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 SO32-, 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 reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO32- 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 flavoprotein, carries out a 6-electron reduction of SO32- to S2-. 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 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. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/51423 |
| Date | 22 August 2013 |
| Creators | Susanti, Dwi |
| Contributors | Animal and Poultry Sciences, Mukhopadhyay, Biswarup, Popham, David L., Dean, Dennis R., White, Robert H., Murali, T. M., Bevan, David R. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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