We have isolated a DNA fragment from BaciUus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities fbr the exoprotease production in the N-terninal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

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...

Studies were performed on the prtR gene which enhances the production of the Bacillus subtilis extracellular proteases and levansucrase, but not the a-amylase, RNase, and alkaline phosphatase. To investigate the mode of action of prtR, the Escherichia coli bla gene was placed under the control of two promoters. One was the promoter of the alkaline protease gene (aprE), and the other was the promoter of B. subtilis dihydrofolate reductase gene (dfrA). Expression of the bla gene was enhanced by prtR only when the apr promoter was used. From these results, it was concluded that the apr promoter or its vicinity was the target of pftR and that prtR does not affect the process after transcription. The mRNA levels of aprE and nprE (the neutral proteae gene) were significantly increased by prtR, but the half-life of the aprE mRNA was not affected. These results show that the prtR gene product enhances protease production by increasing the rate of transcription initiation.

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