Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis

Abstract

The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx–RNA polymerase (Spx–RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx–RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx–RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended −35 box in which nucleotide composition at the −43/−44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Evidence that Oxidative Stress Induces spxA2 Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

“…When the ratios of transcript band intensities of reactions with or without added SpxA1/SpxA2 were measured (see Table S5 in the supplemental material), stimulation of transcript synthesis was generally stronger in reaction mixtures containing the Ϫ100 PspxA2 template compared to transcription from the Ϫ50 PspxA2 DNA, although there was considerable variation in the level of transcript measured in the Ϫ50 PspxA2 reactions due to the lower activity of the shortened template DNA. In sharp contrast to the previously reported results of B. subtilis Spx activity in IVT reaction mixtures containing B. subtilis RNAP, which showed the requirement for the Ϫ43 AGCA element (10,30,31), no such element is found in the region between positions Ϫ35 and Ϫ50 in PspxA2. Removal of the upstream AGCA motif by deletion to Ϫ90 did not affect transcription activated in vitro by either SpxA1 or SpxA2.…”

“…The promoter region of the major transcriptional start site of spxA2 resembles those recognized by the A form of RNAP, with the start site of transcription located 223 bp from the ATG start codon (41). Previous studies of Spx in B. subtilis uncovered the importance of an AGCA motif centered at approximately position Ϫ44 of Spx-controlled promoters as a possible cis-acting element (30,31), which was supported by genomewide Spx-DNA binding studies (10) and by nucleotide-specific protein-DNA cross-linking studies (37). The spxA2 promoter region does not possess the AGCA element in the vicinity of the Ϫ35 core promoter element.…”

“…Previous studies of B. subtilis Spx showed that the likely target of Spx-promoter interaction is the AGCA motif, which is usually centered around position Ϫ43 with respect to the start site of Spx-activated transcription (10,30,31). No sequence upstream of the spxA2 promoter in the region between positions Ϫ50 and Ϫ35 was detected that resembled an Spx targeted cis-acting element, although an AGCA was detected between positions Ϫ100 and Ϫ90.…”

“…For example, in Bacillus anthracis, the causative agent of the zoonotic infectious disease, anthrax, the plasmid-borne atxA gene encodes a transcription factor required for virulence, since it is necessary for the activation of genes for toxin and protective capsule production (5,6). Other Grampositive bacteria possess genes encoding one or more forms of the ArsC (arsenate reductase) family protein, Spx, which is an RNA polymerase (RNAP)-binding protein directing the expression of large regulons that include genes necessary for pathogenesis (7)(8)(9)(10)(11). Studies of streptococci, Listeria monocytogenes, Staphylococcus aureus, and Enterococcus spp.…”

“…However, mutational analysis of Spx-controlled promoter DNA and nucleotide-specific protein-DNA cross-linking provided evidence that RNAP-bound Spx contacts a conserved cisacting element in B. subtilis (10,30,31,37). The Spx-targeted cis-acting site, AGCA centered around position Ϫ44 upstream of the transcription start site, was detected in genome-wide chromatin immunoprecipitation studies (10).…”