Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues

BMC Microbiology

Elizabeth A Karr²  Daniel J Lessner¹  Jessica L Turner²  Catherine E Isom² 

[1] Department of Biological Sciences, University of Arkansas-Fayetteville, Fayetteville, USA;Department of Microbiology and Plant Biology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK, 73019, USA

关键词: Regulation;    Archaea;    Transcription;    Methanogens;   

Others  :  1143421
DOI  :  10.1186/1471-2180-13-163

 received in 2013-05-11, accepted in 2013-07-12,  发布年份 2013

【 摘 要 】

Background

Methanoarchaea 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.

Results

An 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.

Conclusions

Results presented herein suggest that homodimeric MaMsvR acts as a transcriptional repressor by binding Ma P_msvR under non-reducing conditions. Changing redox conditions promote conformational changes that abrogate binding to Ma P_msvR which likely leads to de-repression.

【 授权许可】

   
2013 Isom et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jarrell KF: Extreme oxygen sensitivity in methanogenic archaebacteria. Bioscience 1985, 35(5):298-302.
• [2]Kato MT, Field JA, Lettinga G: High tolerance of methanogens in granular sludge to oxygen. Biotechnol Bioeng 1993, 42(11):1360-1366.
• [3]Fetzer S, Bak F, Conrad R: Sensitivity of methanogenic bacteria from paddy soil to oxygen and desiccation. FEMS Microbiol Ecol 1993, 12(2):107-115.
• [4]Peters V, Conrad R: Methanogenic and other strictly anaerobic bacteria in desert soil and other oxic soils. Appl Environ Microbiol 1995, 61(4):1673-1676.
• [5]Kato S, Kosaka T, Watanabe K: Comparative transcriptome analysis of responses of Methanothermobacter thermautotrophicus to different environmental stimuli. Environ Microbiol 2008, 10(4):893-905.
• [6]Lumppio HL, Shenvi NV, Summers AO, Voordouw G, Kurtz DM: Rubrerythrin and rubredoxin oxidoreductase in Desulfovibrio vulgaris: a novel oxidative stress protection system. J Bacteriol 2001, 183(1):101-108.
• [7]Jenney FE, Verhagen MFJM, Cui X, Adams MWW: Anaerobic microbes: oxygen detoxification without superoxide dismutase. Science 1999, 286(5438):306-309.
• [8]Seedorf H, Dreisbach A, Hedderich R, Shima S, Thauer RK: F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification. Arch Microbiol 2004, 182:126-137.
• [9]Karr EA: The methanogen-specific transcription factor MsvR regulates the fpaA-rlp-rub oxidative stress operon adjacent to msvR in Methanothermobacter thermautotrophicus. J Bacteriol 2010, 192(22):5914-5922.
• [10]Geiduschek EP, Ouhammouch M: Archaeal transcription and its regulators. Mol Microbiol 2005, 56(6):1397-1407.
• [11]Ouhammouch M, Dewhurst RE, Hausner W, Thomm M, Geiduschek EP: Activation of archaeal transcription by recruitment of the TATA-binding protein. Proc Natl Acad Sci USA 2003, 100(9):5097-5102.
• [12]Podar A, Wall MA, Makarova KS, Koonin EV: The prokaryotic V4R domain is the likely ancestor of a key component of the eukaryotic vesicle transport system. Biol Direct 2008., 3(2) BioMed Central Full Text
• [13]Darcy TJ, Hausner W, Awery DE, Edwards AM, Thomm M, Reeve JN: Methanobacterium thermoautotrophicum RNA polymerase and transcription in vitro. J Bacteriol 1999, 181(14):4424-4429.
• [14]Moore BC, Leigh JA: Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease. J Bacteriol 2005, 187(3):972-979.
• [15]Pritchett MA, Zhang JK, Metcalf WW: Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic Archaea. Appl Environ Microbiol 2004, 70(3):1425-1433.
• [16]Guss AM, Rother M, Zhang JK, Kulkkarni G, Metcalf WW: New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species. Archaea 2008, 2(3):193-203.
• [17]Rother M, Metcalf WW: Genetic technologies for Archaea. Curr Opin Microbiol 2005, 8(6):745-751.
• [18]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.
• [19]Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, et al.: CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 2011, 39(suppl 1):D225-D229.
• [20]Zdanowski K, Doughty P, Jakimowicz P, O'Hara L, Buttner MJ, Paget MSB, Kleanthous C: Assignment of the zinc ligands in RsrA, a Redox-Sensing ZAS Protein from Streptomyces coelicolor. Biochemistry 2006, 45(27):8294-8300.
• [21]Jäger D, Sharma CM, Thomsen J, Ehlers C, Vogel J, Schmitz RA: Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability. Proc Natl Acad Sci USA 2009, 106(51):21878-21882.
• [22]Karr EA, Sandman K, Lurz R, Reeve JN: TrpY Regulation of trpB2 transcription in Methanothermobacter thermautotrophicus. J Bacteriol 2008, 190(7):2637-2641.
• [23]Bell SD: Archaeal transcriptional regulation – variation on a bacterial theme? Trends Microbiol 2005, 13(6):262-265.
• [24]Xie Y, Reeve JN: Transcription by an archaeal RNA Polymerase is slowed but not blocked by an archaeal nucleosome. J Bacteriol 2004, 186(11):3492-3498.
• [25]Santangelo TJ, Reeve JN: Archaeal RNA polymerase is sensitive to intrinsic termination directed by transcribed and remote sequences. J Mol Biol 2006, 355:196-210.
• [26]Storz G, Tartaglia LA, Ames BN: Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science 1990, 248(4952):189-194.
• [27]Hellman LM, Fried MG: Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protocols 2007, 2(8):1849-1861.
• [28]Lessner DJ, Ferry JG: The archaeon Methanosarcina acetivorans contains a protein disulfide reductase with an iron-sulfur cluster. J Bacteriol 2007, 189(20):7475-7484.
• [29]Pryor EE Jr, Waligora EA, Xu B, Dellos-Nolan S, Wozniak DJ, Hollis T: The transcription factor AmrZ utilizes multiple DNA binding modes to recognize activator and repressor sequences of Pseudomonas aeruginosa virulence genes. PLoS Path 2012, 8(4):e1002648.
• [30]Lundin M, Nehlin JO, Ronne H: Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1. Mol Cell Biol 1994, 14(3):1979-1985.
• [31]Cook WJ, Kar SR, Taylor KB, Hall LM: Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins. J Mol Biol 1998, 275(2):337-346.
• [32]Liu Y, Yang Y, Qi J, Peng H, Zhang J-T: Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2. J Pharmacol Exp Ther 2008, 326(1):33-40.
• [33]Paget MSB, Buttner MJ: Thiol-based regulatory switches. Annu Rev Genet 2003, 37:91-121.
• [34]Sidorova NY, Hung S, Rau DC: Stabilizing labile DNA–protein complexes in polyacrylamide gels. Electrophoresis 2010, 31(4):648-653.
• [35]Barbirz S, Jakob U, Glocker MO: Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone. J Biol Chem 2000, 275(25):18759-18766.
• [36]Geneious v4.8. http://www.geneious.com/ webcite
• [37]Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by Krawetz S, Misener S. Totowa, NJ: Humana Press; 2000:365-386.
• [38]Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72(1–2):248-254.
• [39]Laemmli UK: Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 1970, 227(5259):680-685.