Search

Spores of wild-type Bacillus subtilis and some isogenic mutant strains were treated by high pressure CO 2 (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. • High pressure CO 2 (HPCD) at innocuous high temperature (HT) (∼85 °C) could effectively inactivate Bacillus subtilis spores. • HPCD + HT inactivated B. subtilis spores by damaging spores' inner membrane, thus blocking spore outgrowth after germination. • The damage to the GRs of HPCD + HT inactivated spores was probably occurred after inactivation. [ABSTRACT FROM AUTHOR]

The effect of water activity (a w ), as lowered by different dietary humectants, on the germination of Bacillus subtilis, Bacillus megaterium and Bacillus cereus spores with germinants that act by different mechanisms has been investigated and compared. Germination of spores of these species by all of the germinants investigated was inhibited as a w decreased, with the general order of efficacy for these non-ionic humectants being sucrose > trehalose > glycerol. The effect of lowering a w on germination by germinant receptor (GR)-dependent germinants was not appreciably altered by varying germinant concentrations, was generally not much more effective with spores lacking coats or an outer membrane, and was less pronounced with heat-activated spores. Analysis of the effect of a w on spore germination via different mechanisms showed that GR-dependent germination was least sensitive to a w , while germination via activation of spore cortex peptidoglycan hydrolysis or dipicolinic acid release was more sensitive. However, germination by high hydrostatic pressure was less sensitive to inhibition by low a w , than was germination by other germinants. Examination of the GR-dependent germination of individual spores indicated that a w acted most strongly in inhibiting the commitment step of germination, while exerting smaller effects on dipicolinic acid release or cortex peptidoglycan hydrolysis. [ABSTRACT FROM AUTHOR]

Under stressful conditions, bacteria can enter viable but non-culturable (VBNC) state to survive. VBNC cells lost ability to grow on routine culture medium but are still alive and may revive in suitable conditions. The revived cells can consume nutrients or produce toxins, leading to food spoilage or human illness, posing great risk to food safety and public health. Previously, we have reported that high pressure carbon dioxide (HPCD), an environment-friendly sterilization technology, can induce VBNC formation. However, the underlying mechanism is unclear. By performing a comprehensive transcriptomic analysis, we revealed that HPCD initiated high expression of asr , encoding an acid shock protein, could promote VBNC formation of E. coli O157:H7. Quantitative reverse transcription PCR analysis suggested that high expression of asr (i) inhibited acid resistance (AR) systems, resulting in endogenous proton accumulation; (ii) inhibited hchA expression, a protein stabilizing factor. The two effects resulted in endogenous protein aggregation, which was highly correlated to VBNC formation. Accordingly, HPCD-stressed cells exhibited decreased efficiency of electron transfer chain and ATP production, which was also contributory to cytoplasmic protein aggregation. Taken together, HPCD-initiated high expression of Asr coupled with decreased ATP production led to protein aggregation, finally promoted the cells to enter VBNC state. • Molecular responses of E. coli O157:H7 to HPCD were investigated to explore crucial factors regulating VBNC formation. • Acid shock protein Asr was found to be involved in regulating HPCD induced VBNC formation. • Asr inhibited AR systems / protein stabilizer HchA to facilitate endogenous protein aggregation to promote VBNC formation. [ABSTRACT FROM AUTHOR]