Transcription of Bacillus subtilis degR is sigma D dependent and suppressed by multicopy proB through sigma D

Abstract

Production of Bacillus subtilis exoproteases is positively regulated by the DegS-DegU two-component regulatory system and other regulatory factors including DegR and ProB. It was shown that the expression of degR was virtually abolished in a sigD mutant and that the transcriptional initiation site in vivo is preceded by a sequence very similar to the consensus sequence of D -recognized promoters. Alteration of the ؊10 sequence of the putative promoter greatly reduced the expression of degR. These results show that degR expression is driven by the alternative sigma factor, D . It was found that degR expression was suppressed by multiple copies of proB on plasmid pLC1 and that this suppression was exerted at the transcriptional level through a target in the vicinity of the degR promoter. Furthermore, it was shown that the expression of another D -directed gene, hag, was suppressed by pLC1. Suppression by pLC1 diminished when the sequence of the ؊10 element of the degR promoter was changed to a A -like promoter sequence. pLC1, however, did not suppress sigD expression. On the basis of these results, we conclude that multicopy proB on pLC1 inhibits transcription from D -driven promoters by affecting some posttranscriptional process of D .Bacteria grow exponentially in optimal conditions and finally reach the steady-state growth phase because of starvation of nutrients or high cell density. At the transition state from the exponential phase to the steady-state phase, bacterial cells are forced to choose one of several ways for their further survival.Bacillus subtilis has several choices to make, such as competence development, acquisition of motility, sporulation, and production of extracellular degradative enzymes, toward the end of the exponential growth phase (7,10,17,23). Such differentiation has been shown to be mediated by several specific regulatory proteins.One of these phenomena, the production of extracellular proteases, is positively regulated by a two-component regulatory system, DegS-DegU (2, 3, 10, 17, 18). In the essential degS-degU system, the sensor kinase, DegS, is thought to accept certain environmental stimuli, autophosphorylate on its own histidine residue, and transfer the phosphate to the aspartate residue of the cognate response regulator DegU (2,17,18,24). DegS is also involved in the dephosphorylation of the phosphorylated DegU (3,27). In the regulation of the exoprotease production, other regulatory factors including DegR (19,20,28,31) and ProB (21) act in concert with the DegS-DegU system. We have reported that multiple copies of the B. subtilis proB gene on pLC1 encoding ␥-glutamyl kinase show a synergistic effect on the production of the exoproteases when degR is carried on the multicopy plasmid pNC61 (21). This effect of multicopy proB on plasmid pLC1 is dependent on degS, and we postulated that the metabolic intermediate, ␥-glutamyl phosphate synthesized by ProB, might transmit a signal to DegS, resulting in a higher level of phosphorylated DegU (21).Acquisition of motility requires co...

A new Bacillus subtilis gene, med, encodes a positive regulator of comK

“…The transcription of degR is dependent on the alternative sigma factor D (35). We have shown that neither mecA nor mecB null mutations affect the expression of sigD, the gene for D , and the reduced expression of degR in mec null mutants is recovered in mecA comK or mecB comK double mutants, indicating that ComK is responsible for the repression of degR (32).…”

“…DegR is a 60-amino-acid polypeptide with homology to the N-terminal region of DegS (36), and it has been shown that multiple copies of degR stabilize the phosphorylated form of DegU (28). Transcription of degR is dependent on D , a sigma factor involved in chemotaxis, suggesting interaction between the pathways controlling induction of chemotaxis genes and regulation of exoprotease production (35).…”

“…RNA was isolated as previously described (35). DNA labeling by PCR was done by using a PCR DIG (digoxigenin) Probe Synthesis kit (Boehringer Mannheim) and oligonucleotides Med-1 and Med-2 ( Table 2) as primers.…”

“…The procedure for primer extension analysis of degR and the primer used were described previously (35).…”

Identification of the Sequences Recognized by theBacillus subtilisResponse Regulator YrkP

“…The pIS-yrkOM3 plasmid was constructed by site-directed mutagenesis using an oligonucleotide-based PCR method described previously (yrkO-pISB1, yrkO-M1, yrkO-M2 and yrkOpISH). 22) To construct pIS-yrkP1, pIS-yrkP2, pIS-yrkP3, (18). b Transcription of yrkP on pDG-yrkP is induced by IPTG.…”