Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis

Abstract

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands Al to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin proffle was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands Al and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.Despite abundant speculation, the role of bacterial autolysins during cell growth and division has remained elusive. All bacteria apparently possess a complement of potentially lethal autolysins capable of hydrolyzing the cell wall peptidoglycan (13). Bacillus subtilis 168 has two major vegetative autolysins, a 50-kDa amidase and a 90-kDa glucosaminidase, which have been purified and characterized (17, 31). During sporulation, two distinct lytic enzymes, an amidase and an endopeptidase, have been identified but not studied in detail (14). A 30-kDa amidase has also been identified, and the gene has been cloned and studied at the molecular level (9, 22). The specific function of all of these enzymes is at present unknown, as is the total number of B. subtilis 168 autolytic enzymes. Work mainly using regulatory mutants with reduced autolysin levels (lyt) has implicated them in several important cellular functions, including cell wall turnover, cell separation, flagellation, competence, and the lytic action of penicillin (8,28,30). The regulatory mutants sigD and sin share a common filamentous and Lyt-phenotype, and the lack of cell separation has been proposed to be due to reduced expression of autolysin genes (12,16,25,32). In some lyt mutants, however, the rate of wall turnover was dependent on the salt concentration, and the mutants showed levels of wall turnover comparable to that of the wild type when they were grown in high salt concentrations (38). Because of the possible functional redundancy (9) and compensatory effect of multiple autolysins, it is important to identify and characterize the total set of such enzymes present during growth and development in order to be able to assess their involvement in any given cellular process.The ability of autolysins to renature after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has allowed their activity to be studied ...

Selective Roles for Toll-Like Receptor (TLR)2 and TLR4 in the Regulation of Neutrophil Activation and Life Span

“…Purified LPS (pLPS) from E. coli strain K235 was prepared as described (4). Peptidoglycans from Staphylococcus aureus 8325-4 and Bacillus subtilis 168 HR were made as previously described (22,23), quantified by dry weight, and stored at Ϫ20°C. Pathway inhibitors were from Calbiochem (CN Biosciences, Beeston, U.K.) and comprised SB203580 (p38 MAPK inhibitor), SN50 (NF-B inhibitor) and SN50 M control peptide, PD98059 (MAPK kinase inhibitor preventing extracellular signal-regulated kinase activation), herbimycin A (tyrosine kinase inhibitor), and LY294002 (phosphatidylinositol 3-kinase (PI-3K) inhibitor).…”

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

“…A 30-kDa amidase gene, cwl4, has been cloned, but its expression is very low under normal growth conditions (11,23). Foster demonstrated novel cell wall lytic activities of A3 and A4 , which increases during sporulation, and also sporulation-specific activities of A5 and A6 by means of renaturing gel electrophoresis in substrate-containing gels (12). The physiological roles of these multiple autolysins, i.e., cell wall turnover (2,5), cell separation (5, 10, 37), flagellation (10), and competence (3), have been suggested by many investigators, but there was little evidence because they used regulatory mutants [flaD (sin, lyt) and sacU (Hy)] with reduced autolysin levels (26).…”

“…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). A 30-kDa amidase gene, cwl4, has been cloned, but its expression is very low under normal growth conditions (11,23).…”

“…The physiological roles of these multiple autolysins, i.e., cell wall turnover (2,5), cell separation (5, 10, 37), flagellation (10), and competence (3), have been suggested by many investigators, but there was little evidence because they used regulatory mutants [flaD (sin, lyt) and sacU (Hy)] with reduced autolysin levels (26). Asymmetric septum peptidoglycan hydrolysis (17,30), which is a morphogenic transition between sporulation stages II and III, cortex maturation (12,15), mother-cell lysis (12), and germination (12) would be subject to the actions of these autolysins. However, the specific functions of individual autolysins of B. subtilis remained unknown except that vegetative cell wall turnover is apparently decreased in a cwlB(lytC)-…”

Staphylococcus aureus peptidoglycan impairs fracture healing: An experimental study in rats

“…The rats were allocated to three groups: (1) local group; injection of purified SaPG in a dose of 1 mg/kg 12 at the fracture site, and injection of a corresponding amount (0.2 mL) isotonic saline intraperitoneally each day during 7 days. (2) Intraperitoneal group; injection of purified SaPG in a dose of 1 mg/kg intraperitoneally (systemically) and 0.2 mL saline at the fracture site each day during 7 days.…”