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. 2010 Nov;192(22):5914-22.
doi: 10.1128/JB.00816-10. Epub 2010 Sep 17.

The methanogen-specific transcription factor MsvR regulates the fpaA-rlp-rub oxidative stress operon adjacent to msvR in Methanothermobacter thermautotrophicus

Elizabeth A Karr  1
Affiliations

The methanogen-specific transcription factor MsvR regulates the fpaA-rlp-rub oxidative stress operon adjacent to msvR in Methanothermobacter thermautotrophicus

Elizabeth A Karr. J Bacteriol. 2010 Nov.

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.

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Figures

FIG. 1.
FIG. 1.
(A) Diagram of the M. thermautotrophicus genome region encoding the fpaA-rlp-rub operon (MTH1350 to -1352) and an adjacent transcription regulator (msvR). The sequence of the msvR-fpaA intergenic region is displayed. The PfpaA TATA box is indicated by a solid single underline and bold text, with its corresponding transcription start site identified by a solid black arrow. The PfpaA2 TATA box is represented by a single dashed underline with its putative corresponding transcription start site identified with a dotted arrow. The translation start site for FpaA is shown in italics. The putative PmsvR TATA box is indicated by the thick gray underline, and its putative corresponding transcription start site is identified by a gray arrow. The translation start site for MsvR is shown in gray italics. Potential transcription regulator binding sites are represented by gray boxes. The coverage of templates T203, GS203, T1349, and T1350 is displayed. The overlapping sequence coverage of T1349 and T1350 is double underlined. (B) The amino acid sequence for the putative transcriptional regulator MsvR. Boxes indicate domains identified by the NCBI CDD. The carboxy-terminal amino acid residues that may be important for serving as redox sensors are underlined with a solid black line. N-terminal residues that may be involved in metal binding are represented by a dashed underline. (C) Alignment of the msvR-fpaA intergenic sequence with those of other methanogens (Methanobacteriales and Methanomicrobiales) that have a homologue of the MsvR regulator sharing an intergenic region with a homologue of FpaA. Sequences below the horizontal line are from the promoter regions of msvR genes in members of the Methanosarcinales. The binding regions identified in panel A are displayed here, and conserved nucleotides within these regions are underlined.
FIG. 2.
FIG. 2.
(A) The fpaA and msvR transcripts generated using the T203 template in the absence (−) or presence (+; 200 nM) of MsvR. Mutations were made in two putative TATA boxes (T255 and T256) for fpaA, and transcripts generated using those templates are displayed (with and without MsvR). (B) The msvR and fpaA transcripts generated from T203 in the presence of increasing concentrations (0 nM, 20 nM, 40 nM, 100 nM, and 200 nM) of MsvR in both standard in vitro transcription assays (solid lines) as well as in an order-of-addition assay (dotted lines), in which the general transcription factors (TBP/TFB) were incubated with the template prior to the addition of MsvR. The amount of transcript produced from promoters in each reaction was quantitated and compared to the respective control reaction (taken as 100% transcript). The data displayed are averages of two independent experiments.
FIG. 3.
FIG. 3.
(A) EMSAs with the GS203 template (contains only the intergenic region) in the presence of increasing concentrations (0 nM, 20 nM, 40 nM, 100 nM, and 200 nM) of MsvRox or MsvRred. The binding reaction mixtures contained no DTT. Shifted species are identified as S1, S2, S3, and S4. These species do not necessarily represent the same complex in each reaction but are species with similar overall molecular weights. The gel wells and positions of free DNA are also indicated. (B) EMSAs with T1350 and T1349 in the absence (−) or presence of oxidized (O) or reduced (R) MsvR (200 nM). (C) Mutations generated in each of the three binding boxes as well as combinations of the three binding boxes. (D) EMSAs performed using templates containing various mutations in the binding boxes in the presence of preoxidized or prereduced MsvR (200 nM). A representative EMSA using GS203 is displayed; however, all experiments/gels included binding reactions with GS203 as a positive control to ensure that differences in binding patterns were not due to technical differences between experiments. (E) EMSA with annealed oligonucleotides corresponding to the centralized region containing the three binding boxes identified in panel C, with a 20-fold excess of MsvR. MsvR preparations used are the same as those indicated in panel D.
FIG. 4.
FIG. 4.
(A) The msvR and fpaA intergenic region, displayed with corresponding DNase I footprints. The footprints for each strand are displayed with a dashed line for those generated with MsvRox and with a solid line for those generated with MsvRred. The PfpaA TATA box is shown in green, and its transcription start site is identified. The putative binding site sequences identified in Fig. 1 are shown in red. The region of sequence overlap between T1349 and T1350 is shown in blue. Vertical arrows above and below each strand depict hypersensitive sites. Hypersensitive sites present in the oxidized, reduced, or both footprints are depicted by a dotted arrow, solid black arrow, and a solid gray arrow, respectively. (B) Aligned chromatograms of MsvRox and MsvRred footprints on T203 (plus strand) and the MsvRred protection site on T264 (plus strand). The black trace corresponds to reaction mixtures containing only DNA, whereas the red trace corresponds to reaction mixtures containing DNA and MsvR. MsvR protected regions are boxed using the same scheme as for T203 in panel A, and the asterisks depict the hypersensitive sites. The protected region on T264 is boxed in blue. (C) Diagram of MsvR protected regions on fragments containing the mutations identified in Fig. 3C. The diagram is aligned to the chromatograms in panel B. The gray boxes represent the regions protected from DNase I digestion when MsvR is bound to the indicated template. The orange box represents the position of the overlap for templates T1349 and T1350. The red boxes represent binding boxes 1, 2, and 3, and the green box represents the PfpaA TATA box.
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References

    1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. - PubMed
    1. Aravind, L., and E. V. Koonin. 1999. DNA-binding proteins and evolution of transcription regulation in the Archaea. Nucleic Acids Res. 27:4658-4670. - PMC - PubMed
    1. Bae, J.-B., J.-H. Park, M.-Y. Hahn, M.-S. Kim, and J.-H. Roe. 2004. Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor: zinc release and disulfide bond formation. J. Mol. Biol. 335:425-435. - PubMed
    1. Darcy, T. J., W. Hausner, D. E. Awery, A. M. Edwards, M. Thomm, and J. N. Reeve. 1999. Methanobacterium thermoautotrophicum RNA polymerase and transcription in vitro. J. Bacteriol. 181:4424-4429. - PMC - PubMed
    1. Deppenmeier, U. 2002. The unique biochemistry of methanogenesis. Proc. Natl. Acad. Sci. U. S. A. 71:223-283. - PubMed

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