Mechanism of inactivation of Bacillus subtilis spores by high pressure CO2 at high temperature.

Spores of wild-type Bacillus subtilis and some isogenic mutant strains were treated by high pressure CO2 (HPCD) at high temperature (HT) (HPCD + HT) at 20 MPa and 84-86 °C for 0-60 min, and centrifuged on a high density solution to obtain pelleted spores that retained CaDPA and light spores that lost CaDPA. All treated spores were analyzed for viability, and tested for germination, outgrowth, core protein damage, mutagenesis and inner membrane (IM) properties. The results showed that (i) with HPCD + HT treated spores, most pelleted spores and all light spores were dead; ii) a significant amount of dead HPCD + HT-treated spores that retained CaDPA germinated, but outgrowth was blocked; (iii) minimal mutants were generated in survivors of HPCD + HT treatment; (iv) the GFP fluorescence decrease in HPCD + HT-treated spores with high GFP levels was slower than spore inactivation; (v) the IM of HPCD + HT-treated spores that retained CaDPA lost its ability to retain CaDPA at 85 °C, and almost all of these spores' outgrowth in high salt was blocked; and (vi) HPCD + HT-treated spores that retained CaDPA germinated with l-valine or AGFK were almost all stained with propidium iodide. These results indicated that HPCD + HT inactivated B. subtilis spores by damaging spores' IM, thus blocking spore outgrowth after germination.