Non-uniform Assembly of the Exosporium and Its Role in Sporulation and Germination

of Bacillus anthracis

 

Christopher T. Steichen, Jeremy A. Boydston, Olga N. Chesnokova, Sylvia A. McPherson, John F. Kearney, and Charles L. Turnbough, Jr.

 

Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294

 

Spores of Bacillus anthracis , the causative agent of anthrax, are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains approximately twenty different proteins. One of the basal layer proteins is ExsY, which is required for complete exosporium formation. Using a ?exsY mutant strain, we showed that in the absence of ExsY, exosporium assembly is arrested after the formation of a cap-like fragment that covers one end of the developing forespore. The basal layer also includes a spore-specific alanine racemase (Alr), which converts the spore germinant L-alanine to the potent germination inhibitor D-alanine. Employing fluorescence microscopy, we demonstrated that the binding of a fluorescently labeled anti-Alr mAb (AR-1) occurred over approximately three-quarters of the exosporium. The mAb did not bind within a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We determined that the AR-1-negative region corresponds to the exosporium cap assembled in a ?exsY mutant strain, which is also the first part of the exosporium assembled within the mother cell during sporulation of B. anthracis . These results provided the first direct evidence for discontinuous and non-uniform assembly of the exosporium. Additionally, we demonstrated that after spore germination, the outgrowing cell always escapes from its exosporium shell by popping through the cap end of the exosporium. This pattern suggests that the cap is designed to facilitate the emergence of the outgrowing cell and therefore acts like a cap on a bottle, which we term the bottle cap model of outgrowth. Finally, we constructed a ?alr mutant of B. anthracis , which is devoid of Alr, and examined the properties of the spores produced by this strain. As expected, mature ?alr spores germinate more efficiently in the presence of L-alanine and respond normally to other germinants. Surprisingly, the production of mature spores by the ?alr mutant was defective because approximately half of the nascent spores germinated and lost their resistance properties before their release from the mother cell. These results indicate that an important function of Alr is to produce D-alanine during the late stages of sporulation to suppress premature germination of the developing spore.