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Journal of Bacteriology, August 2005, p. 5310-5317, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5310-5317.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Orientation within the Exosporium and Structural Stability of the Collagen-Like Glycoprotein BclA of Bacillus anthracis

Jeremy A. Boydston, Ping Chen, Christopher T. Steichen, and Charles L. Turnbough Jr.*

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

Received 16 March 2005/ Accepted 22 April 2005

Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (Tm) of 37°C, like mammalian collagen. However, rBclA trimers exhibited Tm values of 84°C and 95°C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same Tm values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.

* Corresponding author. Mailing address: UAB Department of Microbiology, BBRB 409, 1530 3rd Ave. S, Birmingham, AL 35294-2170. Phone: (205) 934-6289. Fax: (205) 975-5479. E-mail: ChuckT{at}uab.edu.

Journal of Bacteriology, August 2005, p. 5310-5317, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5310-5317.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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