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Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore

Rebecca Giorno^1,

¹ Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153, USA
² Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA
³ Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA
⁴ Headquarters, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA

Correspondence
Adam Driks
adriks{at}lumc.edu

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine–uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB–GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB–GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB–GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH–GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB–GFP and IunH–GFP resides in the region between the coat and the exosporium, called the interspace.

Present address: College of Biological Sciences, Louisiana Tech University, Ruston, LA 71270, USA.

A supplementary table listing PCR primers used in this study is available with the online version of this paper.

This article has been cited by other articles:

				K. M. Severson, M. Mallozzi, J. Bozue, S. L. Welkos, C. K. Cote, K. L. Knight, and A. Driks Roles of the Bacillus anthracis Spore Protein ExsK in Exosporium Maturation and Germination J. Bacteriol., December 15, 2009; 191(24): 7587 - 7596. [Abstract] [Full Text] [PDF]