i MERCURY TOXICITY AND DETOXIFICATION IN THERMUS THERMOPHILUS HB27 By JAVIERA A. NORAMBUENA MORALES A dissertation submitted to the School of Graduate Studies Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Microbial Biology Written under the direction of Tamar Barkay And approved by ___________________________ ___________________________ ___________________________ ___________________________ New Brunswick, New Jersey October 2018 i ii ABSTRACT OF THE DISSERTATION Mercury toxicity and detoxification in Thermus thermophilus HB27 by Javiera A. Norambuena Morales Dissertation Director: Tamar Barkay, Ph.D. Mercury (Hg) is one of the most toxic and widely distributed heavy metals. To detoxify this metal the mercury (mer) resistance operon is present some Bacteria and Archaea. This is the most studied mechanism of Hg-detoxification. This dissertation describes how the mer operon is regulated in Thermus thermophilus HB27, how two of the genes present in this operon (oah2 and merR) are involved in Hg(II) resistance, as well as low molecular weight (LMW) thiol and reactive oxygen species (ROS) responsive systems. T. thermophilus HB27 is a Gram-negative thermophile that has a very peculiar mer operon, it consists of merA (mercuric reductase), an hypothetical protein (hp), merR (regulator), and oah2, which encodes for an enzyme that synthesizes homocysteine. Therefore, the mer operon in T. thermophilus HB27 links mercury resistance to low-molecular weight (LMW) thiols biosynthesis. To determine the role of each gene in Hg-detoxification, mutant strains were constructed and their response to Hg was analyzed. I found that the mer operon has two promoters, one appears to be independent of regulation by MerR and in the other, MerR mediates response to Hg(II) as a repressor/activator. It was also determined that oah2 as well as other LMW thiols biosynthetic genes (oah1, bshA, bshB and oas) and the thioredoxin system are involved in Hg resistance. I discovered that ii iii bacillithiol (BSH) is the major LMW thiol in strain HB27 and showed that Hg(II) caused depletion of the reduced BSH pool. This depletion was associated with an increase in ROS upon Hg(II) exposure and free iron concentration in the cytoplasm. I showed that ROS were triggered by Hg(II) and that superoxide dismutase and pseudocatalase, both known ROS detoxifying enzymes that maintain intracellular redox state, are involved in Hg(II) resistance. These results suggest that small thiols play a role in T. thermophilus HB27’s response to Hg stress, possibly providing a buffer for Hg that is later removed by MerA. If thiols are oxidized by Hg(II), oxidative stress is produced leading to an increase in ROS. Collectively, the research presented in this dissertation describes how Hg(II) regulates the mer operon, interacts with LMW thiols and produces ROS in the extremophile T. thermophilus. Studying the physiology of T. thermophilus provides clues about the origin and evolution of mechanisms for mercury resistance and toxicity, as we as the oxidative stress response. iii iv ACKNOWLEDGEMENTS I would like to thank everyone that helped me during this process. First and foremost, I would like to thank my advisor Dr. Tamar Barkay for allowing me to work with her and be part of her lab. I would like to thank her and my co-advisor Dr. Jeff Boyd for all their feedback, encouragement and time spent discussing my research and new ideas. I am forever in debt with them. I would also like to thanks to the Barkay’s and Boyd’s Lab members, especially Zuelay, Spencer, Ameya, and Nicole, for their help and company through the years. I would specially like to thank Dr. Tom Hanson, for all his invaluable feedback and help with my research; as well as life. To Dr. Gerben Zylstra for all his time and invaluable feedback through the years and on this dissertation, but I would also like to thank him for all his guidance navigating the microbial biology program. I would like to thank people that helped to make this research possible: Dr. Peter Kahn for providing access to and guidance in the use of the spectrophotometer used to measure MerA activity, Dr. Alexei M. Tyryshkin for his help with the EPR experiments and Dr. Akira Nakamura for kindly providing us with a plasmid harboring the Hygromycin B resistance which was fundamental for complementation and construction of double knock out strains. Many thanks to all the different sources of funding that support me through the years, especially Fulbright and Becas Chile that made this possible. To the Graduate School of New Brunswick, as well as, the many Alumni and friends of the Department that, through their generous donations, made fellowships, scholarships, and travel awards possible. I am deeply grateful for the all their financial support. A special thanks to iv v Department of Biochemistry and Microbiology for providing me with the opportunity to be a teaching assistant over the year. It was an honor and pleasure to work with Dr. Natalya Voloshchuk. Finally, I would like to thank to all family and friends that were routing for me from far away; especially to my mom, my aunt Nany, my brother, Pablo, Pamela and Isabel. Without you this would not have been possible. v vi TABLE OF CONTENTS Abstract ii Acknowledgements iv List of Tables vii List of Figures ix Preface xii Introduction 1 Chapter 1 – Expression and regulation of the mer operon by mercury in 7 Thermus thermophilus HB27 Chapter 2 – Low-molecular-weight thiols and thioredoxins are important 32 players in Hg(II) resistance in Thermus thermophilus HB27 Chapter 3 – Superoxide dismutase and pseudocatalase promote Hg(II) 66 resistance in Thermus thermophilus HB27 by maintaining the reduced bacillithiol pool Concluding Remarks 88 Appendix A – Supplementary Material for Chapter 1 93 Appendix B – Supplementary Material for Chapter 2 101 Appendix C – Supplementary Material for Chapter 3 178 References 123 vi vii LIST OF TABLES CHAPTER 1 Table 1.1. mer operon transcript stability following cessation of de-novo 17 transcription. CHAPTER 2 Table 2.1 PCR primers used for construction of T. thermophilus HB27 knockout 39 mutants in Chapter 2. Table 2.2 qPCR primers used to measure gene expression of the listed genes in 42 Chapter 2. APPENDIX A Table 1.S1 Primers used on Chapter 1. 97 Table 1.S2 Primers used for knockout construction on Chapter 1. 99 APPENDIX B Table 2.S1. gyrA Ct values obtained in qPCR experiments for the WT strain of T. 110 thermophilus HB27. Table 2.S2. Fold induction of trx genes in T. thermophilus HB27 at different times 111 after exposure to 1 µM Hg(II). Table 2.S3. Concentrations of LMW thiols and effect of exposure to Hg(II) in T. 112 thermophilus HB27 and in some of its mutants. Table 2.S4. Structure of mer operons in Deinococcus-Thermus. 113 Table 2.S5. Alphaproteobacterial mer operons containing gor, the gene encoding 114 for glutathione reductase (GR). Table 2.S6. Locus tags of the proteins used to construct Thermus phylogenies. 116 vii viii Table 2.S7 Locus tag used to construct alphaproteobacterial phylogenies. 117 APPENDIX C Table 3.S1. Primers used to construct mutant strains for Chapter 3. 121 Table 3.S2. Primers and parameters used for qPCR in Chapter 3. 122 viii ix LIST OF FIGURES INTRODUCTION Figure 1. Metal uptake and resistance mechanisms in prokaryotes. 3 CHAPTER 1 Figure 1.1. Hg-dependent induction of the mer operon. 16 Figure 1.2. Multiple promoter sequences are present in the mer operon. 18 Figure 1.3. Two different transcripts are synthesized from the mer operon. 19 Figure 1.4. T. thermophilus’s MerR acts as a repressor/activator for Pmer. 22 Figure 1.5. The mer operon is transcribed constitutively from Poah2. 24 Figure 1.6. Mercury affects mer operon operon expression. 30 CHAPTER 2 Figure 2.1. The mer and met operons, and the methionine metabolism pathways in 35 T. thermophilus. Figure 2.2. Thiol systems are induced by Hg(II). 48 Figure 2.3. Sensitivity to Hg(II) increased in mutant strains lacking LMW thiol 52 synthesis genes or thiol homeostasis systems. Figure 2.4. BSH is the primary LMW thiol in T. thermophilus and LMW thiols are 55 responsive to Hg(II). Figure 2.5. Decreased thiol availability enhances Hg(II) toxicity. 57 Figure 2.6. Molecular phylogenetic analysis of Oah proteins in Thermus spp. by 60 the Maximum Likelihood method. Figure 2.7. Proposed model for a two-tiered response to Hg(II) toxicity in T. 63 ix x thermophilus. CHAPTER 3 Figure 3.1. Mercury exposure induces ROS and increased SOD and Pcat 75 expression. Figure 3.2. ROS mutants are more sensitive to Hg(II) and have increased ROS 78 levels upon Hg(II) exposure. Figure 3.3. T. thermophilus strains lacking Sod or Pcat have decreased reduced 80 BSH. Figure 3.4. Mercury stress results in aconitase inactivation and increased 82 intracellular free iron. Figure 3.5. Working model for ROS generation by Hg(II). 85 CONCLUDING REMARKS Figure 4.1. Proposed mechanism of Hg(II) toxicity and detoxification in T. 89 thermophilus. APPENDIX A Figure 1.S1. Schematics of the mer operons and rrsB loci of the different strains 93 used on Chapter 1. Figure 1.S2. Differential expression of mer operon’s genes in response to Hg(II) 94 exposure. Figure 1.S3. Control reactions for gene junction PCR. 95 Figure 1.S4. Convergent mer operons possess multiple promoters. 96 APPENDIX B Figure 2.S1. LMW thiol biosynthesis genes are not induced by ROS or disulfide 101 x xi stress. Figure 2.S2. Thiol related genes are not induced by Hg(II) in E.