Catabolite repression in Streptomycin-dependent Escherichia coli
Creators: name: Coukell, Milton Barrie
Publisher: name: University of British Columbia
Year: 2011

description: Biosynthetic reactions in micro-organisms normally are under precise control, usually by a feed-back mechanism in which the end product inhibits the activity and/or represses the formation of the enzyme which initiates the biosynthetic pathway. Streptomycin (Sm)-dependent mutants of Escherichia coli, unlike the parent wild-type strains excrete the amino acid, L-valine, thereby indicating a loss of precise regulation in the biosynthesis of this amino acid (Tirunarayanan, Vischer and Renner, 1962; Bragg and Polglase, 1962). The initiating enzyme for valine biosynthesis, acetohydroxy acid (AHA) synthetase, is derepressed in Sm-dependent E. coli (Coukell and Polglase, 1965), thus providing an explanation for the excretion of valine by this mutant. However, it was apparent that the extent and cause of regulatory insufficiency in Sm-dependent E. coli required further investigation. A study of the effect of the carbon source used for growth on AHA synthetase formation revealed that in wild-type E. coli B this enzyme was subject to catabolite repression. End-product inhibition of AHA synthetase by L-valine in E. coli B attained a maximum at 60-70% inhibition. These previously unreported properties of AHA synthetase (sensitivity to catabolite repression and incomplete end-product inhibition) are significant in the regulation of the biosynthetic pathway leading to the aliphatic amino acids and pantothenate. In Sm-dependent E. coli B, growing with non-limiting antibiotic, catabolite repression of AHA synthetase was relaxed. Additional evidence for relaxation of catabolite repression in Sm-dependent E. coli was provided by the observation that Sm-dependent mutants of E. coli (strains B and E) were inducible for β-galactosidase in the presence of glucose Furthermore, several glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and iso-citrate dehydrogenase) were found to be insensitive to variation in the nature of the carbon source in a Sm-dependent mutant. Cell yield experiments revealed that aerobic glucose metabolism in the Sm-dependent mutant was one-third less efficient than in the Sm-sensitive strain, although the two strains were equally efficient under anaerobic conditions. Moreover, the rate of ATP synthesis in the Sm-dependent mutant was less than that of the wild-type parent organism. Therefore, relaxation of catabolite repression in Sm-dependent E. coli B appears to result from an impairment of aerobic energy metabolism in this mutant. Catabolite repression in Sm-dependent E. coli B under conditions of antibiotic-limitation was investigated. The growth rate of Sm-dependent E. coli B on limiting concentrations of dihydrostreptomycin (DHSm) was evaluated by means of a constant (K DHSm ) relating half-maximal growth rate to antibiotic concentration. K DHSm varied with the nature of the carbon source being highest with energy-rich compounds (e.g. gluconate) and lowest with energy-poor compounds (e.g. lactate). Glucose-sensitive enzymes of Sm- dependent E. coli B were specifically repressed by antibiotic-limitation and exhibited specific activities lower than those observed for the same enzymes in glucose-grown extracts of wild-type E. coli B. Parallelism was observed between decreasing antibiotic concentration, decreasing growth rate, and increasing catabolite repression of certain glucose-sensitive enzymes (notably AHA synthetase and fumarase). The decreased efficiency of aerobic glucose metabolism of Sm-dependent E. coli B was not affected by variation in the concentration of antibiotic. Thus, it is improbable that carbohydrate metabolism is the antibiotic dependent site in the Sm-dependent mutant. The results are compatible with the hypothesis of Spotts and Stanier (1961) that the primary site of action of DHSm in the Sm-dependent organism is the ribosome (i.e . , protein synthesis). However, the growth-limiting effect of antibiotic deprival appears to be augmented by catabolite repression. Additionally, the Sm-dependent mutant is deficient in energy metabolism which can explain the relaxation of control by catabolite repression when antibiotic is present in non-limiting concentration.