and the start codon sequences of the etrA gene . The sequence in boldface corresponds to the loxP residual. 103 Growth comparisons of Shewanella oneidensis MR-l and EtrA7-1. A grth curve comparing the wild type with EtrA7-l (the MR-l AetrA mutant chosen at random) was conducted (FIG. 4.3). The growth curves of EtrA7-1 complemented with the etrA gene (this complement harbors pCCG02c) and the EtrA7-1 harboring the pCM62 were also included. The growth of EtrA7-1 is approximately 20% of that of the wild type. The EtrA7-l complement grew slower than the wild type but after 23 h of incubation it reached an optical density similar to that of the wild type. The EtrA7-1 harboring the pCM62 was used to account for any differences observed due to the presence of the vector and not caused by the complementation. There are no differences between the growth of the mutant and this control indicating that the presence of pCM62 in the mutant does not affect its growth. After 10 h incubation, all samples show reduction of nitrate (FIG. 4.4). The MR-l AetrA complement samples present less accumulation of nitrite when compared to the wild type, which explains the delay in growth observed. This is reasonable since the gene has not been transcribed using its natural promoter, which can cause a delay in expression and differences in protein concentration. However, samples taken after 23 h incubation showed concentrations in the complement similar to those in the wild type (FIG. 4.5). EtrA7-1 and the control harboring pCM62 grew similarly. In both cultures, there is reduction of most of the nitrate and nitrite accumulation. Ammonium production is limited. In these cases, nitrate reduction hardly improved grth (F 16.4. 3). 104 Incubation Time (h) FIG. 4.3. Growth of Shewanella oneidensis MR-l wild type (0), EtrA7-1 (I), EtrA7-l complement ( ),and EtrA7-I harboring pCM62 (1') under anaerobic conditions with 3 mM KN03. Each time point is an average of three biological replicates. 3 g 2.5 v 2 g I N03- 3 1.5 I N02- C 1 D NH4+ o 0 S 5. o 0. 0 - . MR-1 EtrA7-1 EtrA7-1 EtrA7-1 comp comp w/vector FIG. 4.4. Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensis MR-l wild type , EtrA 7-1, Etra7-l complement (harboring pCCGOZc) and EtrA7-1 harboring pCM62 during growth curve after 10 h incubation period. Each measurement is an average of three biological replicates. 105 3.5 2.5 I N03. I NO2' r3 NH,+ 1.5 Concentration (mM) N 0.5 MR-1 EtrA7-1 EtrA7-1 comp EtrA7-1 w/ pCM62 FIG. 4.5. Concentrations of nitrate, nitrite and ammonium in cultures of Shewanella oneidensis MR-l wild type, EtrA 7-1, Etra7-1 complement (harboring pCCGOZc) and EtrA7-1 harboring pCM62 during grth curve after 23 h incubation period. Each measurement is an average of three biological replicates. 106 Gene expression profile of wild type versus EtrA7-l. The global expression pattern of EtrA7-1 was compared to that of the wild type when grown anaerobically with 2 mM KNO3. Out of 627 differentially expressed genes in EtrA7-1 relative to the wild type, there are 302 up-regulated and 325 down-regulated genes. The differentially expressed genes were classified in 20 functional “TIGR Role” categories (FIG. 4.6). “Conserved hypothetical proteins” was the predominant category in both up-regulated (17.8%) and down-regulated genes (15.9%). Other categories under the up-regulated genes include “hypothetical” (14.2%), “protein synthesis” (9.9%), “energy metabolism” (8.6%) and “unknown function” (7.6%). Many of the up-regulated genes that are grouped in the “energy metabolism” category include genes that encode a formate dehydrogenase ($04509-4511), a cytochrome c oxidase ccoPQN (802361-2362, $02364), NADH:ubiquinone oxidoreductases nqrA-2, nqu-2, nqu-2, nqu-2, nqrE-2, nqu-2 (801103-1108), genes of proteins involved in gluconeogenesis like pckA ($00162) and in glycogen synthesis like gIgX, glgC and glgA ($01495, $Ol498-1499). In this group there was also activation of the succinate dehydrogenase gene sdhC ($01927), the succinyl-CoA synthase operon sucABCD ($Ol930-1933) and the acetate CoA- transferase, synthase (801891-1892) (TABLE 4.2). A complete list of all the genes induced two-fold or higher is provided (SUPPLEMENTAL TABLE B3). The category “transport and binding proteins” contained 6.3% of the up-regulated genes. In this category there are genes encoding heavy metal efflux pumps and systems ($00520, $O4597-4598, $OA0153), and ABC transporters ($01690), especially those specific for phosphate transport ($01560, 801723-1724), pstB-I ($01725), pstB-Z 107 100% , 80% E: 2 i 2 S g 60% g. 3 E 40% i '6 g 20% Down-regulated genes Up-regulated genes 0 Unknown function :1 Transport and binding proteins 121 Transcription I Signal Transduction I Regulatory Functions I Purines, pyrimidines. nucleosides. and nucleotides I Protein synthesis I Protein fate I Other categories In Hypothetical I Fatty Acid and Phospholipid Metabolism I Energy metabolism a DNA metabolism I Disrupted Reading Frame I Consened Hypothetical Proteins I Central Intermediary Metmolism 13 Cellular Processes I: Call Emelope I Biosynthesis of Ceiectors, Prosthetic Groups and Carriers I Amino Acid Biosynthesis FIG. 4.6. Distribution of differentially expressed genes (> 2-fold change) grouped in 20 functional categories in anaerobic cultures of EtrA7-1 with 2 mM KNO3 as the sole electron acceptor relative to the wild type (reference strain). The total of genes down- regulated is 325 and the up-regulated is 302. 108 (804289) and pstA (804290). There was up-regulation of genes encoding the pho regulon, which regulates these phosphate transporters genes, such as phoB (801558), phoR ($01559), which belong to the “signal transduction” category and phoU ($01726) that belongs to the “regulatory functions” category. Also, the genes encoding proteins involved in long-chain fatty acid transport (803099) and HlyD family secretion proteins (801925 and 804319) that also belong to the “transport and binding proteins” category were activated. The induction of genes from various categories in response to stress conditions was observed. Some of these categories include “cellular processes”, “cell envelope”, “protein fate”, “other categories”, “regulatory functions”, “transcription” and “DNA metabolism”. The “cellular processes” category include a stringent starvation protein encoded by the sspAB genes (800611-0612), a cold shock protein (801648), a phage shock protein operon pspABC (801807-1809), and a virulence regulator encoded by bipA (804408). There was up-regulation of the RTX toxin operon (804317-4320), which codes for the toxin, the toxin secretion ATP-binding protein, the HlyD family secretion protein (mentioned above), an agglutination protein (aggA) and an OmpA family protein. Up-regulated genes in the “cell envelope” category are mostly involved in the synthesis of structural proteins (800004, 800300, dacA-l or 801164, 801166, rodA or 801167, $01245, 803933, 804321, and 804377). There was also induction of genes encoding transferases (803172 and 803176) as well as the mrdA gene (801168) that codes for a penicillin-binding protein 2. In the “fatty acid and phospholipid metabolism” induction of genes fabF-l (802774), fabD (802777), fabH-I ($02778), fabB ($03072), fabG-2 (804382), and fabF -2 (804383) involved in the synthesis of membrane components was 109 detected. In the “protein fate” category there was up-regulation of genes lepB (801347), dsdB (801887) and of the hle gene that code for a chaperone (800163) and the export protein genes secD-l and secF-1(801193-1194). Other up-regulated genes involved in stress response include rpoD (801284), era (801349), recO (801350), cinA (800272) and a gene that encodes a site-specific recombinase (80A0086). Another stress response observed was the induction of genes involved in the activation of the MR-l prophages. There was induction of 25 genes of the LambdaSo phage (802940-2974) and 2 of the late genes of the MuSol (800674-0675) and Mu802 (802684-2685) phages. There is induction of host genes that are also required for activation of the LambdaSo phage such as nusA (800219) and nusG (800219). The down-regulated genes show a different pattern (FIG. 6). “Energy metabolism” associated proteins are the second largest category (16.9%) of down- regulated genes. In this category there are down-regulated genes involved in anaerobic metabolism such as the napBHGAD operon (800845-0849), the cymA (804591) and the nrfA (803980) genes, the fumarate reductase genes fidAB (800398-0399), the nqu-l, C-l, D-l, E-l and F -1 operon (800903-0907), the nuoECDB operon (801018-1020), the cydAB genes (803285-3286), the outer membrane protein genes mtrAB (801776-1777) and ochB (801778-1779), the prismane protein hcp gene (801363), and the alcohol dehydrogenease genes ath (801490), and adhE (802136)(TABLE 4.3). The genes that encode the anaerobic dimethyl sulfoxide reductase dmaA-l (801429) and dmsB-l (801430) as well as genes of a quinine-reactive Ni/Fe hydrogenase the hydC, hyaB, and hoxK (802097-2099) are down-regulated. Other down-regulated genes in this category (“energy metabolism”) include some electron transfer flavoproteins (eth or 803144, 110 804453), a formate dehydrogenase (804513 and 804515), as well as genes involved in metabolism of carbon containing compounds such as the pflAB (802912-2913), ackA (802915), pta (802916) and the ppc (800274) genes. Another category with a high percentage of down-regulated genes is the “transport and binding proteins” which contain genes that code for ABC transporters (cydCD or 803285-3286), nosF or 800487, 800821, 804446-4448), TonB-dependent receptors (nosA or 800630), and two L-lactate permeases (lldP, 801522). Genes that encode secretion proteins such as the HlyD family secretion protein (800820, 803483), as well as efflux proteins (800822, 802045, 804475) and transporters such as the formate transporter (802911), an ammonium transporter (803820), and an outer membrane porin (803896) were also down-regulated. In the “regulatory functions” category there is down-regulation of etrA (as expected since it was deleted), and repression of the LambdaSo phage transcriptional regulator of the Cro/Cl family (802990) was also apparent (ratio of 0.43). A complete list of all the genes repressed two-fold or higher is provided (SUPPLEMENTAL TABLE B.4). 111 TABLE 4.2. Genes induced in anaerobic cultures of EtrA7-1 relative to the wild type (reference strain). Gene Relative Gene ID name expression' COG Annotation 1) Energy metabolism 800162 pckA 2.21 (i 0.48)b phosphoenolpyruvate carboxykinase (ATP) 800747 fpr 2.17 (i 1.01) ferredoxinuNADP reductase NADH:ubiquinone oxidoreductase, Na translocating, alpha 801103 nqrA-2 2.25 (:t 0.54) subunit NADH:ubiquinone oxidoreductase, Na translocating, 801104 nqu-2 2.70 (i 1.01) hydrophobic membrane protein quB NADH:ubiquinone oxidoreductase, Na translocating, gamma 801105 nqu-2 3.15 (i .080) subunit NADH:ubiquinone oxidoreductase, Na translocating, 801106 nqu-2 4.65 (:t 2.07) hydrophobic membrane protein quD NADH:ubiquinone oxidoreductase, Na translocating, 801107 nqrE-2 3.63 (:i: 1.61) hydrophobic membrane protein quE NADH:ubiquinone oxidoreductase, Na translocating, beta 801108 nqu-2 4.21 (:t 2.05) subunit 801495 gng 2.03 (i 0.50) glycogen operon protein 801498 glgC 6.86 (:t 4.90) glucose-1 -phosphate adenylyltransferase 801499 glgA 5.42 (:i: 5.26) glycogen synthase 801891 3.77 (i 1.80) 3-oxoadipate CoA-succinyl transferase, beta subunit 801892 atoD 3.21 (d: 2.14) acetate CoA-transferase, subunit A 801927 sdhC 2.47 (i 1.26) succinate dehydrogenase, cytochrome b556 subunit 801930 sucA 3.02 (i 1.22) 2-oxoglutarate dehydrogenase, El component 2-oxoglutarate dehydrogenase, E2 component, 801931 sucB 3.60 (d: 1.58) dihydrolipoamide succinyltransferase 801932 sucC 3.29 (i 0.98) succinyl-CoA synthase, beta subunit 801933 sucD 3.28 (at 1.24) succinyl-CoA synthase, alpha subunit 802361 ccoP 2.30 (i 0.92) cytochrome c oxidase, cbb3-type, subunit [[1 802362 ccoQ 3.44 (i: 1.16) cytochrome c oxidase, cbb3-type, CcoQ subunit 802364 ccoN 2.76 (i 1.07) cytochrome c oxidase, cbb3-type, subunit 1 804509 2.33 (:l: 0.56) formate dehydrogenase, alpha subunit 804510 fth-l 4.03 (i 1.57) formate dehydrogenase, iron-sulfur subunit 804511 2.53 (:1: 0.31) formate dehydrogenase, C subunit, putative 2) Transport and binding proteins ‘ 801723 7.86 (:i: 3.75) phosphate ABC transporter, perrnease protein, putative 804319 6.21 (i 1.58) 11in family secretion protein 801724 4.75 (d: 2.78) phosphate ABC transporter, perrnease protein, putative 801560 4.51 (:i: 3.45) phosphate-binding protein 801725 pstB-l 3.56 (:i: 2.74) phosphate ABC transporter, ATP-binding protein 802750 tolR 2.95 (i 1.06) tolr protein 804598 2.82 (:h 1.85) heavy metal efflux pump, Cch family 801925 2.68 (d: 1.43) HlyD family secretion protein 804597 2.47 (i 1.55) heavy metal efflux system protein, putative 80A0153 2.20 (i 0.65) heavy metal efflux pump, Cch family 800520 2.08 (i 0.34) heavy metal efflux pump, Cch family 804289 pstB-2 2.08 (i 1.02) phosphate ABC transporter, ATP-binding protein 804290 pstA 2.04 (d: 1.06) phosphate ABC transporter, perrnease protein (Continued) 112 TABLE 4.2. (Cont’d) Genes induced in anaerobic cultures with nitrate of EtrA7-1 relative to the wild type (reference strain). Gene Relative Gene 1]) name expression‘ COG Annotation 3) Cellular processes 800611 sspA 2.60 (d: 0.58) stringent starvation protein a 800612 sspB 2.09 (i 0.32) stringent starvation protein b 801648 2.46 (i 1.75) cold shock domain family protein 801807 pspA 3.11 (:i: 1.64) phage shock protein A 801808 pspB 3.49 (t 0.42) phage shock protein B 801809 pspC 3.45 (:1: 1.84) phage shock protein C 802355 2.30 (d: 0.31) universal stress protein family 803582 2.95 (i 0.52) methyl-accepting chemotaxis protein 804317 2.55 (i 0.63) RTX toxin, putative 804320 aggA 7.18 (:t 2.36) agglutination protein 804408 bipA 2.45 (:t 0.3 8) virulence regulator BipA 4) Cell envelope 804321 7.10 (i 1.77) OmpA family protein 801245 5.26 (:1: 0.92) membrane protein, putative 801164 dacA-l 2.67 (i 0.69) D-alanyl-D-alanine carboxypeptidase 801166 2.03 (:i: 0.92) membrane-bound lytic transglycosylase, putative 801167 rodA 2.93 (i 0.69) rod shape-determining protein RodA 801168 mrdA 2.94 (:1: 1.02) penicillin-binding protein 2 5) Fatty acid and phodpholipid metabolism 802774 fabF-l 3.07 (d: 0.27) 3-oxoacy1-(acyl-carrier-protein) synthase 11 802777 fabD 3.35 (i: 0.61) malonyl CoA-acyl carrier protein transacylase 802778 fabH-l 2.14 (i 0.29) 3-oxoacyl-(acy1-carrier-protein) synthase 111 803072 fabB 2.44 (i 0.88) 3-oxoacyl-(acyl-carrier-protein) synthase 1 804382 fabG-Z 2.13 (i 0.51) 3-oxoacy1-(acyl-carrier-protein) reductase 804383 fabF-2 2.09 (i: 0.99) 3-oxoacyl-(acyl-carrier-protein) synthase II 6) Protein Fate 801193 secD-l 6.40 (d: 2.28) protein-export membrane protein SecD 801194 secF-l 6.25 (:i: 0.91) protein-export membrane protein SecF 802964 6.17 (at 0.85) ClpP protease family proteinc 802887 dsbB 6.14 (:i: 3.76) disulfide bond formation protein b 800218 secE 3.31 (:t 1.04) preprotein translocase, SecE subunit 800163 hle 2.82 (:1: 0.45) chaperonin Hle 7) Biosynthesis of co-factors, prosthetic groups and carriers 801109 apr 6.44 (d: 2.17) thiamin biosynthesis lipoprotein Apr 8) Signal transduction 801558 phoB 5.22 (i 2.59) phosphate regulon response regulator PhoB 801559 phoR 3.68 (:1: 1.86) phosphate regulon sensor protein PhoR 9) Regulatory functions 801726 phoU 2.59 (i 1.77) phosphate transport system regulatory protein PhoU 801349 Era 2.70 (i: 0.51) GTP-binding protein Era 804312 2.11 (a: 0.69) adenylate cyclase CyaA, putative (Continued) 113 TABLE 4.2. (Cont’d) Genes induced in anaerobic cultures with nitrate of EtrA7-l relative to the wild type (reference strain). Gene Relative Gene ID name mression' COG Annotation 10) Transcription 800219 nusG 801203 nusA 801284 rpoD 801348 me 11) DNA metabolism 801350 recO 5.00 (:i: 1.76) DNA repair protein RecO SOA0086 3.00 (d: 0.94) site-specific recombinase, resolvase family 800272 cinA 2.11 (i 0.77) competence/damage-inducible protein CinA 12) Conserved hypothetical and hypothetical proteins 2.09 (d: 0.64) transcription antiterrnination protein NusG 2.50 (:1: 0.36) N utilization substance protein A 2.70 (i 1.22) RNA polymerase sigma-70 factor 3.02 (:t 0.88) ribonuclease 111 804322 7.24 (:1: 2.46) conserved hypothetical protein 800005 7.00 (i 3.12) conserved hypothetical protein TIGR00278 802967 6.01 (:k 0.69) conserved hypothetical protein 802972 5.31 (d: 1.67) hypothetical protein 13) Other cathegories‘ 800674 1.89 (i 0.92) prophage MuSol , protein Gp32, putative 800675 2.06 (d: 0.82) prophage MuSol , major head subunit, putative 802684 2.54 (i 0.30) prophage Mu802, protein Gp32, putative 802685 2.23 (:t 0.63) prophage Mu802, major head subunit, putative 802940 3.58 (:i: 1.10) prophage LambdaSo, host specificity protein J, putative 802941 2.70 (:1: 0.72) prophage LambdaSo, tail assembly protein I 802948 2.53 (t 0.44) prophage LambdaSo, tail assembly protein K, putative 802953 3.61 (i 1.02) prophage LambdaSo, tail length tape meausure protein 802956 2.67 (i 1.47) prophage LambdaSo, major tail protein V, putative 802963 4.57 (d: 0.65) prophage LambdaSo, major capsid protein, HK97 family 802965 7.02 (i 0.93) prophage LambdaSo, portal protein, HK97 family 802969 5.60 (:l: 1.27) prophage LambdaSo, holin, putative 802973 4.43 (:t 0.93) prophage LambdaSo, lysozyme, putative ‘ The relative expression is presented as the ratio of the dye intensity of the anaerobic cultures with 2 mM KNO; of EtrA7-1 to that of MR-l (reference). ”The standard deviation was calculated from six data points, which included three independent biological samples and two technical samples for each biological sample. cThese genes are of prophage origin. 114 TABLE 4.3. Genes repressed in anaerobic cultures with nitrate of EtrA7-1 relative to the wild type (reference strain). Gene Gene Relative [D name expression‘ COG Annotation 1) Energy Metabolism 800398 frdA 0.30 (21:0.16)b fumarate reductase flavoprotein subunit 800399 frdB 0.39 (i 0.06) fumarate reductase iron-sulfur protein 800274 ppc 0.48 (i 0.19) phosphoenolpyruvate carboxylase 800845 napB 0.15 (a: 0.04) cytochrome c-type protein NapB 800846 napH 0.18 (i 0.11) iron-sulfur cluster-binding protein napH 800847 napG 0.14 (i 0.07) iron-sulfur cluster-binding protein NapG 800848 napA 0.18 (i 0.13) periplasmic nitrate reductase 800849 napD 0.30 (:t 0.04) napD protein NADH:ubiquinone oxidoreductase, Na translocating, hydrophobic 800903 nqu-l 0.34 (i 0.15) membrane protein quB NADH:ubiquinone oxidoreductase, Na translocating, gamma 800904 nqu-l 0.28 (:t 0.09) subunit NADH:ubiquinone oxidoreductase, Na translocating, hydrophobic 800905 nqu-l 0.27 (:1: 0.14) membrane protein quD NADH:ubiquinone oxidoreductase, Na translocating, hydrophobic 800906 nqrE-l 0.23 (i: 0.07) membrane protein quE 800907 nqu—l 0.23 (i 0.08) NADH:ubiquinone oxidoreductase, Na translocating, beta subunit 800970 0.31 (i 0.17) fumarate reductase flavoprotein subunit precursor 801018 nuoE 0.44 (i 0.17) NADH dehydrogenase I, E subunit 801019 nuoCD 0.35 (i 0.13) NADH dehydrogenase I, C/D subunits 801020 nuoB 0.40 (i 0.10) NADH dehydrogenase I, B subunit 801363 hcp 0.13 (i 0.08) prismane protein 801364 0.12 (:1: 0.07) iron-sulfur cluster-binding protein 801429 dmaA-l 0.43 (t 0.09) anaerobic dimethyl sulfoxide reductase, A subunit 801430 dmsB-l 0.29 (a: 0.04) anaerobic dimethyl sulfoxide reductase, B subunit 801490 ath 0.28 (i 0.12) alcohol dehydrogenase 11 801776 mtrB 0.22 (:i: 0.04) outer membrane protein precursor MtrB 801777 mm 0.25 (:1: 0.06) decaheme cytochrome c MtrA 801778 och 0.30 (i 0.09) decaheme cytochrome c 801779 och 0.30 (:1: 0.05) decaheme cytochrome c 802097 hydC 0.07 (d: 0.04) quinone—reactive Ni/Fe hydrogenase, cytochrome b subunit 802098 hyaB 0.11 (:t 0.10) quinone-reactive Ni/Fe hydrogenase, large subunit 802099 hoxK 0.07 (i 0.11) quinone-reactive Ni/F e hydrogenase, small subunit precursor 802136 adhE 0.40 (d: 0.10) aldehyde-alcohol dehydrogenase 802727 0.32 (a; 0.23) cytochrome c3 802912 pflB 0.18 (i 0.11) formate acetyltransferase 802913 prA 0.20 (i: 0.13) pyruvate fonnate-lyase 1 activating enzyme 802915 ackA 0.23 ($0.16) acetate kinase 802916 pta 0.23 (i 0.14) phosphate acetyltransferase 803117 0.41 (d: 0.11) thioredoxin, putative 803144 eth 0.36 (i 0.13) electron transfer flavoprotein, alpha subunit 803285 cydB 0.21 (d: 0.06) cytochrome d ubiquinol oxidase, subunit 11 803286 cydA 0.22 (i 0.10) cytochrome d ubiquinol oxidase, subunit 1 803980 nrfA 0.18 (:1: 0.06) cytochrome c552 nitrite reductase 804453 0.40 (:t 0.13) electron transfer flavoprotein-ubiquinone oxidoreductase, putative (Continued) 115 TABLE 4.3. (Cont’d) Genes repressed in anaerobic cultures with nitrate EtrA7-l relative to the wild type (reference strain). Gene Relative Gene ID name expression‘| COG Annotation 804513 0.06 (:t 0.02) formate dehydrogenase, alpha subunit 804515 0.07 (i 0.01) formate dehydrogenase, C subunit, putative 804591 cymA 0.39 (:t 0.27) tetraheme cytochrome c 2) Transport and binding proteins 800487 nosF 0.28 (i 0.05) copper ABC transporter, ATP-binding protein 800630 nosA 0.30 (i 0.06) TonB-dependent receptor 800820 0.20 (i 0.05) HlyD family secretion protein 800821 0.14 (i 0.05) ABC transporter, ATP-binding/permease protein 800822 0.10 (i 0.04) outer membrane efflux family protein 800827 lldP 0.31 (i 0.07) L-lactate perrnease 801522 0.47 (d: 0.07) L-lactate perrnease, putative 802045 0.45 (i 0.08) cation efflux family protein 802911 0.40 (d: 0.20) formate transporter, putative 803483 0.22 (i 0.08) HIyD family secretion protein 803779 cydC 0.44 (i 0.12) ABC transporter, ATP-binding protein CydC 803780 cydD 0.32 (:t 0.08) ABC transporter, ATP-binding protein CydD 803820 0.16 (:i: 0.26) ammonium transporter, degenerate 803896 0.26 (d: 0.17) outer membrane porin, putative 804446 0.24 (d: 0.12) molybdenum ABC transporter, ATP-binding protein 804447 0.32 (:i: 0.20) molybdenum ABC transporter, perrnease protein molybdenum ABC transporter, periplasmic molybdenum-binding 804448 0.35 (:1: 0.17) protein 804475 0.30 (a: 0.10) cation efflux family protein ORF3506 0.19 (i 0.06) ammonium transporter (tpt) 3) Regulatory functions 802356 etrA 0.05 (i 0.01) electron transport regulator A 802990 0.43 (:t 0.16) prophage LambdaSo, transcriptional regulator, Cro/CI family 804603 lexA 0.47 (i 0.12) LexA repressor 4) Cellular processes 804226 ftsL 0.48 (i 0.06) cell division protein FtsL 804299 cat 0.50 (a; 0.06) chloramphenicol acetyltransferase 804405 katG-2 0.31 (a: 0.09) catalase/peroxidase HPI 5) Signal transduction 804477 cpxR 0.32 (at 0.10) transcriptional regulatory protein CpxR 804478 cpr 0.34 (i 0.15) sensor protein Cpr 804633 ompR 0.38 (i 0.19) transcriptional regulatory protein OmpR 804634 envZ 0.33 (:k 0.20) osmolarity sensor protein EnvZ ' The relative expression is presented as the ratio of the dye intensity of the anaerobic cultures with 2 mM KNO3 of EtrA7-1 to that of MR-l (reference). t’I‘he standard deviation was calculated from six data points, which included three independent biological samples and two technical samples for each biological sample. 116 dr Comparison of the gene expression profile of anaerobic cultures of EtrA7-1 grown at 1 mM KNO; and at 40 mM KNO3. A total of 358 genes were differentially expressed when anaerobic growth of EtrA7-1 at a low nitrate concentration was compared to that at a high concentration. This total was divided in two groups, 154 genes up-regulated (TABLE 4.4) and 204 genes down-regulated (TABLE 4.5). A complete list of all the genes induced and repressed two-fold or higher is provided (SUPPLEMENTAL TABLES B5 and B6). Among the up-regulated genes there are genes involved in energy metabolism, specifically in the regeneration of acetyl CoA, the pyruvate dehydrogenase multi-enzyme complex E1, E2 and E3 (800424-0426). In addition, the formate acetyltransferase operon (802912-2916) is up-regulated, which is involved in the conversion of acetyl CoA and formate into CoA and pyruvate. There is also induction of genes involved in carbohydrate metabolism such as ppc (800274), tkt (800930), maIQ (801493), glgB (801494), glgA (801499), eda (802486), edd (802487), pg! (802488), zwf (802489), and glmS (804741) genes. Also genes involved in anaerobic metabolism are up-regulated such as the nqu-I, C-I, D-I, F -1 operon (800903-0907), and the genes encoding cytochromes scyA (800264), and cydB (803285). There is also activation of genes involved in amino acid scavenging and biosynthesis such as glnB-I, gInA, ntrB, and ntrC. Induction of the amt (800760) and (pi gene (ORF03506), which encode two ammonium transporters, was detected. The down-regulated genes include ABC transporters (800821, 801042-1044, and 801959), genes involved in oxidative stress response katG-l (800725), katB (801070), dnaK (801126), and dmaA-l (801429) and the phage shock protein genes pspABC (801807-1809). Also, the ilvADMGC operon (804344-4349) is highly down-regulated. 117 TABLE 4.4. Genes induced in anaerobic cultures of EtrA7-1 at 1 mM (reference) versus 40 mM KNO3. Gene Relative Gene ID name expression' COG Annotation 1) Energy metabolism 800264 scyA 2.35 (i 0.74)b cytochrome c 800274 ppc 4.17 (i: 1.61) phosphoenolpyruvate carboxylase pyruvate dehydrogenase complex E1, pyruvate 800424 aceE 2.72 (d: 0.88) dehydrogenase pyruvate dehydrogenase complex E2, dihydrolipoamide 800425 aceF 2.68 (:1: 0.44) acetyltransferase pyruvate dehydrogenase complex E3, lipoamide 800426 lpdA 1.99 (i 0.34) dehydrogenase NADH:ubiquinone oxidoreductase, Na translocating, 800903 nqu-l 2.05 (i 0.31) hydrophobic membrane protein quB NADH:ubiquinone oxidoreductase, Na translocating, gamma 800904 nqu-l 2.39 (i 1.05) subunit NADH:ubiquinone oxidoreductase, Na translocating, 800906 nqrE-l 2.61 (:1: 1.29) hydrophobic membrane protein quE NADH:ubiquinone oxidoreductase, Na translocating, beta 800907 nqu-l 2.62 (d: 1.06) subunit 801493 malQ 6.56 (:i: 8.84) 4-alpha-glucanotransferase 801494 glgB 6.28 (d: 8.81) 1,4-alpha-glucan branching enzyme 801499 glgA 6.54 (i 9.60) glycogen synthase 2-deydro-3deoxyphosphogluconate aldolase/4-hydroxy-2- 802486 eda 3.45 (at 0.96) oxoglutarate aldolase 802487 edd 4.01 (:1: 0.77) 6-phosphogluconate dehydratase 802488 pgl 3.30 (d: 1.00) 6-phosphog1uconolactonase 802489 zwf 2.60 (:h 0.44) glucose-6-phosphate l-dehydrogenase 802912 pflB 2.59 (i 0.80) formate acetyltransferase 802913 pflA 3.14 (:1: 1.01) pyruvate formate-lyase 1 activating enzyme 802915 ackA 3.28 (:i: 1.35) acetate kinase 802916 pta 2.36 (i 1.12) phosphate acetyltransferase 803285 cydB 2.68 (i 1.41) cytochrome d ubiquinol oxidase, subunit 11 804509 2.69 (:1: 1.19) formate dehydrogenase, alpha subunit 804511 2.45 (d: 1.49) formate dehydrogenase, C subunit, putative 804741 glmS 3.44 (i 0.87) glucosamine--fi'uctose-6-phosphate aminotransferase 2) Amino acid biosynthesis 804410 glnA 6.18 (:t 3.91) glutamine synthetase, type 1 801121 proB 2.26 (:t 0.57) glutamate 5-kinase 801122 proA 2.50 (i 0.42) gamma-glutamyl phosphate reductase 3) Regulatory functions 800761 glnB-l 3.37 (i 3.05) nitrogen regulatory protein P-11 1 4) Signal transduction 804471 ntrB 2.89 (i 1.40) nitrogen regulation protein 804472 ntrC 2.76 (i 1.37) nitrogen regulation protein NR(I) 5) Transport and binding proteins ORF03506 3.38 (d: 1.44) 800760 amt 7.79 (a: 6.39) ammonium transporter (tpt) ammonium transporter ‘ The relative expression is presented as the ratio of the dye intensity of the anaerobic cultures of EtrA7-1 own at 40 mM KNO; to that of the anaerobically grown at 1 mM KNO; (reference). he standard deviation was calculated from six data points, which included three independent biological samples and two technical samples for each biological sample. 118 TABLE 4.5. Genes repressed in anaerobic cultures of EtrA7-1 at 1 mM (reference) versus 40 mM KN03. Gene Gene Relative ID name expression‘ COG Annotation 1) Energy Metabolism 801427 0.35(d: 0.12)b decaheme cytochrome c 801429 dmaA-l 0.47 (:1: 0.24) anaerobic dimethyl sulfoxide reductase, A subunit 804513 0.26 (d: 0.34) formate dehydrogenase, alpha subunit 804515 0.29 (:h 0.26) formate dehydrogenase, C subunit, putative 2) Cellular processes 800725 katG-l 0.24 (i 0.10) catalase/peroxidase HPI 801070 katB 0.22 (:1: 0.21) catalase 801807 pspA 0.18 (i 0.07) phage shock protein A 801808 pspB 0.12 (at 0.05) phage shock protein B 801809 pspC 0.14 (d: 0.05) phage shock protein C 3) Cell envelope 800150 0.28 (i 0.11) lipoprotein, putative 802194 0.02 (:t 0.00) OmpA family protein 804334 0.03 (i 0.01) inner membrane protein, putative 4) Transport and binding proteins 800519 0.34 (i 0.10) cation efflux protein, putative 800822 0.52 (i 0.22) outer membrane efflux family protein 801042 0.42 (i 0.09) amino acid ABC transporter, ATP-binding protein 801043 0.37 (:t 0.07) amino acid ABC transporter, perrnease protein 801044 0.20 (i 0.07) amino acid ABC transporter, periplasmic amino acid-binding protein 801557 0.30 (:1: 0.09) outer membrane porin, putative 801560 0.10 (i 0.04) phosphate-binding protein 801689 0.32 (:1: 0.21) cation transport ATPase, E1-E2 family 801723 0.12 (i 0.05) phosphate ABC transporter, perrnease protein, putative 801724 0.45 (:1: 0.14) phosphate ABC transporter, perrnease protein, putative 801925 0.17 (i 0.08) HlyD family secretion protein 5) Protein fate 801126 dnaK 0.47 (i 0.26) chaperone protein DnaK 6) Amino acid biosynthesis 804344 ilvA 0.16 (i 0.05) threonine dehydratase 804345 ilvD 0.23 (i: 0.05) dihydroxy-acid dehydratase 804346 ilvM 0.26 (d: 0.09) acetolactate synthase 11, small subunit 804347 ilvG 0.27 (i 0.08) acetolactate synthase 1], large subunit 804349 iva 0.18 (i 0.10) ketol-acid reductoisomerase 7) Signal Transduction 801558 phoB 0.11 (:t 0.04) phosphate regulon response regulator PhoB 801945 pth 0.41 (a; 0.10) sensor protein Pth 801946 phoP 0.34 (:t 0.10) transcriptional regulatory protein PhoP 804477 cpxR 0.33 (:t 0.12) transcriptional regulatory protein CpxR 804478 cpr 0.49 (:1: 0.17) sensor protein Cpr 8) Regulatory proteins 801937 fur 0.38 (:1: 1.14) ferric uptake Legulation protein ‘ The relative expression is presented as the ratio of the dye intensity of the anaerobic cultures of EtrA7-1 rown at 40 mM KNO; to that of the anaerobically grown at 1 mM KNO; (reference). e standard deviation was calculated from six data points, which included three independent biological samples and two technical samples for each biological sample. 119 DISCUSSION This study shows a genetic stress response caused by the deletion of the etrA gene in Shewanella oneidensis MR-l. The MR-l AetrA mutant (EtrA7-l) was confirmed using PCR diagnostic techniques, DNA sequencing and expression analysis. As it has been observed previously for various etrA mutants in MR-l, EtrA7-1 retained its ability to reduce nitrate, however, the anaerobic growth when nitrate was the only electron acceptor was significantly lower than that of the wild type. The inability of EtrA7-1 to grow despite its ability to reduce nitrate could be due to stress factors caused or enhanced by the mutation. The genetic expression pattern of EtrA7-l when compared to that of the wild type afier anaerobic cultivation with nitrate revealed the expressin of various genes that have been previously reported to respond to stressful conditions (i.e. starvation). Among these genes there is up-regulation of the sspAB genes that encode the stringent starvation protein. This protein in E. coli is highly expressed during nutrient starvation conditions and the SspA protein has been found to be required for the transcription of bacterial phage late genes (17). Up-regulation of the pspABC operon was also detected. These genes encode the phage shock protein, which in E. coli is secreted to the periplasm and maintains the proton motive force under stress conditions provoked by filamentous phage infection (23). There was induction of a virulence factor (bipA) found in various E. coli enteric pathogens. This protein is a chaperone and it has been associated with rearrangements in the cytoskeleton of the infected host cells, in regulation of cell motility by flagellae and in the regulation of the expression of capsular genes (29). In addition, there is up-regulation of the genes involved in activation of the S. oneidensis MR-l prophage LambdaSo as well as two 120 genes that encode late genes of MuSol and Mu802, suggesting activation of their lytic cycle. There is also induction of bacterial genes (nusA and nusG) that are required to stabilize the Lambda protein antitermination complex in E. coli (4, 48) as well as a membrane-bound lytic transglycosylase (801166) that has the potential to help the process of lyses. Conversely, there is repression of the LambdaSo transcription regulator Cro/CI family, which represses the transcription of the Lambda genes in E. coli (49). This expression pattern supports the activation of the lytic cycle of these phages. Induction of these prophages by stressful conditions, specifically irradiation exposure, has been previously reported in S. oneidensis MR-l (36). In this study induction of the early genes was observed for cells collected after a short incubation period of 5-20 min, whilst activation of late genes was observed when cells were incubated for a longer time (60 min). I also reported previously induction of some of these prophage genes in response to accumulation of nitrite and of other probable intermediaries of the nitrate reduction pathway, which could cause oxidative stress and DNA damage to the cell (Chapter 3). Moreover, it is known that starvation can activate the lytic cycle of prophages in other bacteria (49). The induction of these genes suggests that the lytic cycle of these phages is compromising the survival of this organism under starvation conditions created by the absence of etrA. The EtrA protein is a global regulator that activates the expression of various genes involved in metabolism when oxygen is not present. In this study the growth conditions were optimal for normal growth, however, despite the availability of sufficient nutrient concentrations in the growth medium the bacterium was not able to use them, generating a starvation response. Another piece of evidence for the “internal 121 starvation condition” is the significant induction observed for the pho genes. The pho regulon (phoBR U), which has been very well studied in E. coli, activates and induces the expression of other genes when the cell experiences phosphate starvation (21, 50, 51). In E. coli, this regulator system induces the expression of genes that encode transport proteins for inorganic phosphate and other phosphorus sources, and of genes involved in phosphorus utilization. In this study there is induction of a variety of genes involved in the transport and metabolism of phosphate indicating phosphate starvation conditions in EtrA7-l. There are also genes induced such as era, which regulates the TCA cycle and responds to starvation (34), and recO that is involved in repair of DNA damage (36). Among the physical starvation responses described in bacteria, there is the reduction of cellular size and biofilm dissolution. Cell size reduction has been observed for some bacteria of the Shewanella genus (2, 6, 16). This particular response has been of interest in the study of S. algae since it has been observed that when bacteria are inoculated in contaminated sites, its transport is limited to the surroundings of the injection wells and very low bacterial numbers travel downstream the plume (6). Thus, if cells are cultivated in starvation conditions, the culture is more resistant to the harsh environmental conditions and the size of the cells is small enough enabling the inocula to penetrate deeper and travel further. The results presented herein show a significant percentage of genes up-regulated that belong to the “cell envelope” and to the “fatty acid and phospholipid metabolism” categories. These categories include genes involved in cell membrane composition, and shape determination (rodA), which can be involved in rearranging the cell membrane composition and in reducing the cellular size, which in 122 turn conserves energy. A rearrangement in the cell membrane composition can also increase the membrane permeability to the substrates needed. Detachment of bacteria has been observed for biofilms exposed to long periods of starvation (47). In the EtrA7-1 cultures examined there is down-regulation of genes associated with biofilm formation such as the cprB genes that in E. coli are described as part of a signal transduction pathway for the adherence process (13). In addition to these proteins, E. coli possesses a second signal transduction pathway, the EnvZ-OmpR two- component system, which operates the same process. In EtrA7-l these genes (envZ and ompR) are down-regulated as well. Repression of these genes in the mutant suggests an impediment to biofilm formation, which represents a starvation response. However, in studies of MR-l induction of biofilm detachment was associated with oxygen limiting conditions (47). To determine the regulators involved in this process, genes encoding possible regulators such as etrA, crp, and arcA were deleted. The MR-l AetrA mutant was reported to be defective in its detachment response (47) but to a lesser extent when compared to the other regulators examined (Crp and ArcA). The authors suggested an EtrA involvement in the regulation of biofilm detachment in MR-l, however a direct linkage of these regulators with detachment of biofilms could not be concluded. In EtrA7-l, high induction levels of a gene annotated as aggA were detected. This gene encodes an agglutination protein, which is involved in the fimbrial biogenesis system of pathogenic gram-negative bacteria (22). The fimbriae mediate the aggregation of cells adhered to the epithelial host cells. In MR-I, this agglutination protein has been associated with biofilm formation since it was the most up-regulated protein in MR-l biofilm forming cells (11). Therefore, our results suggest an involvement of EtrA in the 123 repression of aggA during anaerobic conditions, which might trigger an activation of detachment in MR-l. Conversely, the repression of the cprB genes might be due to starvation conditions and not to regulation by EtrA, however the lack of these transcripts can induced detachment in the mutant. This response might counteract the effects of AggA in adherence, which might explain the variable detachment phenotype observed for the MR-l AetrA mutant by Thorrnann et a1. (47). Accumulation of nitrite is another stressful situation observed in the cultures of EtrA7-1. Nitrite can trigger a stress response since, as previously discussed, high concentrations are toxic to the cell. This type of stress can be responsible for the up- regulation of genes that help alleviate the damage associated with it. Among these genes, there is hle that encodes a chaperone that assist the cells in the folding of proteins and repair, and rpoD, which codes for the sigma factor 70 that regulates many of these genes including those regulated by the pho regulon (24). There are also genes that encode export proteins and efflux systems that can help the cell in detoxification. It is also important to mention that other genes that encode for proteins that protect the cell against oxidative damage are down-regulated such as the uvrC, recC, hemB-I, hemB-Z, hemH-I, katG-Z, parE and lexA (36). The cause of down-regulation in these genes is unclear. Induction of various genes that are involved in carbon metabolism was detected. This suggests a regulatory role of EtrA in their expression. Some of these genes are involved in the TCA cycle and in some aerobic metabolism processes. In E. coli, Fnr not only induces the expression of genes necessary for anaerobic metabolism but also represses genes involved in the TCA cycle and some other genes involved in aerobic metabolism (39). Since EtrA is structurally similar to Pm and since it has been associated 124 with oxygen sensing and regulation of anaerobic metabolism it is possible that EtrA is repressing the expression of some of these genes in MR-I in the absence of oxygen. These results were observed previously in experiments performed in an MR-l etrA mutant (3). In addition, some of these genes and even a higher number of up-regulated genes in this category were observed when the growth of the mutant (EtrA7-1) was compared under two nitrate concentrations (1 mM versus 40 mM KNO3). Various global regulators control the expression of these genes, thus this regulation cannot be expected to be an ON/OFF expression regulation. Therefore, if there is an increase in the induction factor it is reasonable to expect an increase in the genetic expression response. In this case, at higher nitrate concentrations there is an increase in the expression of these genes. Down-regulation in the expression of some genes in EtrA7-1 in response to anaerobic conditions on nitrate might represent an involvement of EtrA in their regulation. Among these, the ones of greater interest in this study were those involved in the nitrate reduction pathway. Those include, the genes in the nap operon (napBHGAD), the cymA gene (napC homolog), and the nrfA gene, which encodes the cytochrome c552 nitrite reductase. Down-regulation of other genes that has been associated with the nitrate reduction pathway includes the hop gene, which encodes the prismane protein associated in other bacteria with the reduction of hydroxylamine (5, 14). Among the down-regulated genes, there are genes that encode proteins necessary for anaerobic processes other than nitrate reduction. These genes include the fumarate reductase (frdAB), the anaerobic dimethyl sulfoxide reductase genes dmaA-l and dmsB-l and the quinone—reactive Ni/Fe hydrogenase genes the hydC, hyaB, and hoxK. These genes have been considered candidates for EtrA regulation (3, 26, 37, 38), and these results are consistent with those 125 observed by Beliaev, et al. (3). Moreover, the latter identified possible recognition sites of EtrA for some of these genes including napDAHGB, nrfA, frdAB, hop, and hydC. The genetic expression differences observed for EtrA7-l when its growth was compared anaerobically at a low versus a high nitrate concentration showed the down- regulation of genes that are oxidative stress inducible as well as down-regulation of the pspABC operon (mentioned previously). In addition, down-regulation in the expression of the pho genes (phoBPQ) was detected Up-regulation was observed for genes involved in the transport and metabolism of carbohydrate such as the TCA cycle as mentioned previously. In addition, there was induction of genes involved in ammonium assimilation (glnB-I, gInA, and ntrBC) as well as two ammonium transporters, amt (800760) and tpt (ORF03506) and the glutamate kinase proAB (801121-1122). These genes are activated in amino acid starvation conditions. This ammonium starvation condition might be created by an imbalance in the utilization of the carbon source versus the low levels of ammonium produced. The reduction of nitrite into ammonium occurs very slowly in the mutant due to the low levels of nitrite reductase. This creates a deficiency of ammonium and subsequently of amino acids, which in turn activate the expression of these genes. The same occurs with the activation of the pho genes. Since the reduction of nitrite into ammonium, which is the one that gives more energy to the cell (compare with the reduction of nitrate into nitrite), is slowed down to a minimum, a deficiency of phosphate (ATP) is generated. Moreover, genes involved in storage of carbohydrates such as the glycogen synthase gene glgAC (801498-1499) and gng (801495) are up-regulated. This was also observed when the genetic expression pattern of the mutant was compared to that of the wild type. These genes are activated in response to high concentrations of 126 glucose indicating an excess of carbon in the cell relative to the concentration of ammonium, which was limited in EtrA7-l. EtrA is a global regulator that might be acting in cooperation with other proteins to control various anaerobic metabolism processes in MR-I (3, 26, 38). Therefore, the expression of these genes cannot be expected to be under an “all or none” regulation mechanism but rather, it is regulated in a gradual fashion that depends on many factors that accumulate to increase or decrease its expression. In this study there is a decrease in the rate of nitrate and nitrite reduction. Nevertheless, the activity is not halted indicating that even when EtrA is not present there are other regulators stimulating the expression of the genes involved in this pathway such as the genes that encode the only nitrate reductase in MR-l, NapA. In E. coli the nap and nrf genes are positively regulated by Pm and NarP. MR-l possesses the genes for a homolog of the two-component regulatory system in E. coli NarQ/NarP (803981-3982). This can explain the decrease in the expression of these genes rather than a complete shut down. A double mutant defective in NarP and EtrA will determine whether these regulators act jointly to control the expression of these genes. A positive regulatory role of EtrA in the expression of the napBGHAD genes, the nrfA and the cymA genes is suggested. In studies where the cymA gene was deleted in 8. oneidensis MR-l, researchers observed that nitrate reduction, among other anaerobic processes, was abolished indicating a requirement for cymA in this process (42, 43). CymA is a homologue of the NapC (one of the components of the NapABC multi- enzyme complex) of E. coli. This protein, CymA, has been suggested to be part of the electron transport complex of the nitrate reduction pathway in MR-l (31, 42, 43). 127 Even though, this study was not designed to investigate starvation in MR-l, the deletion of the etrA gene in this organism stimulated a massive starvation response. However, the results obtained suggest that even when MR-l can activate genes in response to starvation to increase its survival, the activation of the lytic cycle of three prophages may provoke an aggressive infection. 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In vivo effect of NusB and NusG on rRNA transcription antitermination. J. Bacteriol. 186:1304-1310. 49. Voyles, B.A. 1993. The biology of viruses. Mosby-Year Book, Inc., St. Louis, MO. 50. White, A.K., and W.W. Metcalf. 2004. The htx and ptx operons of Pseudomonas stutzeri WM88 are new members of the pho regulon. J. Bacteriol. 186:5876-5882. 51. White, A.K., and W.W. Metcalf. 2002. Isolation and biochemical characterization of hypophosphite/2-oxoglutarate dioxygenase. A novel phosphorus-oxidizing enzyme from Psuedomonas stutzeri WM88. J. Biol. Chem. 277:38262-3 8271 . 52. Wolin, E.A., M.J. Wolin, and R.S. Wolfe. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-2886. 133 CHAPTER V Summary and Future Research 134 SUMMARY The goal of this study was to clarify and examine the nitrate reduction pathway in Shewanella oneidensis MR-l. This was performed by combining classical microbiology approaches with innovative molecular biotechnology. Physiological and stoichiometric analyses indicated that nitrate is reduced to nitrite and nitrite is completely reduced to ammonium. This concludes that DNRA is the nitrate reduction pathway in MR-l and not denitrification. MR-l demonstrated greater cytotoxicity to nitrite as compared to nitrate. Even though nitrate concentrations higher than 2 mM were proven to be toxic, the growth rate of MR-l remained steady beyond 2 mM. Gene expression analyses in MR-l indicated an oxidative stress response to high nitrate concentrations. Also, induction of prophage related genes was observed. Oxidative stress and activation of the prophage lytic cycle are potential causes of growth inhibition at high nitrate concentrations. On the other hand, up-regulation of genes encoding transport and efflux systems as well as enzymes and proteins that metabolize toxic intermediates was observed. These proteins and other stress response chaperones protect the cell against oxidative damage and help in its survival and its internal stability. This in turns might decrease the expression of the prophage related genes giving the bacterium a chance to live in these environmental conditions. In MR-l, tolerance to nitrite is also obtained via a higher rate of consumption of nitrite as compared to the reduction of nitrate. This was observed in physiological experiments and could be inferred from the nrfA expression analyses. The transcription of the nrfA gene reached a plateau at a lower nitrate concentration as compared to napA, which might be a strategy in MR-l to immediately reduce the nitrite in the cell for detoxification purposes. 135 Expression studies for napA and nrfA genes demonstrated that increasing nitrate concentrations do not cause repression in their expression as occurs in E. coli. This implies that there is no alternative mechanism for the reduction of nitrate in MR-l. Further experiments where the napA gene was completely deleted from the genome of MR-l concluded that NapA is the only nitrate reductase and therefore responsible for nitrate reduction pathway in MR-l. In addition, gene expression studies in MR-l where nitrate was supplied as the only electron acceptor and ammonium was not added suggested nitrate assimilation in MR-l. Since nitrate assimilation was beyond the scope of this study, this needs further investigation. Regulation of the nitrate reduction pathway was examined by mutational analyses. Studies with a MR-l AetrA deletion mutant indicated a decrease in biomass and in the rate of nitrate reduction when compared to the wild type. Since reduction of nitrate was not abolished, this suggests a partial but not absolute regulatory role of EtrA in the nitrate reduction pathway of MR-l. This has been observed in E. coli where F nr and the two-component regulatory system NarP/NarQ regulate the expression of napA and nrfA. As stated in Chapter III, MR-l possesses homologues for the narP and narQ genes, which increases the chances in MR-l to have a similar regulatory mechanism controlling this pathway. A starvation stress response was suggested by the gene expression analyses of the etrA mutant. In addition, induction of the prophage related genes was detected. However, in this case, contrary to the induction observed in response to a high nitrate concentration, the up-regulation of the prophage related genes was massive including genes for all three prophages described in MR-l. This implies a more aggressive phage infection which 136 could have caused the low biomass observed in MR-l AetrA deletion mutant. This indicates that starvation stress is more detrimental to MR-l than the oxidative stress caused by high concentrations of nitrate. The cell response is similar to that observed for MR-l after exposure to radiation (6). This is the first time starvation has been examined in MR-l. This study not only shed light to the nitrate reduction pathway of this organism but it also advances the understanding of the internal response to stress conditions and its degree of tolerance. MR-l has the potential to be an excellent bioremediator with a very unique anaerobic metabolism. However, its sensitivity to environmental stress might limit its performance in the field. Understanding its biology and its genetic machinery will help increase its chances as a bioremediator. This work also demonstrates the potential of the microarray technology in the formulation of hypotheses and as a screening method to identified and examine genes of unknown function that can help us explain some of the cellular processes. FUTURE RESEARCH Future studies in the area should focus on the examination of nitrate assimilation in MR-l. Assimilation of nitrate is defined as the reduction of nitrate to ammonium, which is subsequently incorporated into cell material for the synthesis of nucleic acids and proteins (4). This process does not occur in all bacteria although it is widely spread among different species. Since NapA is the only nitrate reductase in Shewanella oneidensis MR-l, nitrate assimilation will be a product of the activity of NapA. The aim 137 of this study was not to evaluate nitrate assimilation, thus this was not examined. However, if this process indeed takes place in MR-I, this will be the first time a role in nitrate assimilation is attributed to NapA. This will denote that MR-l is even more powerful than it is known and other bacteria that posses NapA and similar genetic capabilities might also be able to assimilate nitrate. Nitrate assimilation could be address via nitrogen isotopic fractionation (3). Starvation in Shewanella oneidensis MR-l has not been directly investigated. More studies to better understand its effects in MR-l will help elucidate ways to counteract some of the obstacles it might encountered as bioremediator. Starvation is a critical stress often experienced by bacteria when applied as a bioremediator in contaminated sites. Tolerance and survival mechanisms to this kind of stress increase the potential of a bacterium as a bioremediator. MR-l has been proven to grow and effectively consumed nitrate at high concentrations (this study) as well as other harmful compounds (8), however, its survival is threatened by other type of stresses such as radiation exposure and possibly starvation (6). These types of stresses are well known to cause activation of the lytic cycle of prophages in MR-l and in other bacteria (9), which compromise its survival. This is a significant obstacle in the effective performance of MR-l as a bioremediator. However, bacteria that posses the metabolic machinery but that as MR-l are susceptible to other stresses could be genetically manipulated to improve their capabilities in such harsh conditions. Therefore, studies to examine the regulation of the transcription of the prophage related genes in MR-l are crucial to improve its performance. Mutation analyses to investigate the regulation of genes such as the LambdaSo prophage Cro/CI repressor family should be undertaken. A homologue of this 138 gene in E. coli is known to repress the expression of the prophage genes required to initialize activation of the Lambda prophage lytic cycle in E. coli. Also, point mutation of enzyme recognition sites and deletion of key genes will help in the development of a fi‘ee-phage strain. Unfortunately, little is known about the prophage related genes in MR-l. Therefore, research should focus in studying the biology and more specific, its genome. The majority of these genes are conserved hypothetical or hypothetical proteins. Once the function of these genes is characterized, genetic engineering approaches can be undertaken to repress transcription. The same is true for many of the genes differentially expressed in response to high concentrations of nitrate. A dramatic genetic expression difference was observed when the growth of MR-l was compared on a low versus a high nitrate concentration. Many of these genes belong to the conserved hypothetical and hypothetical proteins category, which have not been characterized (its function is unknown). The elucidation of the role of these genes in the cell will explain the tolerance of MR-l to some of this toxic metabolites and its unique metabolic versatility. This study also identified genes that are directly or indirectly regulated by EtrA. Several studies have attempted to elucidate the role of EtrA in MR-l, however, since it does not act alone, its study is more complicated (I, 5, 7). Therefore, none of these studies prove direct regulation of EtrA on some of these genes. This can be achieved by cloning the promoter region of the candidate genes into a lacZ expression vector (2). This vector can be transformed in MR-l wild type and in the MR-l etrA- mutant. Expression of lacZ can then be compared to determine whether or not EtrA will bind the promoter affecting the transcription of this gene. An increase in the concentration of lacZ 139 transcripts in the strain lacking EtrA as compared to the wild type will indicate a repression role by EtrA for the gene regulated by the operon under examination. Whilst, increasing concentration of lacZ transcripts in the wild type as compared to the etrA mutant will represent an induction role by EtrA. Also, generation of double mutants defective in EtrA and NarP will determined the regulatory mechanism of the expression of the genes associated to the nitrate reduction pathway. 140 REFERENCES . Beliaev, A.S., D.K. Thompson, M.W. Fields, L. Wu, D.P. Lies, K.H. Nealson, and J. Zhou. 2002. Microarray transcription profiling of a Shewanella oneidensis etrA mutant. J. Bacteriol. 184:4612-4616. . Boston, T. and T. Atlung. 2003. FNR-mediated oxygen-responsive regulation to the nrdDG operon of Escherichia coli. J. Bact. 185:5310-5313. . Cadish, G., M. Espana, R. Causey, M. Ritcher, E. Shaw, J.A. Morgan, C. Rahn, and GD. Bending. 2005. Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial activity in soils. Rapid Commun Mass Spectrom. 19:1424-1428. . Lin, J. T., and V. Stewart. 1998. Nitrate assimilation by bacteria. Adv. Microb. Physiol. 39: 1-30. . Maier, T.M., and C.R. Myers. 2001. Isolation and characterization of a Shewanella putrefaciens MR-l electron transport regulator etrA mutant: reassessment of the role of EtrA. J. Bacteriol. 183:4918-26. . Qiu, X., G.W. Sundin, L. Wu, J. Zhou, and J.M. Tiedje. 2005. Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-l following exposure to UVC, UVB, and UVA radiation. J. Bacteriol. 187:3556-64. . Saffarini, D.A., and K.H. Nealson. 1993. Sequence and genetic characterization of etrA, an firr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-I. J. Bacteriol. 175:7938-7944. . Tiedje, J.M. 2002. Shewanella-the environmentally versatile genome. Nat. Biotechnol. 20: 1093-1094. . Voyles, B.A. 1993. The biology of viruses. Mosby-Year Book, Inc., St. Louis, MO. 141 APPENDIX A Shewanella oneidensis MR-l deletion mutants 142 Additional MR-l deletion mutants I encountered several obstacles in the generation of the deletion mutants presented in this work but fortunately I was able to solve them. Due to time limitations, I could not complete the final steps of construction of the ones described in this appendix. These mutations were generated as described in Chapters III and IV and the primer sequences used are provided (TABLE A.1). Some of the difficulties encountered included non-specific insertion of the construct in the genome, replication of the suicide vector in MR-l and contamination of the mutant with the wild type. To select the colonies with the insertion in the correct location in the genome, primers for PCR targeting the sequences upstream and downstream of the construct were designed. The size of the product of this reaction will indicate whether or not the construct replaced the gene of interest. To solve the replication of the suicide vector in MR-l it was necessary to transfer the constructs to a plasmid that possessed a different origin. MR-l can replicate plasmids that possess origins from pUC plasmids, which were present in the pCMl84. The constructs were transferred to the pKNOCK-Gm plasmid as described in Chapters III and IV, which has an R6K origin that requires a 1: protein that is not present in MR-l. This vector worked as a suicide vector in MR-l. Once the constructs were introduced by conjugation in MR-l, candidate colonies for the deletion were screened by PCR. The PCR reaction revealed two fragments, one that showed removal of the gene and a band of the size of the wild type phenotype. Furthermore, sequencing attempts showed mixed product. These results indicated that the mutant colony was contaminated with the wild type. This could be due to the excessive production of exopolysaccharide (EPS), which has been described in 143 MR-l as a protection against oxygen toxicity. The EPS protects the wild type from the antibiotic selection and it makes its elimination difficult. After many, many attempts to dilute out the wild type by serial liquid growth and plating, I could not succeed in recovering only the mutant. The mutants presented in this appendix were left at this point (TABLE A7.1). To solve this, these cultures need to be transfer three times on LB agar and screened by PCR. If no clean colonies (free of wild type) are selected, a colony needs to be transfer to LB broth, incubated overnight and then transfer three times on LB agar. These steps need to be repeated until a clean mutant can be selected. To prevent further undesirable mutations it is important that the colonies are transfer only three times on agar and then in LB broth. 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