High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation

Abstract

Transcription of the major BaciUlus subtilis autolysin gene (cwlB) was investigated. Deletion of the region upstream of the gene cluster 1ppX-cwbA-cwlB led to a loss of promoter activity. Primer extension analysis suggested that the cwlB operon is transcribed by Eo&E and Eo'A, the former transcripts being predominants at the exponential growth phase. Expression of the lppX-lacZ fusion gene was reduced by about 90% in a sigD-null mutant. A sin (flaDi) mutation caused a severe defect in transcription of the lppX-cwbA-cwlB operon. The sin (flaDi) mutation also reduced expression of a sigD-lacZ fusion gene constructed in the B. subtilis chromosome. Since the sigD-null mutant exhibits motility and autolysin deficiencies and filamentation, similar phenotypes in the sin (faDI) mutant may be caused by reduction in expression of the o'r protein.Peptidoglycan hydrolases are believed to be involved in important biological processes such as cell wall turnover (4,7,18,51) and cell separation (7,9,39) and to act as pacemaker and spacemaker enzymes for cell growth (51). Some of the peptidoglycan hydrolases can act as autolysins, thereby appearing to be potential suicide enzymes. This life and death dichotomy of function demands efficient and strict regulation of peptidoglycan hydrolase activity (18).Bacillus subtilis produces two autolysins, N-acetylmuramoyl-L-alanine amidase (the major autolysin) and endo-p-N-acetylglucosaminidase (glucosaminidase) (16,40). During sporulation, two other cell wall hydrolases, sporulationspecific amidase (s-amidase) and -y-D-glutamyl-L-mesodiaminopimelyl endopeptidase, are formed; the hydrolases are thought to be associated with the formation of cortex peptidoglycans (13). Some B. subtilis strains (YT-25 and K-77) produce extracellular endo-,B-N-acetylmuramidases (21, 34, 35). We previously reported the cloning of the cell wall hydrolase gene, cwlA (24). The product of this gene is not a major autolysin but an N-acetylmuramoyl-L-alanine amidase (10, 22) that is highly active on spore cortex peptidoglycans (10). Recently, we cloned and sequenced the major autolysin gene, cwlB, a mutant deficient in which is extremely resistant to cell lysis even after prolonged incubation (25 (6,9,30,41,47,50). The sigD and div-341 mutations are identical to the flaB and secA mutations, respectively (30, 42). The sacU(Hy) mutations, which map to adjacent genes (degU and degS), cause an increase in the production of degradative enzymes, poor development of competence, and lack of flagella (loss of motility) (8,33,37). The flaD (lyt) mutation causes similar * Corresponding author.phenotypes (an increase in alkaline protease, filamentous cell morphology, poor development of competence, and lack of flagella) (2, 36). Recently, we demonstrated that the flaD (lyt) gene is identical to the sin (sporulation inhibition) gene (11,45,46). Smith and colleagues found that Sin is a 14-kDa dual-function DNA-binding protein which has a negative function in sporulation (spoIL4, spoIIF, and spoIIG) and in aprE (major alkaline pr...

Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis

“…In particular, several autolysins such as lytC (cwlB), lytD (cwlG), and lytF (cwlE) are transcribed by Eσ D RNA polymerase (Kuroda and Sekiguchi 1993;Lazarevic et al 1992;Margot et al 1994;Ohnishi et al 1999;Rashid et al 1995). Thus, it is predicted that the transcription of cwlK may depend on Eσ A and/or Eσ D RNA polymerases because cwlK is transcribed during the vegetative growth phase (Fig.…”

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

“…Overexpression of sinR inhibits the production of extracellular proteases and represses sporulation by preventing expression of spo0A and stage II sporulation genes (spoIIA, spoIIG and spoIIE) (Gaur et al, 1986 ;Mandic-Mulec et al, 1992. Inactivation of sinR results in a loss of competence and motility, in a deficiency of autolysin production, and in filamentation (Kuroda & Sekiguchi, 1993 ;Olmos et al, 1997). The SinI protein regulates SinR activity by protein-protein interaction, preventing SinR from binding to its target DNA sequence (Bai et al, 1993).…”

Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis

“…The cwlB operon consists of three genes encoding a putative lipoprotein (LppX), a modifier protein (CwbA) that stimulates amidase activities, and CwlB, in that order (22,24,26,29). The transcription of the cwlB operon mainly depends on expression of the eD protein, which is responsible for cell motility and chemotaxis (26,32). Other cell wall hydrolases of B. subtilis have been described previously by us (23) and Foster (11,12).…”

“…The former occurs in large amounts (16,24), and the gene, cwlB, has recently been cloned and studied at the molecular level (24,29). The cwlB operon consists of three genes encoding a putative lipoprotein (LppX), a modifier protein (CwbA) that stimulates amidase activities, and CwlB, in that order (22,24,26,29). The transcription of the cwlB operon mainly depends on expression of the eD protein, which is responsible for cell motility and chemotaxis (26,32).…”