The Swelling of Bacterial Spores during Germination and Outgrowth

Abstract

SUMMARYThe swelling of spores of Bacillus cereus and B . subtilis during germination and outgrowth was followed by measuring changes in packed cell volume (pcv) and by photomicrographic measurements of single organisms. Three stages of swelling were recognized : (1) germination swelling involving a rapid increase of about 20% in pcv as the spore germinated; (2) preemergence swelling of up to 100 yo increase in pcv before emergence from the spore coat; (3) elongation. Germination swelling was due mainly to an increase in the breadth of the spore. It occurred in the absence of oxygen and was not inhibited by nisin whereas pre-emergence swelling was oxygen-dependent and was inhibited by nisin. Post-emergence swelling occurred during elongation of the new vegetative form. The significance of these findings is discussed,

Function and Location of a "Germination Enzyme" in Spores of Bacillus Cereus

“…The response was most rapid at about pH 7.8 and was similar to the response of spores to a germinant such as L-alanine or inosine (Gould & Hitchins, 1965), however, it occurred with sensitized spores which were non-viable. The enzyme did not cause germination of spores which had not been previously sensitized, in these respects germination induced by the enzyme was similar to germination induced by lysozyme or hydrogen peroxide (Gould & Hitchins, 1963 …”

“…The substrate of the enzyme is probably the structural material of the spore cortex, which is predominantly mucopeptide (Warth, Ohye & Murrell, 1963). The importance of this mucopeptide in maintaining the dormant resistant structure of spores of various bacillus and clostridium species was emphasized by the observation of the germinative action of lysozyme (Gould & Hitchins, 1963), because lysozyme is known to hydrolyse mucopeptide. A search for an enzyme-release mechanism which could be initiated by a germinant or some product of its metabolism by a spore, will clearly be worthwhile.…”

“…Spores of Bacillus cereus, strain PX were grown and cleaned, as described previously (Hitchins, Gould & Hurst, 1963).…”

“…How a ' germinant ' initiates the normal germination reaction is unknown. One possible mechanism of germination was suggested when it was found that lysozyme made spores germinate, probably by hydrolysing p 1-4 glycosidic bonds in mucopeptide in the spore cortex (Gould & Hitchins, 1963). To show germination by lysozyme, spores were first treated with reagents which ruptured disulphide bonds : this opened the spore coat structure, presumably by attacking the keratin-like (Vinter, 1962; Kadota, Iijima & Uchida, 1965), and allowed lysozyme to reach lysozyme-sensitive mucopeptide in the underlying cortex (Gould, Georgala & Hitchins, 1964).…”

“…It can also be simulated by what appear to be less natural means; i.e. by mechanical abrasion (Rode & Foster, 1960a), by surface active substances (Rode & Foster, 1960b), by alkanes (Rode & Foster, 1965), by enzymes like lysozyme (Gould & Hitchins, 1963), a spore enzyme (Gould & Hitchins, 1965) and by a heat-activated factor+a cofactor in Bacillus cereus strain T spore extracts (Vary, 1965). How a ' germinant ' initiates the normal germination reaction is unknown.…”