A gene encoding alanine racemase is involved in spore germination in Bacillus thuringiensis

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted L-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of L-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, D-alanine was an effective and competitive inhibitor of L-alanine-induced germination of B. thuringiensis spores. D-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing L-alanine-induced germination by inhibiting alanine racemase. D-alanine also increased the rate of inosine-induced germination of wild-type spores. However, D-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted D-alanine to L-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.