REGULATION OF A NEW CELL-WALL HYDROLASE GENE, CWLF, WHICH AFFECTS CELL-SEPARATION IN BACILLUS-SUBTILIS

Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwl B sigD mutant cells and separated by sodium dodecyl sulfate-polyacryla mide gel electrophoresis. N-terminal amino acid sequencing revealed th at its sequence is completely identical to that of the internal region of the papQ gene product. Disruption of the papQ gene in the B. subti lis chromosome led to the complete loss of CwlF, indicating that papQ is identical to cwlF. CwlF exhibits high sequence similarity to the p6 0 proteins of Listeria species, NlpC proteins of Escherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The P -galactosidase activity of the cwlF-lacZ transcriptional fusion and No rthern blot analysis of the cwlF gene indicated that the gene is expre ssed as a monocistronic operon during the exponential growth phase, an d primer extension analysis suggested that the crt IF gene is transcri bed mainly by E sigma A RNA polymerase and weakly by E sigma H RNA pol ymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to th e filamentation of the sigD mutant. The CwlF production was not affect ed by the pleiotropic mutations flaD1 and degU32(Hy), which endow cell s with the ability of extensive filamentation.