Heat

Studies were carried out on the inhibitory effect of NH4+ on germi- Germination proceeds well in the presence of both L-alanine and inosine. Previously we suggested that germination of unactivated spores of Bacillus cereus T induced by L-alanine and inosine may proceed by at least two steps, the first step induced by L-alanine and the second by inosine (14).Inorganic ions are also required for complete germination : Levinson and Sevag (8) reported that manganese ions stimulate germination, and Rode and Foster (9-11) reported that germination was poor in the absence of ions or at low ionic strength. Generally, an ionic strength of about 0.1 M is optimal for germination (1, 13).Of the various cations studied previously (6,7,11,17,18), NH4+ has the most interesting effects: it stimulated germination of heat-activated spores of some strains of Bacillus subtilis and it induced germination of the spores of Bacillus megaterium QM B1551 in the presence of nitrate, but it did not facilitate germination of B. cereus T. These findings suggest that the spores of different strains and species require different ions for germination.The present study was conducted to confirm that NH4+ plays a role in regulating the initiation of germination of B. cereus T in the presence of L-alanine and inosine, and also to obtain information on the action of NH4+.

The effect of potassium ion on L-alanine-inosine-induced germination of unactivated spores of Bacillus cereus T was studied. Unactivated spores germinated in 0.1 M sodium phosphate buffer (NaPB), but not 0.1 M potassium phosphate buffer (KPB), at pH 8.0 and at 30 C. Inhibition of germination was also observed on incubation of unactivated spores in NaPB containing potassium chloride. Previously it was demonstrated that germination of unactivated spores involves at least two steps, one induced by L-alanine, and the other by inosine. Potassium ion seems to inhibit the response of the spores to inosine, because: (1) Spores that had been preincubated with L-alanine in NaPB or KPB, germinated in NaPB but not KPB in the presence of inosine. (2) During germination in NaPB, incorporation of L-[14C]alanine showed bimodal kinetics with a rapid first phase and a second continuous phase, but in KPB the second phase of incorporation did not occur.The events occurring before germination of unactivated spores are discussed with reference to the initiation of germination. Dormant bacterial spores change to a metabolically active form during germination. This change is accompanied by certain characteristic events, which have been used as criteria of germination; these events include loss of heat resistance, reduction of the dipicolinate content, acquisition of stainability, and decrease in optical density (2). From the metabolic point of view, however, it can be considered that some factors leading to the initiation of germination have already occurred before these events become apparent.L-Alanine is one of the best known of the substances that initiate germination of spores of some bacterial species. L-Alanine is thought to play two distinct roles in germination (11,13), and this idea is strongly supported by the finding of two distinct profiles of incorporation of L-alanine during germination of Bacillus thiaminolyticus spores (12). We reported previously (10) that unactivated spores of Bacillus cereus T can germinate in the presence of inosine alone if they have been preincubated with L-alanine. This function of L-alanine seems to be quite different from that of the amino acid during L-alanine-inosine-induced germination of heat-activated spores. 443