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The protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of spores of Clostridium perfringens type A food poisoning isolates

¹ Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA
² Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
³ Department of Microbiology, College of Science, Oregon State University, Corvallis, OR 97331, USA

The genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment. cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of Ca²⁺ and dipicolinic acid (DPA); the viability of these spores was 10⁴-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants.

Correspondence
Mahfuzur R. Sarker
sarkerm{at}oregonstate.edu

Two supplementary tables, listing bacterial strains and plasmids and primers used in this study, and a supplementary figure, showing sequence alignment of Csp proteins of C. perfringens strains, are available with the online version of this paper.

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