多效调控基因degU32(Hy),degQa和degR对枯草芽孢杆菌Ki-2-132生长和蛋白酶发酵的影响

通过向枯草芽孢杆菌Ki-2-132染色体和／或细胞质导入来自枯草杆菌168菌株的degU32（Hy）和degR基因,以及来自芽孢杆菌解淀粉菌株（Bacillus amyloliquefaciens）的degQa基因,对上述基因对枯草芽孢杆菌Ki-2-132细胞的生长、孢子发生、蛋白酶发酵的影响进行了研究。尽管上述多效调控基因来自不同的芽孢杆菌种和菌株,它们在枯草芽孢杆菌Ki-2-132中依然表现多效性。枯草杆菌Ki-2-132degU32（Hy）表现出增高了的蛋白酶产量;当和质粒或染色体上的degQa基因协作,可以进一步依赖葡萄糖的水平和degQa的基因剂量影响细胞生长,增加蛋白酶产量,以及影响孢子的形成。与此不同,degR在degU32（Hy）突变体中并不显著影响其蛋白酶的产量,这一发现支持DegR蛋白通常稳定磷酸化的DegU,而其在degU32（Hy）菌株中不再进一步放大该突变体内已被磷酸化的DegU的调控作用。     

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.     

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.     

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

Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR. To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases. The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect. The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination. The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region. It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR. We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU. Possible involvement of DegR in this process is discussed.     

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.     

The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis.

Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.     

Stress related changes of cell surface hydrophilicity in Bacillus subtilis

The changes of cell surface hydrophilicity in Bacillus subtilis were analyzed in response to oxygen-limitation, heat shock, salt stress, pH-shock, phosphate- and carbon-limitation. Although cell surface hydrophilicity varied during growth phases, an increase of surface hydrophilicity was observed under several of these stress conditions. An observed drop in intracellular GTP and/or ATP may be an element of the signal transduction pathway leading to an increase in surface hydrophilicity in response to environmental stresses. Attachment of cells to soil particles under salt stress conditions is strongly influenced by the degS/degU two-component system, which thereby provides a mechanism for the bacteria to escape from the hostile environment.     

Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases

A mutant strain of Bacillus subtilis carrying lesions in the structural genes for extracellular neutral (nprE) and serine (aprA) proteases was constructed by the gene conversion technique. This mutant had less than 4% of the extracellular protease activity of the wild type and sporulated normally, indicating that neither of these sporulation-associated proteases is essential for development.     

Effects of degU32(Hy), degQa and degR Pleiotropic Regulatory Genes on the Growth and Protease Fermentation of Bacillus Subtilis Ki-2-132

Effects of degU32 (Hy), degR genes from Bacillus subtilis 168 and deg Qa gene from Bacillus amyloliquefaciens on Bacillus subtilis Ki-2-132 cell growth, sporulation and protease fermentation were investigated by introducing these genes into B. subtilis Ki-2-132 chromosome and/or cytoplasm. Although the genes come from different species and strains, they showed pleiotropic effects in B. subtilis Ki-2-132. B. subtilis Ki-2-132degU32 (Hy) showed increased protease production, and when cooperating with deg Qa either in plasmid or in chromosome, further altered cell growth, increased protease production and affected the spore formation in a glucose and dosage dependent manner. By contrast, degR did not significantly affect the protease productivity in degU32 (Hy) mutant, consisting with that DegR was used to stabilise DegU-phosphate, which in degU32 (Hy) strain no longer further amplify the DegU-phosphate effect.     

Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin

Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.