Probing the germination kinetics of ethanol-treated Bacillus thuringiensis spores

Bacillus thuringiensis (Bt) is the most widely used microbial insecticide. To clarify the mechanism of bacterial resistance to ethanol toxicity, the present study investigated the effects of 70% (v/v) ethanol at a moderate temperature (65°C) on Bt spore germination by single-cell Raman spectroscopy and differential interference contrast microscopy. We found that over 80% of Bt spores were inviable after 30 min of treatment. Moreover, ethanol treatment affected spore germination; the time for initiation of rapid calcium dipicolinate (CaDPA) release (i.e., lag time, T_lag), time taken for rapid CaDPA release (i.e., ΔT_release), and time required for complete hydrolysis of the peptidoglycan cortex of spores (i.e., ΔT_lys) were increased with longer treatment times. Alanine-initiated germination upon ethanol treatment for 30-90 min showed a 2- to 4-fold longer T_lag, 2- to 3.5-fold longer ΔT_release, and ∼2-fold longer ΔT_lys relative to the control. Dodecylamine-initiated germination treated for 15-30 min had 3- to 5-fold longer T_lag and 1.4- to 1.7-fold longer ΔT_release than the control. Germination induced by exogenous CaDPA was observed only in a small fraction of spores treated with ethanol for 5 min. Single-cell Raman spectroscopy revealed that more than 52% of spores lost CaDPA after 30 min of ethanol treatment; these showed reductions in the intensity of 1280 and 1652  cm^-1 bands (corresponding to protein α-helical structure) and increases in that of 1245 and 1665  cm^-1 bands (attributed to irregularities in protein structure). These results indicate that CaDPA in the core of Bt spores confers resistance to ethanol, and that damage to the spore inner membrane by ethanol treatment results in CaDPA leakage. Additionally, moderate-temperature ethanol treatment and consequent denaturation of germination-related proteins affected spore germination, specifically by inactivating the cortex-lytic enzyme CwlJ. Our findings provide a theoretical basis for the development of more effective methods for killing spore-forming bacteria; microscopy imaging and Raman spectroscopy can provide novel insight into the effects of chemical agents on microbial cells.