Regulation of a New Cell Wall Hydrolase Gene, cwlF , Which Affects Cell Separation in Bacillus subtilis

Abstract

Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of thepapQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical tocwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins ofEscherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The β-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the cwlF gene is transcribed mainly by EςA RNA polymerase and weakly by EςH RNA polymerase. 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 the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutationsflaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

Post‐translational control of vegetative cell separation enzymes through a direct interaction with specific inhibitor IseA in Bacillus subtilis

“…In addition, the cell membrane of the elongated cells stained with FM 4-64 apparently exhibited a cell morphology comprising a chain-like structure with septa, but not the filamentous structure that was observed in septation defective cells. The morphology of long chained cells was similar to that of the D,L-endopeptidase-deficient cells (Ishikawa et al, 1998;Ohnishi et al, 1999;Yamamoto et al, 2003; Fukushima et al, 2006), strongly suggesting that IseA overexpression gives rise to a cell separation defect.…”

“…A slight difference between them is in the number of the LysM repeats, three for LytE, four for CwlS and five for LytF, respectively, and they have highly homologous catalytic domains (65%~72%). Besides, previous reports revealed that the transcription of the genes of these cell separation enzymes is controlled by RNA polymerases s A and s H for lytE, s D for lytF and s H for cwlS (Ishikawa et al, 1998;Ohnishi et al, 1999;Britton et al, 2002). Although these enzymes have similar domain structures, they are controlled by different s factors and expressed at different times.…”

“…In Escherichia coli cells, cell separation occurs concomitantly with septation, but in B. subtilis cells, cell septation and separation are uncoupled in the cell cycle. The vegetative cell separation event in B. subtilis is governed by these three D,L-endopeptidases, as mutants as to these genes exhibited aggregated microfibre formation (Ishikawa et al, 1998;Ohnishi et al, 1999;Fukushima et al, 2006). For the division of vegetative cells, these cell separation enzymes are specifically localized at cell separation sites and poles, which were previous septa (Yamamoto et al, 2003;Fukushima et al, 2006).…”

Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants

“…These hydrolases can be extracted from the cell wall with a high concentration of LiCl [5,6]. Many cell wall hydrolases consist of two domain structures: catalytic and cell wall binding (CWB) ones [7]. Previously, we reported that the CWB portion of the major autolysin, CwlB(LytC), of B. subtilis binds e¤ciently to the B. subtilis cell wall [8], and recently a fused protein (CWB-LipB) comprising the CWB domain and the B. subtilis extracellular lipase B was localized on the cell surface and found to retain lipase activity [9].…”

Production of a recombinant lipase artificially localized on the Bacillus subtilis cell surface

“…Amidases are the most common cell wall hydrolases and, on the basis of the amino acid sequence similarity in the catalytic domain of amidases, it is possible to divide them into three classes: class I, CwlA, CwlL, XlyA, XlyB, BlyA and a Bacillus sp. amidase; class II, CwlB(LytC), CwlC, CwlD and CwlM; class III, SleBs and CwlJ [6]. Non-catalytic domains are often found in amidases, and some of these domains contain two or three repeated amino acid sequences [47 ].…”