Altered phosphorylation of Bacillus subtilis DegU caused by single amino acid changes in DegS

Abstract

The Bacillus subtilis sacU locus consists of the degS and degU genes, which play a major role in controlling the production of degradative enzymes including extraceilular proteases. DegS has been shown to be autophosphorylated and to transfer the phosphoryl group to DegU. In this study, we partially purified the DegS proteins which carry amino acid changes resulting from various mutations and examined the phosphorylation reaction. The mutations used were degS42, causing a reduction in exoprotease production, and degSlOO(Hy) and degS200(Hy), causing overproduction of the enzymes. The following results were obtained. The DegS protein derived from degS42 was deficient in both autophosphorylation and subsequent phosphate transfer to DegU. Compared with wild-type DegS, the DegS proteins derived from the overproduction mutations, degSlOO(Hy) and degS200(Hy), were less active in the autophosphorylation and phosphorylation of DegU. However, the DegU phosphates produced by the mutant DegS proteins were more stable than that produced by the wild-type DegS. These results suggest that phosphorylation is tightly linked to exoprotease production and that the prolonged retention of the phosphoryl moiety on DegU activates the genes for the extracellular proteases. It was also shown that the rate of dephosphorylation of DegU-phosphate was increased as the amount of DegS was increased. All of these results suggest that DegS is involved in the dephosphorylation of DegU-phosphate.Bacillus subtilis secretes a variety of degradative enzymes such as a-amylase, proteases, and levansucrase, etc., during or after the end of the logarithmic phase of growth. The production of extracellular proteases (20,22), an intracellular serine protease (30), and levansucrase (20) is under the control of the sac U locus, which consists of two genes, degS and degU (11,19,34). These two genes constitute an operon and encode the DegS and DegU proteins, whose amino acid sequences have homologies with those of the sensor and effector proteins, respectively, of the bacterial two-component regulatory system (29, 32). In Escherichia coli, the sensor proteins, CheA, EnvZ, NtrB, and PhoR, have been shown to be autophosphorylated in the presence of ATP and transfer the phosphoryl group to the effector proteins, CheY and CheB (12, 13, 37), OmpR (1,7,14), NtrC (18,26,36), and PhoB (23), respectively. Recently, the VirA-VirG system of Agrobacterium tumefaciens was also shown to involve phosphorylation (16). The B. subtilis DegS-DegU system resembles the E. coli EnvZ-OmpR, NtrB-NtrC, and PhoR-PhoB systems in that they all activate transcription of the target genes (9,10,23,26,28).We previously reported the purification of the B. subtilis DegS and DegU proteins and showed that DegS was phosphorylated in the presence of [-y-32P]ATP and transferred the phosphoryl group to DegU (25). It was also demonstrated that a deletion of degS resulted in reduced production of the exoproteases, suggesting that phosphorylation of DegU by DegS is involved in the exoprotease productio...

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

“…We therefore investigated whether the suppression of degR expression by pLC1 also requires DegS. Strain ODM601 is isogenic to strain ODM50 except that it carries the degS42 mutation, a mutation that causes deficiency in autophosphorylation of DegS and subsequent phosphorylation of DegU (27). Results showed that pLC1 had no inhibitory effect on the transcription of degR in the degS42 background (Fig.…”

“…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.…”

Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis

“…DegS is also involved in the dephosphorylation of the phosphorylated DegU (6,28). It has been demonstrated that the phosphorylated form of DegU is stabilized by multiple copies of degR (29) or mutations that cause overproduction of the exoproteases, such as degSlOO(Hy), degS200(Hy), degU32(Hy), and degU9(Hy) (6,7,41,43).…”

A pivotal role for the response regulator DegU in controlling multicellular behaviour

“…DegS is a cytoplasmic bifunctional protein that exhibits kinase and phosphatase activities (Tanaka et al, 1991). DegS interacts with the SMC-ScpA-ScpB complex, which controls DNA condensation and repair.…”