Sulfite Reductase and Thioredoxin in Oxidative Stress Responses of Methanogenic Archaea Dwi Susanti Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Genetics, Bioinformatics and Computational Biology Biswarup Mukhopadhyay, committee chair David R. Bevan Dennis R. Dean David L. Popham Robert H. White T.M. Murali July 2nd, 2013 Blacksburg, Virginia Keywords: sulfite reductase, thioredoxin, Methanocaldococcus jannaschii, methanogens, oxidative stress, redox, regulation Copyright 2013, Dwi Susanti Sulfite Reductase and Thioredoxin in Oxidative Stress Responses of Methanogenic Archaea Dwi Susanti (ABSTRACT) Methanogens are a group of microorganisms that utilize simple compounds such as H2 + CO2, acetate and methanol for the production of methane, an end-product of their metabolism. These obligate anaerobes belonging to the archaeal domain inhabit diverse anoxic environments such as rice paddy fields, human guts, rumen of ruminants, and hydrothermal vents. In these habitats, methanogens are often exposed to O2 and previous studies have shown that many methanogens are able to tolerate O2 exposure. Hence, methanogens must have developed survival strategies to be able to live under oxidative stress conditions. The anaerobic species that lived on Earth during the early oxygenation event were first to face oxidative stress. Presumably some of the strategies employed by extant methanogens for combating oxidative stress were developed on early Earth. Our laboratory is interested in studying the mechanism underlying the oxygen tolerance and oxidative stress responses in methanogenic archaea, which are obligate anaerobe. Our research concerns two aspects of oxidative stress. (i) Responses toward extracellular toxic species such as 2- SO3 , that forms as a result of reactions of O2 with reduced compounds in the environment. These species are mostly seen in anaerobic environments upon O2 exposure due to the abundance of reduced components therein. (ii) Responses toward intracellular toxic species such as superoxide and hydrogen peroxide that are generated upon entry of O2 and subsequent reaction of O2 with reduced component inside the cell. Aerobic microorganisms experience the second problem. Since a large number of microorganisms of Earth are anaerobes and the oxidative defense mechanisms of anaerobes are relatively less studied, the research in our laboratory has focused on this area. My thesis research covers two studies that fall in the above-mentioned two focus areas. In 2005-2007 our laboratory discovered that certain methanogens use an unusual sulfite 2- reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO3 that is produced outside the cell from a reaction between oxygen and sulfide. This reaction occurred during early oxygenation of Earth and continues to occur in deep-sea hydrothermal vents. Fsr, a 2- 2- flavoprotein, carries out a 6-electron reduction of SO3 to S . It is a chimeric protein where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively. We hypothesized that Fsr was developed in a methanogen from pre-existing parts. To begin testing this hypothesis we have carried out bioinformatics analyses of methanogen genomes and found that both Fsr-N homologs and Fsr-C homologs are abundant in methanogens. We called the Fsr-C homolog dissimilatory sulfite reductase-like protein (Dsr-LP). Thus, Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP) in methanogens. During the course of this study, we also identified two new putative F420H2-dependent enzymes, namely F420H2-dependent glutamate synthase and assimilatory sulfite reductase. Another aspect of my research concerns the reactivation of proteins that are deactivated by the entry of oxygen inside the cell. Here I focused specifically on the role of thioredoxin (Trx) in methanogens. Trx, a small redox regulatory protein, is ubiquitous in all living cells. In bacteria and eukarya, Trx regulates a wide variety of cellular processes including cell divison, biosynthesis and oxidative stress response. Though some Trxs of methanogens have been structurally and biochemically characterized, their physiological roles in these organisms are unknown. Our bioinformatics analysis suggested that Trx is ubiquitous in methanogens and the pattern of its distribution in various phylogenetic classes paralleled the respective evolutionary histories and metabolic versatilities. Using a proteomics approach, we have identified 155 Trx targets in a hyperthermophilic phylogenetically deeply-rooted methanogen, Methanocaldococcus jannaschii. Our analysis of two of these targets employing biochemical assays suggested that Trx is needed for reactivation of oxidatively deactivated enzymes in M. jannaschii. To our knowledge, this is the first report on the role of Trx in an organism from the archaeal domain. During the course of our work on methanogen Trxs, we investigated the evolutionary histories of different Trx systems that are composed of Trxs and cognate Trx reductases. In collaboration iii with other laboratories, we conducted bioinformatics analysis for the distribution of one of such systems, ferredoxin-dependent thioredoxin reductase (FTR), in all organisms. We found that FTR was most likely originated in the phylogenetically deeply-rooted microaerophilic bacteria where it regulates CO2 fixation via the reverse citric acid cycle. iv To my parents Endang Prasetyaning Utami and Susarwadi, my husband Bennu Guntoro and my little sunshine Adelia Fathiya Putri Guntoro v ACKNOWLEDGMENTS I would like to thank Dr. Biswarup Mukhopadhyay for providing me opportunities to carry out graduate research in his laboratory. I thank him for his input and guidance in both scientific and personal matters. I respect him for his passion in science, his knowledge as well as his research- and work-ethics. Most importantly, I would like to thank him for the supports, encouragements and advice. I could not ask for a better adviser. I also would like to thank Dr. Endang Purwantini. I would have not been here at Virginia Tech if it was not because of her. She trained me how to perform research during my undergraduate studies. She introduced me to the opportunity of studying abroad for pursuing my Ph.D. I would never have thought that I would be able to continue my education in this country or even in any other country than Indonesia. I enjoyed our discussion about scientific issues and about how we, as a scientist, could contribute to solving problems in Indonesia. I would like to thank my committee members, Drs. David R. Bevan, David L. Popham, Dennis R. Dean, Robert H. White and T.M. Murali for their time, advice and scientific input throughout my Ph.D. training. My thanks to Dennie Munson for all her help from the beginning of the admission process to the final exam. I am really grateful to be part of the Genetics, Bioinformatics and Computational Biology program. Many thanks to my labmates Dr. Lakshmi Dharmarajan, Jason R. Rodriguez, Eric F. Johnson and Usha Loganathan for encouragements, discussions and fun that we had in the lab. My thanks to all my colleagues in the GBCB program and many thanks to the members of Indonesian student association (PERMIAS) and Indonesian community in Blacksburg for all the supports and awesome time that we had. vi I would like to express my deepest love, gratitude and appreciations to my family, my parents (Endang Prasetyaning Utami, Susarwadi, Maryam and Sumardjo), my husband Bennu, my little angel Adelia, my sister and brothers and their families for their unconditional love and endless support. My special thanks are to the love of my life, Bennu and Adelia. vii ATTRIBUTIONS Results presented in chapter 3 have been published in PLOS ONE (1). This study is supported by NASA Astrobiology: Exobiology and Evolutionary Biology grants NNG05GP24G and NNX09AV28G to Dr. Biswarup Mukhopadhyay. I thank Dr. William B. Whitman of University of Georgia, for the genomic data on Methanothermococcus thermolithotrophicus reported in this work. Chapter 4 will soon be submitted for publication in a peer-reviewed journal. I would like to thank the following collaborators who have contributed to the research presented here: Drs. Joshua Wong of University of California Berkeley and William H. Vensel of United States Department of Agriculture for their contributions in 2D-gel electrophoresis and mass spectrometric analysis; Usha Loganathan for her assistance in the generation of Trx and Mtd proteins; Drs. Bob B. Buchanan of University of California Berkeley, Ruth A. Schmitz of Institut Für Allgemeine Mikrobiologie Germany and Monica Balsera of Instituto de Recursos Naturales y Agrobiología de Salamanca Spain for contributing their expertise. The research on thioredoxins of methanogens in our laboratory is supported by the NSF grant MCB # 1020458 to Drs. Biswarup Mukhopadhyay and Bob B. Buchanan. The finding presented in chapter 5 has been published in PLANTA (2). My primary contributions in this paper are shown in Figure 5.2 and Table 5.1. My collaborative work that is outside the main focus of this dissertation has been presented in the Appendices. Appendix A contains results that have been published in the Journal of Bacteriology (3). Our laboratory was the lead for this project. I generated genomic materials for the genome