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Role of outer coat in resistance of Bacillus megaterium spore
T Nishihara1, Y Takubo, E Kawamata
1Faculty of Pharmaceutical Sciences, Osaka University.
Abstract:
The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed of galactosamine-6-phosphate (GalN-P) polymers with polypeptides and calcium. OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer of GalN-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experiments revealed that the outer coat limited the entry of compounds with a molecular weight of more than 2,000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of OC-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainly of GalN-P polymers, and the molecular sieving effect.
Insights
The Bacillus megaterium outer coat fraction (OC-Fr), primarily galactosamine-6-phosphate polymers, protects spores from damage. This structure acts as a barrier, limiting entry and enhancing resistance to various treatments.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Bacterial spores exhibit remarkable resistance to harsh environmental conditions.
- The outer coat of Bacillus megaterium spores is a key component contributing to this resistance.
Purpose of the Study:
- To elucidate the chemical structure and protective mechanisms of the Bacillus megaterium spore outer coat fraction (OC-Fr).
- To investigate the role of OC-Fr in spore resistance against chemical, physical, and enzymatic challenges.
Main Methods:
- Isolation and chemical analysis of OC-Fr from Bacillus megaterium spores.
- Enzymatic digestion using hexosaminidases and lysozyme.
- Molecular sieving experiments to determine pore size limitations.
- Assessment of heat resistance in wild-type and OC-Fr-deficient mutant spores.
Main Results:
- The OC-Fr backbone is composed of galactosamine-6-phosphate (GalN-P) polymers, polypeptides, and calcium.
- OC-Fr and its GalN-P fragments showed resistance to enzymatic degradation, even after N-acetylation.
- Molecular sieving indicated the outer coat restricts molecules >2,000 Da.
- Metal ion exchange affected spore heat resistance; OC-Fr-deficient mutants had reduced, but still significant, resistance.
Conclusions:
- The unique chemical structure of OC-Fr, primarily GalN-P polymers, confers significant protection to Bacillus megaterium spores.
- The outer coat's molecular sieving properties further contribute to spore resistance against external agents.
- OC-Fr plays a crucial role in spore survival under adverse conditions.