Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

Abstract

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores' germination kinetics were heterogeneous; (ii) individual spores' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (Tlag) prior to a period of the rapid release (ΔTrelease) of the spores' dipicolinic acid in a 1:1 chelate with Ca(2+) (CaDPA); (iii) spore germination at 50 MPa had longer average Tlag values than that at ∼150 MPa, but the ΔTrelease values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔTrelease values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.