The Methanogen-Specific Transcription Factor MsvR Regulates the fpaA-rlp-rub Oxidative Stress Operon Adjacent to msvR in Methanothermobacter thermautotrophicus

Abstract

Methanogens represent some of the most oxygen-sensitive organisms in laboratory culture. Recent studies indicate that they have developed mechanisms to deal with brief oxygen exposure. MsvR is a transcriptional regulator that has a domain architecture unique to a select group of methanogens. Here, runoff in vitro transcription assays were used to demonstrate that MsvR regulates transcription of the divergently transcribed fpaA-rlp-rub operon in Methanothermobacter thermautotrophicus in addition to transcription from its own promoter. The protein products of the fpaA-rlp-rub operon have previously been implicated in oxidative stress responses in M. thermautotrophicus. Additionally, electrophoretic mobility shift assays (EMSAs) and DNase I footprinting were used to confirm a binding site inferred by bioinformatic analysis. Sequence mutations within these binding sites did not significantly alter EMSA shifting patterns on longer templates but did on shorter 50-bp fragments encompassing only the region containing the binding sites. Footprinting confirmed that the regions protected for the longer mutant templates are at different positions within the intergenic region compared to those seen in the intact intergenic region. Oxidized and reduced preparations of MsvR demonstrated different EMSA binding patterns and regions of protection on the intergenic sequence, suggesting that MsvR may play a role in detecting the redox state of the cell.Biological methane production is limited to a small group of microorganisms in the domain Archaea that are termed methanogens. These organisms are strict anaerobes that can generate methane from H 2 and CO 2 and/or a limited series of C 1 compounds, depending upon the organism (5). Despite a necessity for growth in environments devoid of oxygen, the genomes of methanogens encode various enzymes (e.g., superoxide dismutase and alkyl hydroperoxide reductase) involved in the detoxification of reactive oxygen species. Previous studies have shown peroxide stress has little effect on methane production and overall growth yield in the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus. The same study identified upregulation of the fpaA-rlp-rub operon (mth1350 to mth1352) believed to be involved in the detoxification of reactive oxygen species (10). This operon encodes a flavoprotein (fpaA), a rubrerythrin-like protein (rlp), and a rubredoxin (rub) (10,19). A homologue of the flavoprotein from the methanogen Methanobrevibacter arboriphilus has been demonstrated to reduce O 2 in the presence of H 2 and was reclassified as an F 420 H 2 oxidase (24). This lends further evidence to support the role of proteins encoded by this operon in the detoxification of reactive oxygen species.The archaeal transcription machinery represents a chimeric system with components reminiscent of features found in the other two domains of life, Bacteria and Eukarya. The multisubunit RNA polymerase is similar to the eukaryotic RNA polymerase II complex and, as is the case for the eukaryotic system, is...

Negative Effect of Glucose onompAmRNA Stability: a Potential Role of Cyclic AMP in the Repression ofhfqin Escherichia coli

“…The pQE30-crp plasmid was transformed into E. coli BL21(DE3), and the CRP recombinant protein was isolated using a His tag column system purchased from Qiagene (Hilden, Germany) (26). The binding reactions (20 l) were performed by incubating 10 ng of labeled DNA fragments with various amounts (5,16,39). The DNA fragment from nucleotide position Ϫ251 to Ϫ32 (as indicated in Fig.…”

The Methanosarcina acetivorans thioredoxin system activates DNA binding of the redox-sensitive transcriptional regulator MsvR

“…Based on results from previous studies [15,17] and herein, we propose the following model (Fig. 2) for the regulation of the P msvR binding activity of MaMsvR by thiol-disulfide exchange involving the thioredoxin system.…”

“…The cysteines within the V4R domain (C206, C225, C232, and C240) are postulated to function in redox-sensing, whereby thiol-disulfide exchange causes conformation changes which alter the ability of MaMsvR to bind an inverted repeat sequence motif (TTCGN 7-9 CGAA) upstream of P msvR . Three of the residues (C206, C232, and C240) are conserved in all MsvR homologs [17]. Specifically, C206 was shown to be critical for redox-sensitive binding of MaMsvR to P msvR , because a MaMsvR C206A variant was able to bind to P msvR under non-reducing conditions, whereas the wild-type MaMsvR is unable [15].…”

“…Recent evidence has revealed the presence of a redox-sensing transcriptional regulator (MsvR) in methanogens. In Methanothermobacter thermautotrophicus , MsvR regulates expression of F 420 H 2 oxidase (FpaA) by redox-sensitive binding to the promoter region of fpaA [17]. MsvR also regulates its own expression in M. thermautotrophicus .…”

A Novel CO-Responsive Transcriptional Regulator and Enhanced H 2 Production by an Engineered Thermococcus onnurineus NA1 Strain

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