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 phasecontrast 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 (T sub lag) prior to a period of the rapid release (delta T(sub release)) of the spores dipicolinic acid in a 1:1 chelate with Ca2+ (CaDPA); (iii) spore germination at 50 MPa had longer average T(sub lag) values than that at ~150 MPa, but the deltaT(sub release) 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 delta T(sub release) 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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2014
- Accession Number
- ADA603352
Entities
People
- Christopher J. Doona
- Lingbo Kong
- Peter Setlow
- Yong-qing Li
Organizations
- University of Connecticut Health Center