Dual Negative Control ofspxTranscription Initiation from the P₃Promoter by Repressors PerR and YodB inBacillus subtilis

Abstract

The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P 3 promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P 3 promoter. Expression of an spx-bgaB fusion carrying the P 3 promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P 3 transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P 3 promoter DNA was used as a template. Point mutations at positions within the P 3 promoter relieved YodB-dependent repression, while a point mutation at position ؉24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P 3 ؊10 and ؊35 regions, while PerR binds to a region downstream of the P 3 transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.Spx is a highly conserved transcriptional regulatory protein of low-GC-content gram-positive bacteria (2,4,7,29,36,40). It does not possess sequence-specific DNA-binding activity but instead directly targets RNA polymerase (RNAP) (26,28), an interaction that interferes with contact between transcriptional activators (26, 39) and RNAP while activating transcription at promoters of genes whose products function in intracellular thiol homeostasis (24,25) and responses to encounters with toxic oxidants (25,29,33,34). Recent studies suggested that Spx plays a role in cellular invasion by a microbial pathogen (2). Spx does not belong to any other known family of transcriptional regulatory proteins but resembles the arsenate reductase ArsC of plasmid R773 in terms of its primary and higher-order structure (18,28,40).The spx gene resides in the yjbC-spx operon of the Bacillus subtilis genome and is transcribed from at least five promoters by four forms of RNAP holoenzyme ( A , B , M , and W ). Induction of spx expression has been associated with phosphate starvation, ethanol, and oxidative stress (1, 25, 35), while induction of the spx regulon is activated by a variety of stress conditions that include heat shock, salt stress, oxidative stress, and toxic phe...

Defining the structure of the general stress regulon of Bacillus subtilis using targeted microarray analysis and random forest classification

“…For 20 of the 37 genes, additional regulation by secondary regulators has already been proven by previous studies. These 20 genes are: bioB, bmrR, purK, radA, yacK, yacM, ydaF, ykrT, yocL, ypuD, yraA, ysnF, ywhH, yxjG and the operon yceC-yceD-yceE-yceF-yceG-yceH (Au et al, 2005;Banse et al, 2008;Baranova et al, 1999;Bower et al, 1996; Cao et al, 2002;Chu et al, 2006;Chumsakul et al, 2011;Comella & Grossman, 2005;Derré et al, 1999;Drzewiecki et al, 1998;Ebbole & Zalkin, 1987;Eiamphungporn & Helmann, 2008;Erwin et al, 2005;Grundy & Henkin, 1998; Höper et al, 2005;Jervis et al, 2007;Kearns et al, 2005; Krüger et al, 1996;Kumaraswami et al, 2010;Leelakriangsak et al, 2007;Lei et al, 2009;Marvasi et al, 2010;Nakano et al, 2003;Nguyen et al, 2009;Perkins et al, 1996; Petersohn et al, 2001;Sekowska & Danchin, 2002;Wang et al, 2006;Weng et al, 1995; You et al, 2008). Details about their secondary regulators are included in Supplementary Table S4.…”

Derepression of the Bacillus subtilis PerR Peroxide Stress Response Leads to Iron Deficiency

“…1). PerR also plays an auxiliary role in regulating expression of spx (48), which coordinates the disulfide stress response, and positively regulates the srfA operon, which encodes enzymes for surfactin biosynthesis (36).…”

Regulation of Quinone Oxidoreductase by the Redox-sensing Transcriptional Regulator QorR in Corynebacterium glutamicum

“…A DUF24 protein family member was shown to function as a transcriptional regulator in the actinobacterium C. glutamicum, as was reported previously for the firmicute B. subtilis (1,3). More than 1,000 proteins of 488 bacterial species are classified into the DUF24 protein family in the Pfam database (26), suggesting that the DUF24 protein family is a regulatory protein family widely spread among bacteria.…”

“…Although HxlR is necessary for formaldehyde-induced expression of the operon, the finer details as to how formaldehyde modulates hxlAB expression are still not clear. YodB negatively controls expression of spx, azoR1, and yodC, genes involved in oxidative stress response (2,3). The DNAbinding activity of YodB is inhibited by thiol-reactive compounds, and its cysteine residues function in redox sensing (2).…”