Bernard S. Strauss
1961 Journal of Bacteriology  
During the course of our studies on the mechanism of action of the mutagen ethyl sulfate (Strauss and Okubo, 1960) we decided to test the effect of incubation in buffers of various composition after treatment with the mutagenic agent. It was found that incubation of treated cells in citrate had a lethal effect and it was decided to investigate the reason for this lethality. As a result of the investigations to be described below, we have concluded that treatment with ethyl sulfate leads to
more » ... lfate leads to changes in the permeability of the treated cells which permit the ready removal of intracellular ions by chelating agents. The removal of these ions, particularly magnesium ion, leads to breakdown of the intracellular nucleic acid and to cell death. MATERIALS AND METHODS Strains td-2, a tryptophan-requiring mutant of Escherichia coli strain K-12 (obtained from C. Yanofsky) and WP-2, a tryptophan-requiring mutant of E. coli strain B/r (obtained from E. Witkin) were used in these studies. Cultures were prepared as follows: a 24-hr slant of the organism was used to inoculate 50 or 100 ml of a mineral salts medium containing 40 g of DL-tryptophan per ml with 0.25% glutamic acid and 0.3% glucose, all brought to pH 7.2. The salts were those in the minimal medium C described by Roberts et al. (1955). Cultures were incubated 22 to 24 hr with aeration, harvested by centrifugation, and then washed twice with 0.15 M saline. Cells were suspended in saline before use. Survival was measured by serial dilution in saline followed by plating 0.1-ml samples on petri dishes containing the growth medium hardened with 1.5% agar. The frequency of 1 Supported in part by grants RG-6171 and RG-7816 from the U. S. Public Health Service and by contract between Syracuse University and the U. S. Atomic Energy Commission. A portion of this work was done in the Department of Zoology, Syracuse University. revertants from tryptophan requirement to nonrequirement was measured by plating 0.1 ml of an undiluted suspension on solidified growth medium with tryptophan omitted. The data are presented so that the average number of revertants per plate can be calculated from the revertant frequency and the viability. In some experiments, cells were harvested from the incubation medium and resuspended in saline before plating, in others, especially for kinetic studies, plating was direct from the incubation medium on the assumption that the small volume of buffer would be sufficiently diluted by the agar medium to stop its action. Platings were in triplicate and, for survival, at different dilutions so that significant numbers of colonies could be counted on plates that were not too crowded. The ethyl sulfate (diethyl sulfate) was obtained from Eastman Organic Chemicals and was purified by vacuum distillation before use. An ethanolic solution was made by adding 0.8 ml of ethyl sulfate to 4.2 ml of ethanol as previously described (Strauss and Okubo, 1960) . Cells were treated by adding 0.4 ml of this solution to 40 ml of a buffered suspension to give a solution approximately 0.0125 M. Rapid killing due to hydrolysis of the ethyl sulfate occurred if the medium was not sufficiently buffered. Ethanol alone is not sufficient to produce the effects reported in this paper; we have routinely added this substance to flasks in which control suspensions are incubated. However, the ethyl sulfate disperses itself over the surface of the suspension as a fine oil only when added as an ethanolic solution. This oil disappears soon after the ethyl sulfate is added to a bacterial suspension which is kept agitated, but the ethyl sulfate coalesces and forms large globules at the bottom of the flask when an ethanolic solution is added to buffer alone. Cells were treated with ethyl sulfate by incubation in buffer plus the alkylating agent for 573 on May 8, 2020 by guest
doi:10.1128/jb.81.4.573-580.1961 fatcat:w627qjxwmzarhj23pz6inc47fy