Bacillus stearothermophilus Cell Shape Determinant Gene, mreC and mreD, and Their Stimulation of Protease Production in Bacillus subtilis

Protease production stimulating genes were isolated from a soybean protein degrading bacterium, Bacillus stearothermophilus HA19. The cloned fragment stimulated production of a 37-kDa protease in B. subtilis. The nucleotide sequence of the genes and their flanking regions were identical to the B. subtilis cell shape determinant genes mreC and mreD [J. Bacteriol., 176, 6729-6742 (1992); J. Bacteriol., 176, 6717-6728 (1992)]. The mreC and mreD genes in B. subtilis stimulate secretion of a neutral protease (37-kDa), and the protease activity in the culture medium reached 2500 U per ml (approximately 10 times higher than the host strain) after 24 h of cultivation in L broth, suggesting the mreCD genes regulate protease expression and the protease is related to the cell shape determination in Bacilli. The protease productions in B. subtilis carrying mreC or mreD deletion plasmids were not elevated, so the 37-kDa protease stimulation requires both mreC and mreD genes. The extracellular protease was purified, and the molecular mass of the enzyme was 37,000 Da by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.0 and 50°C, respectively, and the enzyme was stable at pH 7-10. The enzyme was inactivated by EDTA, but not by phenylmethyl sulfonyl fluoride and diisopropyl fluorophosphate.