Germination of Bacillus cereus Spores: Critical Control by DL-Alanine Racemase

Abstract

Effects of dl-alanine racemase, D-alanine, heat-activation and ammonia on germination of Bacillus cereus T spores were studied. Michaelis-Menten parameters for dl-alanine racemase activity in intact spores were: K m (l-alanine)=3·6 mm; K m (d-alanine) = 1·2 mm; V max (l- to d-alanine) = 1·6 μmol min−1 (mg dry spores)−1; V max (d- to l-alanine) = 0·53 μmol min−1 (mg dry spores)−1. Racemase activity was irreversibly inhibited with pseudo first order kinetics by incubating intact spores with 10 mm-d-cycloserine. (Aminooxy)acetate (50 μm) completely inhibited racemase activity with 100 mm-l-alanine as substrate. d-Cysteine was a moderately effective racemase inhibitor. Comparisons of the effects of racemase inhibitors, d-alanine and spore concentration on germination rates in 1 mm-l-alanine indicated that racemization of l-alanine accounted for nearly total inhibition of germination at spore concentrations exceeding about 50 μg ml−1, in the range conventionally used for spectrophotometric germination assays. Heat-activation decreased the inhibitory effect of d-alanine, increasing the germination rate in 2 mm-racemic alanine by a factor of 26. Using spores with an inhibited alanine racemase, germination rates in 1 mm-l-alanine were stimulated by 40 mm-NH4Cl to an extent comparable to the stimulation obtained by heat-activating the spores. Germination of unactivated spores in 1 mm-l-alanine + 40 mm-NH4Cl was completely inhibited by 1 mm-d-alanine. These results clarified the roles of d-alanine and dl-alanine racemase in controlling the germination of unactivated spores and suggested that the germinative functions of ammonia and the stereoisomers of alanine may be related.