Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism inBacillus subtilisRequires Spx

Abstract

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase ␣ subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfatedependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions.Spx is a global transcriptional regulator of the oxidative stress response in Bacillus subtilis and is highly conserved among low-GϩC-content gram-positive bacteria (34,35,43). It was initially identified as a protein encoded by a gene that was the site of suppressor mutations in clpP and clpX mutants (29). Spx concentration is proteolytically controlled by the ATPdependent protease ClpXP (35,36). The high concentrations of Spx in clpX and clpP mutants result in poor growth and sporulation and the loss of competence development as well as reduced anaerobic metabolism. Spx was discovered to exert global negative transcriptional control by blocking activator-RNA polymerase (RNAP) interaction (35). This it does by binding to the C-terminal domain of the RNAP ␣ subunit at a site bearing Tyr263, which is a highly conserved amino acid position in the RNAP ␣ of gram-positive bacteria. This interaction was determined to be responsible for the defects in growth and development observed in clpX and clpP mutants. Spx exhibited no DNA-binding activity in vitro.High concentrations of Spx also resulted in the induction of genes whose products function in thiol homeostasis and the biosynthesis of cysteine, as shown by probing B. subtilis RNA with genomic microarrays (34). The trxA and trxB genes, encoding thioredoxin and thioredoxin reductase, respectively, are transcriptionally activated under conditions of oxidative stress by a mechanism that involves Spx-RNAP interaction. Reconstitution of transcription initiation in vitro demonstra...

Crystal structure of theBacillus subtilisanti-alpha, global transcriptional regulator, Spx, in complex with the α C-terminal domain of RNA polymerase

“…2 A). The possibility that this sulfate-binding site has biological relevance has been bolstered by recent findings of Zuber and coworkers (30) that reveal that when cells are grown with sulfate as the sole sulfur source, Spx regulates transcription of sulfur assimilation genes. Perhaps this conserved RPI motif is involved in both modulating the reactivity of C10 or C13 and binding sulfate in vivo.…”

The Global Regulator Spx Functions in the Control of Organosulfur Metabolism inBacillus subtilis

“…Spx was found to negatively affect the transcription of genes that function in the utilization of organosulfur compounds as alternative sources of sulfur (13). Such operons are repressed in minimal glucose media containing either of the two preferred sulfur sources, sulfate and cysteine.…”

“…These operons are derepressed when cysteine or sulfate are replaced with methionine or an organosulfonate as sole sulfur source (8,44,46). The ytmI, yxeI, and ssu operons are derepressed in sulfate medium when cells bear an spx null mutation (13).…”

“…1). Both CymR and Spx exert negative control of the ytmI, yxeI, and ssu operons in the presence of sulfate, while only CymR is required for negative control in cysteine medium (13,14) (see below).…”

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.…”