Abstract
BackgroundMethanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. The mechanisms by which they sense and respond to oxidizing conditions are unknown. MsvR is a transcription regulatory protein unique to the methanoarchaea. Initially identified and characterized in the methanogen Methanothermobacter thermautotrophicus (Mth), MthMsvR displays differential DNA binding under either oxidizing or reducing conditions. Since MthMsvR regulates a potential oxidative stress operon in M. thermautotrophicus, it was hypothesized that the MsvR family of proteins were redox-sensitive transcription regulators.ResultsAn MsvR homologue from the methanogen Methanosarcina acetivorans, MaMsvR, was overexpressed and purified. The two MsvR proteins bound the same DNA sequence motif found upstream of all known MsvR encoding genes, but unlike MthMsvR, MaMsvR did not bind the promoters of select genes involved in the oxidative stress response. Unlike MthMsvR that bound DNA under both non-reducing and reducing conditions, MaMsvR bound DNA only under reducing conditions. MaMsvR appeared as a dimer in gel filtration chromatography analysis and site-directed mutagenesis suggested that conserved cysteine residues within the V4R domain were involved in conformational rearrangements that impact DNA binding.ConclusionsResults presented herein suggest that homodimeric MaMsvR acts as a transcriptional repressor by binding Ma PmsvR under non-reducing conditions. Changing redox conditions promote conformational changes that abrogate binding to Ma PmsvR which likely leads to de-repression.
Highlights
Methanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen
Within the DNA binding domain, 48% of the residues indicated by the conserved domain database (CDD) to be involved in DNA binding are conserved (Figure 1a, red boxes) and 45% of residues are conserved throughout the domain (Figure 1a, black box) [19]
This motif is absent in Methanosarcina acetivorans MsvR (MaMsvR), and in other methanogen-specific V4R domain regulator (MsvR) homologues that do carry this motif, the histidine is replaced with a proline
Summary
Methanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. MsvR is a transcription regulatory protein unique to the methanoarchaea. Identified and characterized in the methanogen Methanothermobacter thermautotrophicus (Mth), MthMsvR displays differential DNA binding under either oxidizing or reducing conditions. Since MthMsvR regulates a potential oxidative stress operon in M. thermautotrophicus, it was hypothesized that the MsvR family of proteins were redox-sensitive transcription regulators. Methanoarchaea represent some of the most oxygen sensitive organisms identified to date [1], yet many methanogens can withstand oxygen exposure and resume growth once anaerobic conditions have been restored [2,3,4]. In Methanothermobacter thermautotrophicus, F420H2 oxidase is the product of fpaA (MTH1350) whose promoter, PfpaA, is regulated by the methanogen-specific V4R domain regulator (MsvR). M. thermautotrophicus MsvR (MthMsvR) and its homologues are unique to a subset of methanogens, including the Methanomicrobiales and Methanosarcinales [9]. MthMsvR represses transcription of fpaA and msvR by abrogating the binding of general transcription factors at the promoter, PfpaA or PmsvR, respectively [9]
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