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Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals.

ABSTRACT: sigma B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase. Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine. The sigma B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control sigma B activity in response to environmental stress. However, none of these is sufficient to explain the enhanced stationary-phase activation of sigma B in response to glucose. We show here that the four genes previously identified in the sigB operon constitute the downstream half of an eight-gene operon. The complete sigB operon is preceded by a sigma A-like promoter (PA) and has the order PA-orfR-orfS-orfT-orfU-PB-rsbV-rsbW-sig B-rsbX, where rsb stands for regulator of sigma-B and the previously identified sigma B-dependent promoter (PB) is an internal promoter preceding the downstream four-gene cluster. Although the genes downstream of PB were also transcribed by polymerase activity originating at PA, this transcription into the downstream cluster was not essential for normal induction of a sigma B-dependent ctc-lacZ fusion. However, deletion of all four upstream open reading frames was found to interfere with induction of the ctc-lacZ fusion in response to glucose. Additional deletion analysis and complementation studies showed that orfU was required for full glucose induction of sigma B-dependent genes. orfU encodes a trans-acting, positive factor with significant sequence identity to the RsbX negative regulator of sigma B. On the basis of these results, we rename orfU as rsbU to symbolize the regulatory role of its product.

SUBMITTER: Wise AA

PROVIDER: S-EPMC176564 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals.

Wise A A AA Price C W CW

Journal of bacteriology 19950101 1

sigma B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase. Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine. The sigma B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control sigma B activi ...[more]

PMID: 8002610

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Project description:Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

Project description:Gene arrays containing all currently known open reading frames of Bacillus subtilis were used to examine the general stress response of Bacillus. By proteomics, transcriptional analysis, transposon mutagenesis, and consensus promoter-based screening, 75 genes had previously been described as sigma(B)-dependent general stress genes. The present gene array-based analysis confirmed 62 of these already known general stress genes and detected 63 additional genes subject to control by the stress sigma factor sigma(B). At least 24 of these 125 sigma(B)-dependent genes seemed to be subject to a second, sigma(B)-independent stress induction mechanism. Therefore, this transcriptional profiling revealed almost four times as many regulon members as the proteomic approach, but failure of confirmation of all known members of the sigma(B) regulon indicates that even this approach has not yet elucidated the entire regulon. Most of the sigma(B)-dependent general stress proteins are probably located in the cytoplasm, but 25 contain at least one membrane-spanning domain, and at least 6 proteins appear to be secreted. The functions of most of the newly described genes are still unknown. However, their classification as sigma(B)-dependent stress genes argues that their products most likely perform functions in stress management and help to provide the nongrowing cell with multiple stress resistance. A comprehensive screening program analyzing the multiple stress resistance of mutants with mutations in single stress genes is in progress. The first results of this program, showing the diminished salt resistance of yjbC and yjbD mutants compared to that of the wild type, are presented. Only a few new sigma(B)-dependent proteins with already known functions were found, among them SodA, encoding a superoxide dismutase. In addition to analysis of the sigma(B)-dependent general stress regulon, a comprehensive list of genes induced by heat, salt, or ethanol stress in a sigma(B)-independent manner is presented. Perhaps the most interesting of the sigma(B)-independent stress phenomena was the induction of the extracytoplasmic function sigma factor sigma(W) and its entire regulon by salt shock.

Project description:Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σ(W) regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σ(X), σ(Y), and σ(M) regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σ(W)-regulated. Under these conditions, σ(W) exhibits a basal level of activity. Subsequently, we verified the σ(W)-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σ(W) anti-sigma factor RsiW and subsequent activation of the σ(W)-regulon. Taken together, our studies identify 89 genes as being strictly σ(W)-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σ(W)-dependent genes were relatively mild, which implies that σ(W)-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σ(W), but that this membrane protease also exerts other important post-transcriptional regulatory functions.

Dataset Information

Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals.

Publications

Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals.

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