muramidase and dodecylamine

Previous work reported that decoated Bacillus cereus spores incubated in 4 mol l(-1) CaCl2 are killed at lower temperatures than spores in water. This wet heat sensitization was suggested to support a role for an osmoregulatory peptidoglycan cortex in spore cores' low water content, and their wet heat resistance. Current work has replicated this finding with spores of B. cereus, Bacillus megaterium and Bacillus subtilis. However, this work found that decoated spores apparently killed at 80°C in 4 mol l(-1) CaCl2 : (i) were recovered on plates containing lysozyme; (ii) lost no dipicolinic acid (DPA) and their inner membrane remained impermeable; (iii) released no DPA upon stimulation with nutrient germinants and could not complete germination; and (iv) released DPA relatively normally upon stimulation with dodecylamine. These results indicate that decoated spores treated with 80°C- 4 mol l(-1) CaCl2 are not dead, but some protein(s) essential for spore germination, most likely germinant receptors, are inactivated by this treatment. Thus, the original finding does not support a role for an osmoregulatory cortex in spore wet heat resistance.. Bacillus spores' low core water content is a major factor in their wet heat resistance. One suggested mechanism for achieving low spore core water content is osmoregulated expansion of spores' peptidoglycan cortex. Evidence for this mechanism includes a report that decoated Bacillus cereus spores incubated in 4 mol l(-1) CaCl2 exhibit drastically reduced heat resistance. The current work shows that this heat sensitization of decoated spores of three Bacillus species is most likely due to inactivation of some crucial spore germination protein(s), since while treated spores appear dead, their apparent low viability is rescued by triggering spore germination with lysozyme.

Topics: Amines; Bacillus cereus; Bacillus megaterium; Bacillus subtilis; Bacterial Proteins; Calcium Chloride; Hot Temperature; Muramidase; Osmotic Pressure; Peptidoglycan; Picolinic Acids; Spores, Bacterial; Water

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

Topics: Amines; Bacillus subtilis; Bacterial Proteins; Detergents; Disinfectants; Disinfection; Electrophoresis, Polyacrylamide Gel; Food; Gene Deletion; Hot Temperature; Hydrogen-Ion Concentration; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Microscopy, Interference; Muramidase; Picolinic Acids; Sodium Hypochlorite; Spores, Bacterial

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

Topics: Amines; Bacillus subtilis; Bacterial Proteins; Hydrogen-Ion Concentration; Mercury; Muramidase; Picolinic Acids; Spores, Bacterial; Temperature