Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

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

^* To whom correspondence should be addressed. Email: sarkerm{at}oregonstate.edu.

	Abstract

Spores of Clostridium perfringens possess high heat-resistance and when these spores germinate and return to active growth they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance, and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca²⁺ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little if any Ca²⁺ and DPA, and their core water content was 2-fold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as do DPA-less B. subtilis spores. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of L-asparagine and KCl (AK), KCl alone or a 1:1 chelate of Ca²⁺ and DPA (Ca-DPA). However, the C. perfringens spoVA spores had 20-fold lower viability than wild-type spores. Decoated wild-type and spoVA spores exhibited little if any germination with AK, KCl, or exogenous Ca-DPA, and had 10³- to 10⁴-fold lower colony forming efficiency than intact spores. However, lysozyme treatment rescued these decoated spores. Although the level of DNA protective /-type small, acid-soluble spore proteins in spoVA spores was similar to that in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid and UV radiation. In sum, these results suggest that: (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation; (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination; and (iii) a low core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation and chemicals.