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

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Abstract
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 −2 m d -cycloserine. At concentrations above 2 × 10 −2 m , d -cycloserine was bacteriostatic. Low levels of d -cycloserine (10 −5 m ) 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 −3 m , 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 −3 m 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 met 1 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.