Unbalanced growth and latent killing of Escherichia coli following exposure to sulfur mustard

Abstract

A study has been made of the effects of unbalanced metabolism on the survival of sulfur-mustard-treated Escherichia coli, strains 15T-A-U- (thymine, arginine and uracil requiring) and 15T-PA- (thymine and phenylalanine requiring). The following results were obtained. 1. 1. Pre- and post-treatment growth conditions significantly affected the sensitivity of bacteria to sulfur mustard. The largest resistance was observed when bacteria were cultured before treatment in media containing thymidine, but lacking the required amino acid and uracil. Such bacteria were also immune to thymineless death and more resistant, to other bifunctional alkylating agents, mitomycin C and the decay of incorporated [ 3H]thymidine than were exponentially growing bacteria. 2. 2. The development of the resistant state could not be attributed to the completion of DNA replication, the attainment of the stationary growth phase, or the inhibition of protein synthesis but appeared to be largely due to the low levels of unbalanced metabolism to which the mustard-treated cells were subsequently subjected. Thus, resistant cells, on post-treatment incubation in nutritionally deficient media, were considerably more refractory to latent killing effects and to thymineless death than were exponentially growing bacteria. 3. 3. The latent killing of mustard-treated cells and thymineless death were strikingly similar phenomena. Both processes were often similarly affected by nutritionally induced changes, had the same maximal rates, and were accompanied by a stimulated incorporation of [ 32P]P 1 into a RNA-like product, while unbalanced protein synthesis played only a minor, or indirect role in killing. The most notable difference between latent and thymineless killing was a dose-dependent decrease in the lag before onset of killing. 4. 4. Unbalanced metabolism affected the stability of both DNA and RNA. DNA became increasingly more difficult to isolate in highly polymerized form and some of the DNA was actually degraded to acid-soluble products. Mustard-treated bacteria, during latent killing, rapidly depolymerized 35 % of their DNA, a process which preceded the degradation of RNA, while untreated bacteria, during thymineless death, degraded their DNA much later, following the breakdown of RNA. The depolymerization of DNA did not occur spontaneously but depended on the presence of glucose in the incubation medium, suggesting that the breakdown of bacterial DNA is either the cause or effect of unbalanced metabolism. 5. 5. In contrast to the bifunctional sulfur mustard, the monofunctional analog, β-chloroethyl-β-hydroxyethyl sulfide, was considerably less lethal to bacteria, failed to inhibit bacterial DNA synthesis and did not promote the breakdown of DNA during post-treatment incubation. These results suggest that some cross-alkylating reactions are particularly effective for initiating unbalanced metabolism. 6. 6. The mechanism of death by unbalanced metabolism is discussed with special emphasis on the involvement of nucleases.