Kinetic and Genetic Analyses of d-Cycloserine Inhibition and Resistance in Escherichia coli

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Curtiss, Roy, III (Oak Ridge National Laboratory, Oak Ridge, Tenn.), Leigh J. Charamella, Claire M. Berg, and Paula E. Harris. Kinetic and genetic analyses of d-cycloserine inhibition and resistance in Escherichia coli. J. Bacteriol. 90:1238–1250.1965.—Wild-type cells of Escherichia coli growing at 37 C in mineral salts-glucose medium with vigorous aeration were lysed at maximal exponential rates by 10−4 to 10−2md-cycloserine. At concentrations above 2 × 10−2m, d-cycloserine was bacteriostatic. Low levels of d-cycloserine (10−5m) and pencillin G (10 units per ml) interacted synergistically to cause a rapid exponential rate of lysis. Spontaneous mutations to d-cycloserine resistance occurred in discrete steps at frequencies of 10−6 to 10−7 for each step. First-, second-, and third-step d-cycloserine-resistant mutants were lysed at maximal exponential rates by d-cycloserine concentrations of 10−3, 3 × 10−3, and 5 × 10−3m, respectively. d-Alanine, l-alanine, and dl-alanyl-dl-alanine reversed d-cycloserine-induced lysis, in that order of effectiveness. On the basis of these observations, a d-cycloserine-enrichment cycling technique was developed for isolation of auxotrophic mutants. d-Cycloserine at 2 × 10−3m was as efficient as penicillin G (1,000 units per ml) for mutant enrichment in E. coli and should be useful for isolation of mutants in penicillin-resistant microorganisms. Bacterial conjugation experiments indicated that all three mutations conferring d-cycloserine resistance were linked to the met1 locus. Transduction experiments showed that the mutation conferring first-step resistance was at least 0.5 min away from the mutations conferring second- and third-step resistance. The latter two mutations possibly occurred in the same gene, since they were sometimes carried in the same transducing phage. Studies on expression of d-cycloserine resistance indicated that these mutations were neither dominant nor recessive to each other nor to the d-cycloserine-sensitivity allele. Each allelic state exerted its influence on the phenotype independently of the others. These results are discussed in terms of the known inhibition of alanine racemase and d-alanyl-d-alanine synthetase by d-cycloserine.

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