Date

October 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Environmental and Biomolecular Systems

Institution

Oregon Health & Science University

Abstract

The Bacillus subtilis spx gene encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It resides in the yjbC-spx operon and is transcribed from at least four promoters, three (P[subscript]1, P[subscript]2 and P[subscript]B) residing upstream of yjbC and one (P[subscript]M) located in the intergenic region between yjbC and spx. We uncovered a second intergenic promoter, P[subscript]3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide, by primer extension analysis. P[subscript]3 is recognized by the σ[superscript]A form of RNA polymerase (RNAP) in vitro without the involvement of a transcriptional activator. Deletion analysis together with point mutation analysis uncovered two negative cis-acting control elements within the P[subscript]3 promoter. Previously published studies and transcription factor/transformation array technology uncovered two transcriptional repressors, PerR and YodB that were potential candidates for the missing trans-acting factors affecting P[subscript]3 promoter utilization. PerR was previously characterized as the regulator of the inducible peroxide stress response in B. subtilis, while YodB is a novel DUF24/MarR type repressor that controls genes that are induced in response to phenolic compounds and oxidative stress. The derepression of spx was detected in both perR and yodB mutants by examining the level of spx expression using the spx-bgaB fusion construct. The additive effect was observed in the perR yodB double mutant. The regions of spx P[subscript]3 DNA required for transcriptional repression by YodB and PerR were confirmed by DNase I footprinting analysis. PerR protects an area from approximately position -3 to +35. YodB binds a region from approximately positions -3 to -32. The binding of YodB and PerR proteins to spx P[subscript]3 promoter DNA was impaired by addition of diamide and H[subscript]2O[subscript]2 in vitro as determined by DNase I footprinting analysis. Besides spx, YodB also controls the divergently transcribed yodC gene which encodes a putative nitroreductase that is induced by disulfide stress. Microarray and proteome analyses were performed to identify other genes controlled by YodB. yocJ (azoR1), encoding the putative FMN-dependent NADH-azoreductase, was the most strongly derepressed by yodB null mutation and was induced in response to diamide, catechol, MHQ and nitrofurantoin stress. bsrB encoding a small 6S RNA located downstream of azoR1, is co-transcribed with azoR1 and increased in concentration in response to thiol-reactive compounds. The yodB mutant confers a catechol and MHQ resistance phenotype due to AzoR1 overproduction. In addition, the yodBmhqR double mutant, bearing the deletion of the mhqR gene encoding a MarR-like repressor, that overproduces AzoR1 and MhqR-regulated paralog AzoR2, exhibits hyper-resistance to thiol-reactive compounds. Thus, the detoxification of thiol-reactive substances in YodB and MhqR regulons show overlapping functions. DNase I footprinting analysis, together with promoter sequence alignments, uncovered YodB boxes which contain a common 15 bp consensus sequence for YodB-DNA interaction. The YodB protein contains three cysteine residues Cys6, Cys101 and Cys108. The conserved Cys6 contributes to the repression of spx and azoR1 transcription by YodB. Moreover, mass spectrometry revealed YodB Cys modifications by catechol and MHQ.

Identifier

doi:10.6083/M46W980C

School

OGI School of Science and Engineering

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