Extracellular proteases increase tolerance of Bacillus subtilis to nafcillin

Abstract

Mutants of Bacillus subtilis capable of secreting high amounts of protease were highly tolerant to the lethal and lytic effects of nafcillin. Protease-deficient mutants were more susceptible. However, when subtilisin was added exogenously to a protease-deficient strain, the organism assumed the characteristics of nafcillin tolerance. Similarly, when phenylmethylsulfonyl fluoride, a serine protease inhibitor, was added to the tolerant strains, they became susceptible to nafcillin-induced lysis. The effects of nafcillin on B. subtilis were studied with both viability determinations and assay of cellular lysis. The minimum inhibitory concentrations of nafcillin tended to be higher for the protease hyperproducing strains, but these values could be reduced by the protease inhibitor. No loss of antibiotic activity was observed when nafcillin was incubated with either subtilisin or trypsin. Furthermore, protease and autolysin from B. subtilis were not modified by nafcillin. The results showed that extracellular proteases could render B. subtilis relatively tolerant to the killing and lytic effects of a cell wall antibiotic. The proteases were probably acting on the autolysins of the organism, thereby increasing tolerance to nafcillin.Autolytic enzymes have been implicated in several growth and division events in bacteria. Formation of a septum and subsequent cell separation appear to depend on a functional autolysin (6,18). Additional manifestations of autolytic activity in bacterial cells may be the ability to take up exogenous DNA (transformation) (16) and the turnover of cell wall components. Moreover, lysis in energy-depletedBacillus subtilis is a result of poorly regulated autolytic activity (10).In several different kinds of microorganisms, autolysin activity appears to be necessary for the killing and lytic effects induced by cell wall antibiotics such as penicillin (7,11,14). Other workers suggest that the susceptibility of bacteria to penicillin is unrelated to the level of autolysin (3). To clearly distinguish among penicillin-induced ceL killing, cellular lysis, cell wall turnover, and autolysin levels, a system is required in which the various parameters can be monitored and modified. We found that the addition of proteases and protease inhibitors had a marked effect on the rate of turnover of peptidoglycan in B. subtilis (9). Turnover is an expression of autolytic activity but does not necessarily reflect absolute levels of autolysins t Present address:

Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies

“…These data demonstrate the direct involvement of extracellular proteases in the control of cellular lysis in B. subtilis. The evidence provided here is in agreement with previous observations (Jolliffe et al 1980;Jolliffe et al 1982;Coxon et al 1991) and suggests that of the six extracellular proteases investigated, only NprE and AprE contribute significantly to the control of autolytic activity. These observations have important implications for the use of protease-deficient strains as hosts for the production of heterologous proteins; increased cellular lysis results in the liberation of intracellular proteases (and other macromolecules) which may contribute to degradation of the protein of interest.…”

“…It has been proposed that extracellular proteases are involved in the control of autolysis in B. subtilis by modulating the activity of autolytic enzymes (Jolliffe et al 1980). Mutants deficient in extracellular proteases show increased rates of peptidoglycan turnover and increased susceptibility to cellular lysis due to unchecked autolytic activity (Jolliffe et al 1980;Jolliffe et al 1982;Coxon et al 1991). The absence of an energized cytoplasmic membrane can also lead to cellular autolysis (Jolliffe et al 1981).…”

Characterization of Novel Mycobacterium tuberculosis and Mycobacterium smegmatis Mutants Hypersusceptible to β-Lactam Antibiotics

“…We propose that the ponA2 and pbpX genes encode enzymes used for the formation of a peculiar type of peptidoglycan cross-link whose pathway is not well understood but may be a new drug target. Mycobacteria produce two kinds of peptide cross-links in the cell wall; one is the common DAP-Ala linkage, also called a 4-3 linkage, catalyzed by ␤-lactam-sensitive D,D-transpeptidase enzymes, and the other is an unusual DAP-DAP linkage, also called a 3-3 linkage, believed to be catalyzed by ␤-lactaminsensitive L,D-transpeptidase enzymes (25,56). The role of the 3-3 linkages is unknown, although up to 25% of the peptides are linked in this fashion in the wall of M. smegmatis (56).…”

“…One proposed mechanism was derived from the observation that rates of peptidoglycan turnover in B. subtilis are greater in extracellular-protease-deficient mutants (26). Furthermore, a secreted-protease-hyperproducing strain of B. subtilis is resistant to nafcillin (25), suggesting a protease-dependent downregulation of autolytic enzymes. In this view, ExpA might be a protease that specifically degrades an autolytic enzyme, and the loss of ExpA may result in increased autolysin activity.…”

Optimization and production of salicylic acid by rhizobacterial strain <italic>Bacillus licheniformis </italic> MML2501

“…The above findings show that the possibility of using B. lichenformis MML2501 in a xenobiotic environment is high owing to its tolerance to antibiotics, heavy metals and fungicides. It was reported that the B. subtilis relatively tolerant to the killing and lytic effects of a cell wall antibiotic (Jolliffe et al, 1982). Yilmaz (2003) reported that the Bacillus circulans is very much tolerant to many heavy metals and antibiotics.…”