Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase.

The DegS-DegU protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the DegU protein. Phosphoryl transfer to the wild-type DegU protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the degU24 and degU31 alleles presented a stronger phosphorylation signal compared with that of the wild-type DegU protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the degU24 and degU31 mutants. However, the degU32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified DegU response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the degU32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the degU32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, degU122 and degU146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable DegU proteins, respectively.     

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

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 extracellular 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 degS100(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, degS100(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.     

Stabilization of the phosphorylated form of Bacillus subtilis DegU caused by degU9 mutation

Abstract A Bacillus subtilis response regulator, DegU9, carrying an amino acid alteration caused by the degU9 (Hy) mutation was partially purified, and phosphorylation and dephosphorylation of the protein was studied. The extent of phosphorylation was not as high as the level attained with wild-type DegU, but the DegU9-phosphate once formed was more stable than the wild-type DegU-phosphate. An in vivo study with a degU9 mutant showed that degS was necessary for the overproduction of exoproteases. These results suggest that phosphorylation is necessary for the mutant DegU9 to exert its effect and that the higher stability of phosphorylated DegU9 is responsible for the overproulation phenotype.     

Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU.

The rates of synthesis of a class of both secreted and intracellular degradative enzymes in Bacillus subtilis are controlled by a signal transduction pathway defined by at least four regulatory genes: degS, degU, degQ (formerly sacQ), and degR (formerly prtR). The DegS-DegU proteins show amino acid similarities with two-component procaryotic modulator-effector pairs such as NtrB-NtrC, CheA-CheY, and EnvZ-OmpR. By analogy with these systems, it is possible that DegS is a protein kinase which could catalyze the transfer of a phosphoryl moiety to DegU, which acts as a positive regulator. DegR and DegQ correspond to polypeptides of 60 and 46 amino acids, respectively, which also activate the synthesis of degradative enzymes. We show that the degS and degU genes are organized in an operon. The putative sigma A promoter of the operon was mapped upstream from degS. Mutations in degS and degU were characterized at the molecular level, and their effects on transformability and cell motility were studied. The expression of degQ was shown to be subject both to catabolite repression and DegS-DegU-mediated control, allowing an increase in the rate of synthesis of degQ under conditions of nitrogen starvation. These results are consistent with the hypothesis that this control system responds to an environmental signal such as limitations of nitrogen, carbon, or phosphate sources.     

Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.     

Biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius under salt stress conditions

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3-19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 M NaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32 (Hy), which provides for the over-synthesis of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3-19 increased by 6-10 times. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.     

Bacillus subtilis two-component system sensory kinase DegS is regulated by serine phosphorylation in its input domain

Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity. The phosphorylation state of the response regulator DegU also does not confer a straightforward "on/off" response; it is fine-tuned and at different levels triggers different sub-regulons. Here we describe serine phosphorylation of the DegS sensing domain, which stimulates its kinase activity. We demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number of physiological assays focused on different processes regulated by DegU, DegS S76D phosphomimetic mutant behaved like a strain with intermediate levels of DegU phosphorylation, whereas DegS S76A behaved like a strain with lower levels of DegU phophorylation. These findings suggest a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system.     

Biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius under salt stress conditions

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3–19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 MNaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32(Hy), which provides for the overproduction of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3–19 increased by 6–10 fold. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.     

Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU‐P by ClpCP

The response regulator DegU and its cognate kinase DegS constitute a two‐component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU‐lacZ fusions, we showed that phosphorylated DegU (DegU‐P) binds to two direct repeats (DR1 and DR2) of the consensus DegU‐binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATP‐dependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild‐type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU‐P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU‐P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N‐terminal region of DegU is important for proteolysis.     

The spoOA and degU genes of Bacillus subtilis show genetic homology

Abstract The transformation efficiency of competent Bacillus subtilis degU32 (Hy) strains was found to depend on the marker that was selected. Protrophic transformants were obtained at frequencies similar to those in the wild type control, but Spo⁻ transformants oere rare also when a spoOA::erm insertion that produces a selectable marker (Erm^R) was used. The Erm^R transformants obtained within the degU32 (Hy) background were Spo⁺ and had lost the characteristics of the DegU(Hy) parental recipient strain i.e., secretion of exo-enzymes and sporulation resistance to catabolites. The spoOA::erm insertion was mapped to a location near degU . The similarities between the spoOA and degU sequences and the metabolic interferences between the mutated products which result in this unexpected recombination, are discussed.