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 pro tein degrading bacterium, Bacillus stearothermophilus HA19, The cloned fragment stimulated production of a 37-kDa protease in B. subtilis, T he nucleotide sequence of the genes and their flanking regions were id entical 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 h ost 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. s ubtilis 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 en zyme was 37,000 Da by SDS-polyacrylamide gel electrophoresis and gel f iltration, The optimum pH and temperature for the enzyme activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable at pH 7 -10, The enzyme was inactivated by EDTA, but not by phenylmethyl sulfo nyl fluoride and diisopropyl fluorophosphate.