Alteration in ultrastructure and germination of Clostridium perfringens type A spores following extraction of spore coats

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Abstract
Ultrastructural changes in Clostridium perfringens type A strain FD-1 spores treated with alkali, mercaptoethanol–urea, or dithiothreitol (DTT) were observed by electron microscopy of thin sections. Two prominent coat layers were differentially removed or altered by extraction with 0.1 N NaOH at 4 °C or 50 mM DTT, pH8.5, at 37 °C. Alkali disrupted or completely solubilized an inner, narrow, electron-dense coat without appreciably altering the ultrastructural appearance of the outer, wide, less electron-dense coat. A decrease in electron density of the subcoat region occurred with either an alkali or DTT treatment of the spores. DTT treatment at pH 8.5 substantially removed the outer coat and partially disrupted the inner coat. Germination of alkali-treated spores was completely lysozyme-dependent. DTT-treated spores at pH 8.5 germinated without added lysozyme. The germination system of these DTT-treated spores became heat-sensitive, with lysozyme dependence occurring after heating at 75 °C for 20 min. DTT treatment at pH 10.5 resulted in lysozyme-dependent spores completely devoid of spore coats. Treatment with 10% mercaptoethanol in 7 M urea, pH2.8, resulted in a granular appearance of subcoat material and retarded the rate, but not the extent, of germination. These results indicate a close association of the normal germination lytic system with the subcoat region, possibly on the outer forespore membrane, and indicate a protective role of the spore coats in maintaining the heat resistance of the lytic system.

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