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.