Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 i 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core. MATERLILS AND METHODS Preparation of spores. A phage-resistant strain of B. cereus strain T, isolated from the original strain at the University of Michigan, was grown and sporulated with vigorous aeration in a modified G medium, as reported earlier (6), by use of a Microferm fermentor (model MF-14, New Brunswick Scientific Co., New Brunswick, N.J.). Complete sporulation was usually attained within 10 to 12 hr after inoculation. The free 1011

Clean, intact spores from three enterotoxin positive (ent+) and three enterotoxin-negative (ent-) strains of Clostridium perfringens type A were treated with urealmercaptoethanol, alkaline mercaptoethanol, or alkaline dithiothreitol, reagents known to solubilize spore coat proteins. A protein fraction comprising approximately 15 to 30°/, of the total spore protein was extracted. Protein solubilized by each method conhined a fraction that was precipitated by anti-enterotoxin serum and that had enterotoxin-like biological activity neutralizable with anti-enterotoxin serum but not commercial C. perfringens type A antitoxin. Highly purified C. perfringens type A enterotoxin was precipitated by antiserum prepared against the solubilized spore protein from an ent+ and an ent-strain. These antisera also neutralized the biological activity of C. perfringem type A enterotoxin. Examination of "core" and "coat" fractions prepared from intact spores revealed that the extractable enterotoxin-like protein was associated with the spore coat fraction. It is concluded that the enterotoxin of C. perfringens type A is a structural component ofthe spore coat.Certain strains of Clostridium perfringens type A produce an enterotoxin that is operative in human food poisoning [l-31. One of-the unique features of this toxin is that it is produced only during sporulation and not during vegetative growth [3]. By employing mutants of C. perfringens with an altered ability to spodate, Duncan et al. [a] recently demonstrated that a direct relationship exists between enterotoxin Synthesis and spore formation. The authors therefore proposed that the enterotoxin was a sporulation-specific gene product.The number of known gene products that uniquely appear in the spore or sporulating cell that can confidently be assigned a sporulation-specific function may be limited to some of the enzymes that participate in the synthesis of the cortical peptidoglycan, dipicolonic acid, and the protein(s) of the spore coat [5]. The possibility existed that the enterotoxin was a protein normally associated with or comprising some spore structural component which accumulated in the sporangium as a result of loosely regulated synthesis. Such a relationship seems to exists between the crystal protein of Bacillus thuringiensis that is toxic for certain insect larvae and the spore coat protein of B. thuringiensis. Studies [6-81 have shown that the crystal toxin of B. thuringiensis is immunologically and biochemically similar to a substantial spore fraction which can be removed from spores by extraction with 8 M urert-lo/, mercaptoethanol. Lecadet et al. [9] have recently demonstrated that this protein fraction definitely is present in p d e d spore coat preparations.I n an attempt to determine whether the enterotoxin protein and the spore coat protein of C . perfringens type A exhibited a similar relationship, we have employed procedures known to solubilize protein(s) from spore coats of Bacillus and Clostridium spores to extract intact spores and purified spore coa...

The biphasic nature of germination curves of individual Bacillus cereus T spores was further characterized by assessing the effects of temperature, concentration of germinants, and some inorganic cations on microgermination. Temperature was shown to affect both phases of microgermination as well as the microlag period, whereas the concentration of L-alanine and supplementation with adenosine exerted a significant effect only on the microlag period. The germination curves of individual spores induced by inosine were also biphasic and resembled those of spores induced by L-alanine. High concentrations (0.1 M or higher) of calcium and other inorganic cations prolonged both phases of microgermination, particularly the second phase, and had a less pronounced effect on the microlag period. The second phase of microgermination was completely inhibited when spores were germinated either in the presence of 0.3 M CaC12 or at a temperature of 43 C; this inhibition was reversible. Observations on the germination of spore suspensions (kinetics of the release of dipicolinic acid and mucopeptides, loss of heat resistance, increase in stainability, decrease in turbidity and refractility) were interpreted on the basis of the biphasic nature of microgermination. Dye uptake by individual spores during germination appeared also to be a biphasic process.

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Enterotoxin-like protein was extracted from spores of three enterotoxin-positive and three enterotoxin-negative strains of Clostridium pegringens type A by urea/mercaptoethanol, alkaline mercaptoethanol and alkaline dithiothreitol. Disc immunoelectrophoresis demonstrated that three distinct enterotoxin-like proteins could be extracted. In 7 % acrylamide gels, type I, type 11, and type I11 enterotoxin-like proteins had relative mobilities of 0.52, 0.63, and 0.73 respectively. In contrast to disc immunoelectrophoresis, immunoelectrophoresis in agar gel demonstrated identical electrophoretic properties for the various enterotoxin-like proteins. Immunoelectrofocusing experiments gave isoelectric points of 4.43, 4.43, 4.36, and 4.52 for purified enterotoxin and type I, type 11, and type 111 enterotoxin-like proteins respectively. Ferguson plots ( i e . , log relative mobility versus acrylamide concentration) yielded nonparallel lines which intersected at a nonsieving concentration of acrylamide indicating that the various species of enterotoxin-like protein differed in size.