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

Abstract

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.

Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis

“…After addition of sodium chloride to the medium, B. subtilis cells were shown to accumulate proline (34). Moreover, Ogura and coworkers (26) showed that the presence of multiple copies of the proB gene, which is required for proline biosynthesis, led to enhanced aprE expression. The authors suggested that the proB gene product, ␥-glutamyl phosphate, could serve as a phosphodonor, leading to phosphorylation of DegS and to stimulation of degradative enzyme synthesis.…”

Isolation, characterization, and chromosomal location of a gene encoding the Δ1‐pyrroline‐5‐carboxylate synthetase in Arabidopsis thaliana

“…Sequence comparison with the databases were performed using BLAST through the NCBI E-mail server [18]. Protein secondary structure analysis was determined by using the PredictProtein program (EMBL Heidelberg, Germany) [19,20] with multiple sequence alignments of the seven proteins showing highest similarity, P5CS from V. aconitifolia [8], y-glutamyl kinase and y-glutamyl phosphate reductase from E. coli [13], S. marcescens [14] and Bacillus subtilis [21]. Inclusion of protein homologs increases the prediction accuracy of the secondary structure to 70% [19].…”

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

“…Strain JSB11 [D(proBA : : cat)1] was isolated after transformation of strain JH642 with DNA of plasmid pJS8 (linearized with the restriction enzymes BamHI and EcoRI) and subsequent selection for chloramphenicol-resistant transformants. Strain JSB17 [proA6pEPV1T (tet)] was isolated after transformation of strain JH642 with the circular plasmid pEPV1T (Ogura et al, 1994) and selection for tetracycline-resistant colonies; in this strain, plasmid pEPV1T is inserted into the proA gene via a Campbell-type single-crossover integration event, thereby disrupting the integrity of proA. The proA mutation present in strain JSB17 was introduced into strain FSB1 [(treA : : neo)1] (Spiegelhalter & Bremer, 1998) by DNA transformation with chromosomal DNA from strain JSB17 and subsequent selection for tetracycline-resistant colonies.…”