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Sporulation in Bacillus subtilis. Properties and time of synthesis of alkali-soluble protein of the spore coat.

ABSTRACT: An alkali-extractable protein fraction comprising 6% of the dry weight of the spore can be removed from spores of Bacillus subtilis 168. Three different extraction procedures each yield at least one similar protein. Extracted protein behaved as a single species on ion-exchange chromatography or gel filtration, but two polypeptides were found on electrophoresis. Comparison of molecular weights on electrophoresis and by sucrosegradient analysis suggests that the protein(s) undergo aggregation into multimers. Extracted spores remain viable, but are altered in density and lysozyme sensitivity and they aggregate together. Electron microscopy of extracted spores shows that loss of material seems to occur from the outer coat layers. Extraction therefore probably removes a specific fraction of the spore-coat protein, but without impairment to the spore protoplast. This protein can first be detected immunologically 4h after the initiation of sporulation, and the synthesis of this protein is sensitive to chloramphenicol, actinomycin D and rifamycin. Labelling experiments also show that the protein begins to be synthesized early in sporulation. Examination of the ability of asporogenous mutants to produce cross-reacting material indicates that some event in stage II of sporulation determines its production.

SUBMITTER: Wood DA

PROVIDER: S-EPMC1174430 | biostudies-other | 1972 Nov

REPOSITORIES: biostudies-other

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Project description:Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. 14N spores prepared on solid Schaeffer's-glucose (SG) agar plates and 15N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The 14N:15N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Corroborating the proteomic analyses, electron microscopy analyses show a significantly thinner outer coat layer of the spores cultured on the solid agar medium.

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Sporulation in Bacillus subtilis. Properties and time of synthesis of alkali-soluble protein of the spore coat.

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