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

Abstract

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 x 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 x 10~3 and 5 x 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-enrich-ment cycling technique was developed for isolation of auxotrophic mutants. D-Cycloserine at 2 x 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 met1 locus. Trans-duction 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.