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 Sy-glutamyl kinase was followed by the proA gene encoding glutamyl--y-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part ofproA 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, y-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.
The production of BaciUus subtilis extracellular proteases is under positive and negative regulation. The functional role of degR, one of the positive regulators, was studied in relation to the degS and degU gene products, which belong to the bacterial two-component regulatory system. Studies with a translational fusion between the Escherichia coli lacZ and the Bacillus subtilis subtilisin (aprE) genes indicated that the stimulatory site of DegR lay upstream of position-140, with the region upstream of position-200 being the major target. It was also found that degS and degU were epistatic to degR. These results suggested some relationship among the degR, degS, and degU gene products. The DegR protein was purified to homogeneity, and its in vitro effect on the phosphorylation reaction involving DegS and DegU was studied. For this purpose, a soluble-extract system in which the formation and dephosphorylation of DegU-phosphate could be examined was devised. The addition of DegR to the soluble-extract system enhanced the formation of DegU-phosphate. The enhancing effect was found to be due to the protection of DegU-phosphate from dephosphorylation. From these results, it was concluded that the positive effect of DegR on the production of the extracellular proteases is brought about by the stabilization of DegU-phosphate, which in turn may result in the stimulation of transcription of the exoprotease genes.
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 that 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 overproduction phenotype.
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