Analysis of factors that influence the sensitivity of spores of Bacillus subtilis to DNA damaging chemicals

Abstract

Aims: To elucidate factors influencing the sensitivity of Bacillus subtilis spores to DNA damaging chemicals. Methods and Results: Wild-type spores of B. subtilis made at lower temperatures were more sensitive to the DNA damaging chemicals formaldehyde and nitrous acid than were spores made at higher temperatures, but this was not the case with the DNA alkylating agents ethylmethanesulphonate and methylmethanesulphonate. Spores lacking most DNA protective a ⁄ b-type small, acid-soluble proteins (termed a ) b) spores) made at lower temperatures were also more sensitive to killing through DNA damage by hydrogen peroxide than were a ) b ) spores made at higher temperatures. The spore coat, whose composition varies significantly with sporulation temperature, played only a minor role in spore resistance to these DNA damaging agents. Spores made at lower temperatures exhibited higher permeability to the methylamine and germinated more rapidly with the surfactant dodecylamine than did spores made at higher temperatures. Treatment of spores with the oxidizing agent cumene hydroperoxide sensitized the surviving spores to all these DNA damaging agents. The fatty acid composition of the inner membrane of spores made at different temperatures differed significantly, but levels of unsaturated fatty acids in the inner membrane did not influence spore resistance to DNA damaging agents or the sensitization to such agents by prior treatment with cumene hydroperoxide.Conclusions: The higher rates of methylamine permeation across the inner membrane of spores made at lower temperatures and the greater sensitivity of wild-type spores made at lower temperatures to formaldehyde and nitrous acid and of a ) b ) spores made at lower temperatures to hydrogen peroxide, all agents that must pass through the spore's inner membrane to damage DNA in the spore core, suggest that the permeability of the inner membrane is a significant factor influencing spore sensitivity to these agents. The sensitization of spores to DNA damaging chemicals by pretreatment with an oxidizing agent, a treatment that increases the permeability of the spore's inner membrane, and the more rapid dodecylamine germination of spores made at lower temperatures are consistent with this suggestion. Significance and Impact of the Study: The results in this communication provide new insight into the factors that influence the resistance of spores of Bacillus species to chemicals that kill spores by damaging spore DNA.

Localization of SpoVAD to the Inner Membrane of Spores of Bacillus subtilis

“…Other factors that might modulate DPA movement out of the spore in germination may be the state of the inner and outer membrane lipids. Inner membrane lipids appear to be largely immobile in the dormant spore, and while the precise physical state of this membrane is not known, it has exceedingly low passive permeability to small molecules, even to molecules as small as uncharged methylamine and perhaps even water (6,7,8,37,42). In contrast, the outer membrane does not appear to have these permeability constraints, if this membrane is a permeability barrier at all.…”

Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

“…However, it is possible that the rate-limiting step in dodecylamine germination is the Ca-DPA movement itself and that the initial effect of dodecylamine is not on the channel itself but on the spore's inner membrane. It is also known that oxidative damage to dormant spores, most likely to the spore's inner membrane, greatly increases the rate of Ca-DPA release triggered by dodecylamine (4,6). While the nature of the oxidative damage that leads to this effect is not known, this damage also results in a large increase in the passive permeability of the spore's inner membrane to molecules such as methylamine and decreases the ability of spores to retain Ca-DPA at normally sublethal temperatures (4,6).…”

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

“…Methylamine, a small molecule that can be accumulated at high levels in spores because of the low core pH, is often used to probe the integrity of the IM because its rate of entry into the spore core is lower than that of water. Indeed, IM permeability to methylamine is very low, and this low permeability is even retained in spores that lack a coat and outer membrane (2,14,15). However, damaging the IM with oxidizing agents can significantly increase its permeability to methylamine (16).…”

“…This is because spores are extremely hardy and can survive mild decontamination procedures that kill growing bacteria. While a number of factors are responsible for spores' high resistance, one factor is their low permeability to many toxic chemicals, in particular chemicals that can damage spore DNA that is located in the central spore core (2)(3)(4)(5)(6)(7). There are a number of permeability barriers in the dormant spore.…”