Autolysin regulation in Bacillus subtilis: A study of the influence of protonmotive force on the regulation of cell wall autolytic enzyme activity.

摘要

Respiring cells of Bacillus subtilis maintain cell wall integrity by regulating their autolytic enzymes. The turnover rates of teichoic acid (TA)-containing and teichuronic acid (TUA)-containing walls are comparable, indicating autolysin function is similar. Several independent experiments suggest that cell walls of B. subtilis are protonated during growth. Cationized ferritin (CF) was used to probe cell surface protonation. Suspensions of B. subtilis cells containing either TA or TUA were aggregated with CF only after the addition of a protonmotive force-dissipating agent. Effects of protonmotive force and folding on autolytic activity were studied by adding concentrated autolysin to exponentially growing TA-containing and TUA-containing B. subtilis cells. Both TUA-containing and TA-containing cells lysed only after the addition of sodium azide. Fluorescence activated cell sorter (FACs) analyses of cells, the walls of which were saturated with fluorescein-labeled dextran, revealed cells purged with N₂ to dissipate protonmotive force (pmf) exhibited greater fluorescence intensities than did cells with a functioning pmf. Upon reconstitution of the pmf with phenazine methosulfate (PMS), glucose and oxygen, fluorescence declined. Comparable results were seen using a similar approach with confocal fluorescence microscopy. A different approach utilized pH-dependent chemical modification of cell walls. Results from two distinct pH-dependent reactions suggested the walls of respiring B. subtilis cells were protonated. This is the first study which shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes in both TUA-containing and TA-containing B. subtilis cells.; Finally the influence of protease on autolytic enzymes was investigated, as well as the role of protonmotive force in regulating cell wall proteases in Bacillus subtilis. The chain lengths of several strains of Bacillus subtilis were determined using wild type strains, protease-deficient strains, autolysin-deficient strains, and strains capable of secreting high amounts of protease. Glycan chain lengths of hyperprotease-producing strains and autolysin-deficient strains were determined to be substantially longer than the wild type strains. A crude extract of protease was prepared and its activity measured in a pH range of 3 to 10. Protease exhibited little activity in the pH range of 3–5, whereas activity reached a maximum in the pH range of 7—8 and leveled off near pH 10.