| Physiological interactions between co-germinants of spores of Bacillus cereus were analyzed to clarify the nature of the initial stages of germination. Effects of the novel co-germinant, ammonia, were analyzed in relation to the effect of L-alanine, D-alanine, inosine, adenosine, salts, small amines, and alanine racemase on germination kinetics of unactivated and heat-activated spores. Kinetic parameters of alanine racemase activity in whole spores were: K(,m) (L-alanine), 3.6 mM; K(,m)(D-alanine), 1.2 mM; V(,max) (L- to D-direction), 1.6 (mu)Mol min('-1) mg('-1) spores; V(,max) (D- to L-direction), 0.53 (mu)Mol min('-1) mg('-1) spores. Incubating spores in 10 mM D-cycloserine, 20 (mu)M sodium phosphate, pH 7.5, 30(DEGREES)C specifically and irreversibly inactivated alanine racemase with a half-time for inactivation of 1.2 min. 50 (mu)M aminooxyacetate completely inhibited alanine racemase in whole spores, reversibly, with 100 mM L-alanine as substrate. Inclusion of ammonia in germinant mixtures containing L-alanine and inosine stimulated germination of unactivated spores at rates equal to those obtained using heat-activated spores without ammonia. Studies of the effects of pH, monovalent cations other than NH(,4)('+), and small amines in germination assays in 1 mM L-alanine or 1 mM L-alanine plus 1 mM inosine, using spores with an inactive racemase, indicated that the stimulatory effect of NH(,4)Cl was not a simple ionic effect, since it was correlated with a requirement for non-ionized ammonia or amine rather than with a requirement for salts. Kinetic analysis of germination rates of unactivated spores (with inactive racemase) revealed a complex response to L-alanine having positive cooperativity at concentrations below 2 mM L-alanine. The apparent K(,s) for L-alanine was 100 (mu)M; this K(,s) was increased 2- to 3-fold by 5 (mu)M D-alanine, but was not greatly affected by 5 or 40 mM NH(,4)Cl. Effects of inosine and NH(,4)Cl on germination in 1 mM L-alanine suggested that both co-germinants were affecting the same L-alanine-dependent reaction during germination. Analysis of germination of spores lacking intact spore coats (extracted with mercaptoethanol-urea) revealed that intact coats were not necessary for germination in L-alanine, but were required for stimulation of L-alanine-induced germination by NH(,4)Cl. |
