P-055. Characterization of Spores of Clostridium perfringens that Lack SpoVA Proteins and Dipicolinic Acid

D. Paredes-Sabja1, B. Setlow2, P. Setlow2, M. R. Sarker1;
1Oregon State Univ., Corvallis, OR, 2Univ. of Connecticut Hlth. Ctr., Farmington, CT.

Clostridium perfringens type A isolates carrying a chromosomal enerotoxin (cpe) gene are mainly associated with Clostridium perfringens food poisoning. C. perfringens spores possess high heat-resistance and these spores must germinate and return to active growth to cause GI disease. Previous studies have shown that proteins encoded by the gerK operon are required for both nutrient- and non-nutrient spore germination, and that that α/β-type small, acid-soluble proteins (SASP) play a major role in the resistance of C. perfringens spores to moist heat, UV radiation and some chemicals. We have found that SpoVA proteins are involved in Ca2+ and dipicolinic acid (DPA) uptake during C. perfringens sporulation. Spores of a spoVA mutant had little if any Ca2+ and DPA, conversely, the core water content of spoVA spores was ~ 2-fold higher than in wild type spores. Surprisingly, these spoVA spores did not germinate spontaneously, as do DPA-less Bacillus subtilis spores. C. perfringens wild-type and spoVA spores germinated similarly with a mixture of L-asparagine and KCl (AK), KCl and a 1:1 chelate of Ca2+ and DPA (Ca-DPA). In contrast, decoated wild-type and spoVA spores exhibited little if any germination with AK, KCl, or exogenous Ca-DPA, and had a 103- to 104-fold decreased colony formation efficiency. However, lysozyme treatment rescued these decoated spores. Although the levels of α/β-type small, acid-soluble proteins (SASP) in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited very low resistance to moist heat resistance, hydrogen peroxide, hydrochloride acid, formaldehyde, nitrous acid and UV radiation. In sum, these results suggest that: (i) SpoVA proteins are responsible for Ca-DPA uptake during C. perfringens sporulation; (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens germination, indicating the existence of an novel pathway to trigger cortex peptidoglycan hydrolysis during germination; and (iii) a low core water content is essential in the resistance of C. perfringens spores to moist heat, UV radiation and some chemicals.