Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

Abstract

Aims: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. Methods and Results: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50°C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60°C, in contrast to an optimum temperature of 40°C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23°C to 44°C, and decreased twofold when 1 mol l )1 salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg 2+ , but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. Conclusions: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. Significance and Impact of the Study: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.

Role of GerD in Germination of Bacillus subtilis Spores

“…Germination was also performed on non-heat-activated spores at an OD 600 of 1 in 50 mM Tris-HCl (pH 7.5) buffer either at 37°C with 150 MPa of pressure or at 50°C with 500 MPa of pressure (4,5,24,36). The extent of spore germination due to pressure was analyzed by flow cytometry after staining with Syto 16 (4).…”

“…Heat-activated spores (10-l aliquots [ϳ10 5 spores]) containing kanamycin (50 g/ml) to prevent cell growth were spotted on the slides, a glass coverslip was added and sealed with clear nail polish to prevent the slide from drying, and ϳ100 spores on the slide were examined by phase-contrast microscopy during incubation at 37°C. Control experiments demonstrated that the addition of kanamycin did not affect rates of spore germination.…”

“…6B). Previous work has established that different high pressures trigger spore germination via different pathways (4,5,24,36). Pressures of about 150 MPa induce germination primarily via the nutrient receptor-mediated pathway, while pressures of about 500 MPa induce germination primarily via mechanisms independent of the nutrient receptors.…”

“…While a gerD deletion mutation slowed spore germination triggered by nutrients, there are many nonnutrient agents that can trigger spore germination, including cationic surfactants such as dodecylamine, lysozyme, Ca 2ϩ -DPA, and high pressures (4,5,23,24,26,27,29,30). Many of these agents (dodecylamine, lysozyme, and Ca 2ϩ -DPA) trigger spore germination by pathways different from that for nutrient germination.…”

Superdormant Spores of Bacillus Species Germinate Normally with High Pressure, Peptidoglycan Fragments, and Bryostatin

“…This is consistent with possible roles of nutrient germinant receptor levels and/or heat activation temperature optima in affecting spore superdormancy, since neither dodecylamine nor Ca-DPA triggers Bacillus spore germination through nutrient germinant receptors, and germination with these agents is also not stimulated by heat activation (11,15,17). However, the effects of high pressures, peptidoglycan fragments, and bryostatin, all of which almost certainly trigger spore germination by mechanisms at least somewhat different than triggering of germination by nutrients, dodecylamine, and Ca-DPA (2,3,11,15,20,22,23), have not been tested for their effects on superdormant spores. Consequently, we have compared the germination of dormant and superdormant spores of two Bacillus species by high-pressures, peptidoglycan fragments, and bryostatin.…”