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J. Bacteriol. doi:10.1128/JB.01370-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Analysis of proline reduction in the nosocomial pathogen Clostridium difficile

Department of Molecular Biology and Microbiology, Burnett College of Biomedical Science, University of Central Florida, Orlando, Florida, 32816-2364

^* To whom correspondence should be addressed. Email: wself{at}mail.ucf.edu.

	Abstract

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affects toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase (PR). These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. PR utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from previously studied PR from non-pathogenic species.