ENERGY INTAKE AND RIBONUCLEIC ACID METABOLISM 205porated into RNA is increased by raising energy intake, in one case by an increase in the total RNA per liver without a change in the percentage of phosphorus atoms incorporated in a given time, in the other case by an increase in percentage incorporation rate accompanied by a smaller change in total RNA per liver.We are most grateful to Dr R. M. S. Smellie for instructing us in the ionophoretic method of separating ribonucleotides. We also wish to thank Dr Robb ofthe Statistics Department of Glasgow University for helpful discussions on statistical treatment. One of us (H. N. M.) wishes to acknowledge gratefully receipt of a grant for expenses from the Medical Research Council.

SUMMARY:The rate of germination (defined as loss of heat-resistance accompanied by change in staining properties under specified conditions, with maintenance of viability) of thick puspensions of'Bm*ZZus mbtilis spores in phosphate-buffered L-alanine solution increased with the time from harvesting. The maximum rate of germination was reached after about 20 days' storage in water at 20°. This effect could be retarded, but not reversed, by storage at low temperatures. The rate of germination may be-temporaqly accelerated by heat treatment. Germination was considerably retarded after treatment with mercuric chloride, and was completely inhibited by 8-hydroxyquinoline (oxine) and by 2 :$-dimercaptopropanoI (BAL) at 10 mlcl concentration. The latter effect was partially reversed by the addition of metals.Using dilute spore suspensions of Bacillzls subtilis (104 spores/ml.) Hills (1950) found that 80 yo loss of heat-resistance occurred during 80 min. incubation a t 35' in 0-5 m~ L-alanine buffered with 38 m~ phosphate a t pH 7-8. The present report concerns germination in thicker suspensions, i,e. 5 x lo@ sporea/ml., with a view to the study of the biochemical changes which

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The second step, which involves the acylation of the enzyme and the concomitant liberation of pnitrophenol, is characterized by the rate constant k2= 3-15 sec.-'. The third step involves the liberation of acetate and reactivation of the enzyme; the rate of this reaction is defined by kI3 = 0-0254 sec.-'. 2. The rate constant k2 for the second step is, within experimental error, the same at pH 6-45 and pH 7*75; from this and other available evidence it is suggested that it involves the acetylation of the OH group of a serine residue in the enzyme. The third step has a pH dependence similar to that previously found for the overall rate of reactions catalysed by chymotrypsin, and it is suggested that it involves the imidazole group of a histidine residue ofthe enzyme. The work described in this paper was carried out during the tenure by one of the authors (J.M.S.) of a Fulbright Scholarship and a Guggenheim Fellowship. The other author (H. G.) is an Imperial Chemical Industries Research

SUMMARY : Spore suspensions of Bacillus megaterium germinated spontaneously after heat treatment. The extent of germination depended on the degree of close packing of the suspension, the temperature and duration of heating, and on the presence of water. Spontaneous germination was not inhibited by heating with cyanide, azide, 2 : 4-dinitrophenol or sodium iodoacetate, but was significantly inhibited by sodium fluoride.Freshly harvested spores of a laboratory strain of Bacillus cereus required either inosine or a mixture of alanine + tyrosine + adenosine for optimal germination.After prolonged storage or a short heat treatment, adenosine alone stimulated rapid and complete germination. Heat activation did not occur in the absence of water. It was inhibited to some extent by sodium fluoride. Similar results were obtained with B. cereus, NCTC 8035. After preheating, spores of a virulent and an avirulent strain of Bacitlus anthracis germinated more rapidly and completely in a mixture of adenosine + tyrosine + alanine, but not in adenosine alone. Inoaine stimulated rapid and complete germination of heated suspensions, and was consistently more effective than adenosine. Adenosine deaminase activity has been found in extracts from resting spores of the laboratory B. cereus and the avirulent strain of B. anthracis but not in B. rnegaterium.As in previous studies, the criterion of germination was loss of heat resistance accompanied by a change in staining properties, decrease of refractive index and decrease in dry weight (Powell & Strange, 1953). A laboratory strain of Bacillus megaterium which required glucose specifically for spore germination was described by Powell (1951). Some freshly harvested spore suspensions of this organism germinated very poorly in glucose solution but were activated by heating at 60" for 30 min. Other suspensions, grown on the same medium germinated completely in glucose solution without heat activation. It was later accidentally discovered that when thick spore suspensions (2 x 1010 spores/ml.) were heated at 60" for 1 to 2 hr., then centrifuged and resuspended in water, spontaneous and complete germination occurred without loss of viability. This behaviour provided a means of obtaining concentrated germination exudate (Powell & Strange, 1953). In suspensions thicker than 1 x 1O1O spores/ml. the amount of calcium dipicolinate excreted (Powell, 1953) was actually greater than the saturation concentration, and the substance appeared as a heavy white granular deposit. We have now studied this heat-activated germination in some detail. The modification of germination requirements as a result of preheating has also been studied in two strains of B. cereus and in a virulent and an avirulent strain of B. anthracis. Freshly harvested, unheated spore suspensions of these organisms germinated in a mixture of L-alanine+ tyrosine + adenosine (Hills, 1949).