Aims: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. Methods and Results: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone TM , ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. Conclusions: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. 2004, 97, 838-852 doi:10.1111/j.1365-2672.2004.02370.x Significance and Impact of the Study: The more rapid heat killing and germination with dodecylamine, the g...
Spores of Bacillus species normally initiate germination in response to specific nutrients (for reviews, see references 17 and 25). The identity of these nutrients varies in a species-and strain-specific manner, although common nutrient germinants are amino acids, sugars, and purine nucleosides. Metabolism of nutrient germinants is not what triggers spore germination. Rather, the binding of nutrients to receptors located in the spore's inner membrane triggers subsequent events including (i) the release of monovalent ions, (ii) the release of the spore core's large depot of divalent cations bound to pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), and (iii) hydrolysis of the spore's peptidoglycan cortex. The nutrient receptors are encoded by tricistronic operons. Where studied, these operons are expressed only in the developing spore late in sporulation, and loss of any cistron of a particular nutrient receptor operon eliminates the function of that receptor.B. subtilis spores contain three functional nutrient receptors encoded by the gerA, gerB, and gerK operons, and each receptor or group of receptors responds to different germinants and cogerminants (Fig.
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.
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