Negative Regulation by the Bacillus subtilis GerE Protein

Abstract

GerE is a transcription factor produced in the mother cell compartment of sporulating Bacillus subtilis. It is a critical regulator of cot genes encoding proteins that form the spore coat late in development. Most cot genes, and the gerE gene, are transcribed by K RNA polymerase. Previously, it was shown that the GerE protein inhibits transcription in vitro of the sigK gene encoding K . Here, we show that GerE binds near the sigK transcriptional start site, to act as a repressor. A sigK-lacZ fusion containing the GerE-binding site in the promoter region was expressed at a 2-fold lower level during sporulation of wild-type cells than gerE mutant cells. Likewise, the level of SigK protein (i.e. pro-K and K ) was lower in sporulating wild-type cells than in a gerE mutant. These results demonstrate that K -dependent transcription of gerE initiates a negative feedback loop in which GerE acts as a repressor to limit production of K . In addition, GerE directly represses transcription of particular cot genes. We show that GerE binds to two sites that span the ؊35 region of the cotD promoter. A low level of GerE activated transcription of cotD by K RNA polymerase in vitro, but a higher level of GerE repressed cotD transcription. The upstream GerE-binding site was required for activation but not for repression. These results suggest that a rising level of GerE in sporulating cells may first activate cotD transcription from the upstream site then repress transcription as the downstream site becomes occupied. Negative regulation by GerE, in addition to its positive effects on transcription, presumably ensures that K and spore coat proteins are synthesized at optimal levels to produce a germination-competent spore.

Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

“…Transcription of a cotD-lacZ fusion is sevenfold higher in the wild type than in a gerE mutant (20). Our microarray results showed that whereas AWS144 (wild-type) RNA had cotD transcript at a 7.72-fold-higher level than the reference RNA, AWS133 (citB5 mutant) had cotD transcript at only a 1.12-foldhigher level than the reference.…”

“…Our microarray results showed that the sigK (spoIVCB) transcript was 3.33-fold more abundant in AWS133 (citB5 mutant) than in the reference RNA, while in AWS144 (wild type), spoIVCB was 1.44-fold more abundant than in the reference RNA. This result is reminiscent of the phenotype of a gerE mutant, which has twofold-higher sigK-lacZ expression than does the wild type (20). Since GerE-dependent repression of sigK inhibits accumulation of K during late stages of sporulation (20), we performed immunoblotting to analyze K levels in the wild-type strain and in AWS133.…”

“…GerE is not only a transcriptional activator but also a repressor of some genes (20). GerE represses sigK transcription, for instance.…”

“…First, synthesis of K is regulated not only by the earlier-acting factor, E , and by SpoIIID, a DNA binding protein, but also by excision of a prophage-like element, SKIN, that interrupts the coding sequence of the gene (26,33,41). K is then synthesized in a pro-form, which requires cleavage for activation (28) is required for transcription of gerE and other genes, many of which additionally require GerE as a positive regulator (12,20,21). Late in sporulation, GerE represses sigK transcription and eventually, at a higher protein concentration, represses transcription of many of the genes it previously activated (20,21).…”

“…K is then synthesized in a pro-form, which requires cleavage for activation (28) is required for transcription of gerE and other genes, many of which additionally require GerE as a positive regulator (12,20,21). Late in sporulation, GerE represses sigK transcription and eventually, at a higher protein concentration, represses transcription of many of the genes it previously activated (20,21). Based on in vitro assays, we hypothesize that aconitase binds to the gerE transcript late in sporulation and stabilizes it for translation, thereby increasing the rate and level of GerE protein accumulation.…”