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A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis.

ABSTRACT: To identify genes expressed at intermediate stages of Bacillus subtilis sporulation, we screened for sigma E-dependent promoters. One promoter that we found drives expression of an operon consisting of at least five open reading frames (ORFs). The predicted products of the first three ORFs are very homologous to enzymes involved in fatty acid metabolism, including acetyl coenzyme A (acetyl-CoA) acetyltransferase (thiolase), 3-hydroxybutyryl-CoA dehydrogenase, and acyl-CoA dehydrogenase, respectively. We showed that the fourth ORF encoded a third isozyme of citrate synthase in B. subtilis. Genetic evidence and primer extension results showed that transcription of this operon is directed by the mother cell compartment-specific sigma factor, sigma E, and so the operon was named mmg (for mother cell metabolic genes). Furthermore, we found that a sequence (mmgO) with homology to a catabolite-responsive element mediates glucose repression of mmg promoter activity during sporulation and that this repression was lost in a ccpA mutant.

SUBMITTER: Bryan EM

PROVIDER: S-EPMC178257 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis.

Bryan E M EM Beall B W BW Moran C P CP

Journal of bacteriology 19960801 16

To identify genes expressed at intermediate stages of Bacillus subtilis sporulation, we screened for sigma E-dependent promoters. One promoter that we found drives expression of an operon consisting of at least five open reading frames (ORFs). The predicted products of the first three ORFs are very homologous to enzymes involved in fatty acid metabolism, including acetyl coenzyme A (acetyl-CoA) acetyltransferase (thiolase), 3-hydroxybutyryl-CoA dehydrogenase, and acyl-CoA dehydrogenase, respecti ...[more]

PMID: 8759838

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Project description:The two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P).Growing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase.Under physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.

Project description:UnlabelledSpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell during Bacillus subtilis sporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor ?(G) Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of ?(G) The csfB gene is ordinarily transcribed in the forespore only by the early-acting sigma factor ?(F) However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28, csfB was also aberrantly transcribed later by ?(G), the very target of CsfB inhibition. This regulation of csfB by SpoIIQ Tyr-28 is specific, given that the expression of other ?(F)-dependent genes was unaffected. Moreover, we identified a conserved element within the csfB promoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes ?(G)activity in the forespore as a channel component but also specifically maximizes ?(G)activity as part of a gene regulatory circuit that represses ?(G)-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times.ImportanceCellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developing Bacillus subtilis spore is directed by the alternative sigma factor ?(G) The activity of ?(G)requires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes ?(G)activity as part of a previously unknown regulatory circuit that prevents ?(G)from activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developing B. subtilis spore.

Project description:RNA polymerase sigma factor sigma(F) initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. sigma(F) governs transcription of spoIIIG, encoding the late prespore-specific regulator sigma(G). However, transcription of spoIIIG is delayed relative to other genes under the control of sigma(F), and after synthesis, sigma(G) is initially kept in an inactive form. Activation of sigma(G) requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of sigma(G). We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with sigma(G). Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with sigma(G). The sigma(GE156K) isoform restored sigma(G)-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, beta-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of sigma(GE156K) is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of sigma(G) in the prespore.

Dataset Information

A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis.

Publications

A sigma E dependent operon subject to catabolite repression during sporulation in Bacillus subtilis.

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