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Auxotrophic mutants of Bacillus subtilisneed much higher concentrations of the required adenine, nicotinic acid, riboflavin, thiamine, or tryptophan for optimal sporulation than for maximal growth. Acetate can partially replace thiamine, indicating the importance of the pyruvate dehydrogenase system for differentiation. A glycerol-requiring mutant can sporulate only if its cells contain a small concentration of L-α-glycerol phosphate during development. This can best be achieved by excess (≥5 mM) of extracellular α-glycerol phosphate, which enters B. subtilisvery slowly. The results show that both biosynthetic and catabolic enzymes are often needed to maintain the precise balance of metabolites required for differentiation. Mutants unable to catabolize fructose 6-phosphate, glucose 6-phosphate, or α-glycerol phosphate do not sporulate as long as these compounds accumulate inside the cells; their development is blocked before prespore septa have formed.

The inhibitory effects of anthranilic acid esters (methyl anthranilate and N-methyl anthranilate) on the l-alanine-induced initiation of spore germination was examined in Bacillus subtilis168. Methyl anthranilate irreversibly inhibited alanine initiation by a competitive mechanism. In its presence, the inhibition could be reversed only by the combined addition of d-glucose, d-fructose, and K+. Both l-alanine dehydrogenase and l-glutamate-pyruvate transaminase, enzymes which catalyze the first reaction in l-alanine metabolism, were competitively inhibited by methyl anthranilate. The Kivalues for germination initiation (0.053 mM) and of l-glutamate-pyruvate transaminase (0.068 mM) were similar, whereas that for l-alanine dehydrogenase (0.4 mM) was six to seven times higher. Since a mutant lacking l-alanine dehydrogenase activity germinated normally in l-alanine alone, it is speculated that the major pathway of l-alanine metabolism during initiation may be via transmination reaction.

Mutants deficient in both glucose-6-phosphate dehydrogenase and phosphoglucose isomerase lysed 4 to 5 h after growth in nutrient medium containing glucose, or after prolonged incubation if the medium contained galactose. The lysis could be prevented by the addition of any other rapidly metabolizable carbon source such as fructose, glucosamine, or glycerol. The glucose-induced lysis was also abolished by introduction of a third mutation lacking phospho-glucose mutase activity but not by a third mutation lacking uridine diphosphate-glucose pyrophosphorylase or teichoic acid glucosyl transferase activity. Galactose-induced lysis was prevented only if the additional mutation abolished the uridine diphosphate-glucose pyrophosphorylase activity. The results showed that lysis was caused by the intracellular accumulation of glucose-1-phosphate, which in turn inhibited at least one of the two enzymes that convert glucosamine-6-phosphate to N-acetyl glucosamine-6-phosphate.

1. Two intermediates of tryptophan catabolism were isolated from a sporulating culture of Bacillus cereus and identified as anthranilic acid and kynurenine by their spectral properties. 2. During sporulation the rate of formation of anthranilic acid and kynurenine by whole cells increased and reached a maximum at the pre-spore stage. 3. The specific activities of tryptophan pyrrolase and formylase also increased during sporulation and exhibited a maximal activity at the pre-spore stage. 4. Kynureninase activity reached a maximum during early stages of sporulation and then started to decline. 5. There was a net increase in the activity of tryptophan pyrrolase when cells were grown in the presence of l-tryptophan or dl-kynurenine. 6. The cultures exhibited the maximal activity of kynureninase 2h earlier in the presence of dl-kynurenine whereas l-tryptophan delayed the appearance of the maximal activity by 2h. 7. The omission of glucose from the medium had no effect on the pattern of development of tryptophan pyrrolase during growth and sporulation. 8. On the addition of tryptophan to a chemically defined medium no significant change in the pattern of development of tryptophan pyrrolase was observed.

Enzyme activities of glycolysis and glyconeogenesis are present in spores of Bacillus subtilis, the rate-limiting step of glucose (GLC) metabolism being its phosphorylation. GLC allows initiation of germination in the presence of fructose (FRU) and asparagine (ASN), not because it is used via the Embden-Meyerhof path, but because it is oxidized in the nonphosphorylated form via the spore-specific GLC dehydrogenase. Spores of mutants lacking GLC-phosphoenolpyruvate transferase, FRU-6-P-kinase, or phosphoglucoisomerase activity can still be initiated by the above substrate combination. Furthermore, GLC can be replaced by 2-deoxy-GLC, which is also oxidized by GLC-dehydrogenase, but not by α- or β-methylglucoside, which are not substrates of this enzyme. GLC probably acts by reducing nicotinamide adenine dinucleotide (or nicotinamide adenine dinucleotide phosphate), which is used for some metabolic reaction other than the cytochrome-linked electron transport system, since inhibitors of this system do not inhibit initiation. Spores of a mutant lacking FRU-1-P-kinase activity can no longer be initiated by GLC+FRU+ASN, but they do respond to the combination of GLC+mannose+ASN. Since spores of a FRU-6-P-kinase (or phosphoglucoisomerase) mutant can still respond to either FRU or mannose, FRU-6-P (or some derivative) apparently is needed for initiation (in addition to reduced nicotinamide adenine dinucleotide and an amino donor). Alanine can initiate germination in spores of all of the above mutants, indicating that it can form all required compounds. However, in a mutant lacking P-glycerate kinase activity, alanine initiates only after a long lag and at a slow rate, indicating that some compound in the upper metabolic subdivision is required for initiation, in agreement with the above findings. All initiating agents of B. subtilisprobably produce the same required compound(s) by different metabolic routes.

LYSOZYME has been detected in the milk of donkey, mare, bitch, sow and cow1,2. In order to investigate the possible biological significance of lysozyme an investigation is planned here to compare physical, chemical and physiological characteristics of the lysozymes isolated from bovine and human milk. Although several workers1–4have reported the presence of lysozyme in human milk and colostrum, not much work has been done to obtain information on the quantitative amounts of the enzyme in this milk. In the work recorded here lysozyme contents of a large number of bovine and human milk samples were determined. In an earlier publication2the lysozyme content of bovine milk was reported to vary between 0.0 and 260.0 µg with an average of 13 µg of the enzyme activity per 100 ml. of milk, using egg-white lysozyme (Difco) as the standard. This report deals with the lysozyme content of human milk and colostrum, and the effect of storage and heat on the enzyme.

Methylanthranilate inhibited the germination of spores of aerobic bacilli without affecting growth and sporulation. The inhibition of germination could not be reversed by removal of methylanthranilate.