The Spore-Specific Alanine Racemase of Bacillus anthracis and Its Role in Suppressing Germination during Spore Development

Spores ofBacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinantl -alanine to the germination inhibitord -alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of thealr gene, which encodes Alr, is restricted to sporulating cells and that the bulk ofalr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped twoalr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Δalr mutant ofB. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Δalr spores germinate more efficiently in the presence ofl -alanine, presumably because of their inability to convert exogenousl -alanine tod -alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Δalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produced -alanine during the late stages of sporulation to suppress premature germination of the developing spore.