sigmaK can negatively regulate sigE expression by two different mechanisms during sporulation of Bacillus subtilis

Abstract

Temporal and spatial gene regulation during Bacillus subtilis sporulation involves the activation and inactivation of multiple sigma subunits of RNA polymerase in a cascade. In the mother cell compartment of sporulating cells, expression of the sigE gene, encoding the earlier-acting sigma factor, sigmaE, is negatively regulated by the later-acting sigma factor, sigmaK. Here, it is shown that the negative feedback loop does not require SinR, an inhibitor of sigE transcription. Production of sigmaK about 1 h earlier than normal does affect Spo0A, which when phosphorylated is an activator of sigE transcription. A mutation in the spo0A gene, which bypasses the phosphorelay leading to the phosphorylation of Spo0A, diminished the negative effect of early sigmaK production on sigE expression early in sporulation. Also, early production of sigmaK reduced expression of other Spo0A-dependent genes but not expression of the Spo0A-independent ald gene. In contrast, both sigE and ald were overexpressed late in development of cells that fail to make sigmaK. The ald promoter, like the sigE promoter, is believed to be recognized by sigmaA RNA polymerase, suggesting that sigmaK may inhibit sigmaA activity late in sporulation. To exert this negative effect, sigmaK must be transcriptionally active. A mutant form of sigmaK that associates with core RNA polymerase, but does not direct transcription of a sigmaK-dependent gene, failed to negatively regulate expression of sigE or ald late in development. On the other hand, the negative effect of early sigmaK production on sigE expression early in sporulation did not require transcriptional activity of sigmaK RNA polymerase. These results demonstrate that sigmaK can negatively regulate sigE expression by two different mechanisms, one observed when sigmaK is produced earlier than normal, which does not require sigmaK to be transcriptionally active and affects Spo0A, and the other observed when sigmaK is produced at the normal time, which requires sigmaK RNA polymerase transcriptional activity. The latter mechanism facilitates the switch from sigmaE to sigmaK in the cascade controlling mother cell gene expression.

FIG. 1

Effect of a sinR mutation on sigE-lacZ expression. The sinR null mutation in IS432 was transformed into wild-type cells (PY79 [●]), sigK (spoIVCB23) mutant cells (BK556 [○]), and spoIVCBΔ19 cells that produce ς^K earlier than normal (BZ48 [▵]). The resulting strains were lysogenized with phage SPβ::sigE-lacZ, and expression of lacZ during development was analyzed as described in Materials and Methods. In each of two separate experiments, the β-galactosidase specific activities were normalized to the average maximum specific activity in two isolates containing the sinR mutation in the wild-type background (typically 130 U). Points on the graph are averages of the normalized values (four determinations), and error bars show 1 standard deviation of the data.

FIG. 2

Effect of bypassing the phosphorelay on sigE-lacZ expression. The rvtA11 mutation was introduced into wild-type cells (PY79 [●]), sigK (spoIVCB23) mutant cells (BK556 [○]), and spoIVCBΔ19 cells that produce ς^K earlier than normal (BZ48 [▵]). The resulting strains were lysogenized with phage SPβ::sigE-lacZ, and expression of lacZ during development was analyzed as described in Materials and Methods. In each of two separate experiments, the β-galactosidase specific activities were normalized to the average maximum specific activity in two or three isolates containing the rvtA11 mutation in the wild-type background (typically 70 U). Points on the graph are averages of the normalized values (five determinations), and error bars show 1 standard deviation of the data.

FIG. 3

Effect of altered ς^K production on ald-lacZ expression. Wild-type cells (PY79 [●]), sigK (spoIVCB23) mutant cells (BK556 [○]), and spoIVCBΔ19 cells that produce ς^K earlier than normal (VO48 [▵]) were transformed with DNA from KI220 to introduce ald::Tn917lac. Expression of ald-lacZ was analyzed as described in Materials and Methods. In each of two separate experiments, the β-galactosidase specific activities were normalized to the average maximum specific activity in two isolates containing ald-lacZ in the wild-type background (typically 300 U). Points on the graph are averages of the normalized values (four determinations), and error bars show 1 standard deviation of the data.

FIG. 4

Production of transcriptionally inactive ς^K during sporulation. Wild-type PY79 cells (WT) and BZ410 cells engineered to produce transcriptionally inactive ς^K (C109R) were lysogenized with phage SPβ::sigE-lacZ and induced to sporulate in SM medium. Samples were collected at the indicated hours after the onset of sporulation. (A) Whole-cell extracts (5 μg) were subjected to Western blot analysis using anti-pro-ς^K antibodies as described previously (73). (B) β-Galactosidase specific activity from gerE-lacZ in wild-type (●) and sigKC109R mutant (○) cells.

FIG. 5

Effect of making transcriptionally inactive ς^K during sporulation on sigE-lacZ and ald-lacZ expression. Wild-type cells (PY79 [●]), sigK (spoIVCB23) mutant cells (BK556 [○]), and sigKC109R cells that produce transcriptionally inactive ς^K (BZ410 [□]) were lysogenized with phage SPβ::sigE-lacZ (A) or transformed with DNA from KI220 to introduce ald::Tn917lac (B). Expression of lacZ during development was analyzed as described in Materials and Methods. In each of two separate experiments, the β-galactosidase specific activities were normalized to the average maximum specific activity in two isolates containing the lacZ fusion in the wild-type background (typically 70 U for sigE-lacZ and 300 U for ald-lacZ). Points on each graph are averages of the normalized values (four determinations), and error bars show 1 standard deviation of the data.

FIG. 6

Effect on sigE-lacZ expression of making transcriptionally inactive ς^K earlier than normal during sporulation. The bofA::cat mutation in BSL50 was transformed into wild-type PY79 cells and BZ410 cells engineered to produce transcriptionally inactive ς^K, resulting in PS1 (○) and PS2 (□), respectively. These strains, and wild-type PY79 (●), were lysogenized with phage SPβ::sigE-lacZ, and expression of lacZ during development was analyzed as described in Materials and Methods. In each of two separate experiments, the β-galactosidase specific activities were normalized to the average maximum specific activity in two isolates containing sigE-lacZ in the wild-type background (typically 70 U). Points on the graph are averages of the normalized values (four determinations), and error bars show 1 standard deviation of the data.

FIG. 7

Model showing the mother cell sigma factor cascade and two different mechanisms by which ς^K can negatively regulate sigE expression. ς^K produced earlier than normal may compete with other sigma factors for binding to core RNAP (E) and inhibit formation of Spo0A∼P, an activator of sigE transcription. Transcriptional activity of Eς^K produced at the normal time during sporulation may inhibit Eς^A activity, reducing transcription of sigE, ald, and other early genes late in development.