Wild-type strains of Escherichia coli K-12 cannot grow in media with 'yaminobutyrate (GABA) as the sole source of carbon or nitrogen. Mutants were isolated which could utilize GABA as the sole source of nitrogen. These mutants were found to have sixto ninefold higher activities of 'y-aminobutyrate-a-835 on July 8, 2020 by guest

Active transport of glutamate by Escherichia coli K-12 requires both Na+ and K+ ions. Increasing the concentration of Na+ in the medium results in a decrease in the Km of the uptake system for glutamate; the capacity is not affected. Glutamate uptake by untreated cells is not stimulated by K+. K+-depleted cells show a greatly reduced capacity for glutamate uptake. Preincubation of such cells in the presence of K + fully restores their capacity for glutamate uptake when Na+ ions are also present in the uptake medium. Addition of either K+ or Na+ alone restores glutamate uptake to only about 20% of its maximum capacity in the presence of both cations. Changes in K-+ concentration affect the capacity for glutamate uptake but have no effect on the Km of the glutamate transport system. Ouabain does not inhibit the (Na+-K+)-stimulated glutamate uptake by intact cells or spheroplasts of E. coli K-12.

Mutants of Escherichia coli K-12 isolated for their ability to utilize-y-aminobutyrate (GABA) as the sole source of nitrogen exhibit a concomitant several-fold increase in the activities of 'y-aminobutyrate-a-ketoglutarate transaminase (GSST, EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16). The increase in rate of enzymatic activity is not accompanied by any changes in the affinities of the mutant enzymes for their respective substrates. The synthesis of the two enzymes is highly coordinate under a great variety of conditions, in spite of the wide range of activities observed. In cultures grown in minimal media with ammonium salts as the source of nitrogen, both GSST and SSDH are severely repressed by glucose. Substitution of ammonia with GABA, glutamate, or aspartate greatly reduces the effect of glucose on the synthesis of the GABA utilization enzymes. This escape from catabolite repression is specific for GSST and SSDH and does not involve other enzymes sensitive to catabolite repression (e.g., ,B-galactosidase, EC 3.2.1.23, and aspartase, EC 4.3.1.1). We have recently shown that wild-type Escherichia coli K-12 strains unable to utilize 'y-aminobutyric acid (GABA) as a sole source of nitrogen exhibit very low activity of-y-aminobutyrateaketoglutarate transaminase ultraviolet mutagenesis as described earlier (1). Media and bacteriological techniques. The composition of growth media and the techniques used for growing bacteria and determination of growth rates were as earlier described (1). Preparation of cell extracts. Cell extracts used for GSST and SSDH determinations were prepared by sonic oscillation as described (1).

The control mutation that results in a concomitant severalfold increase in the activities of y-aminobutyrate-a-ketoglutarate transaminase (GSST, EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16), leading to the acquisition of the ability to utilize y-aminobutyrate (GABA) as the sole source of nitrogen by Escherichia coli K-12 mutants, was mapped by mating and transduction with Plkc. The locus affected, gabC, is approximately 48% co-transduced with the thyA gene, located at min 55 of the E. coli K-12 chromosome. The structural gene of the first enzyme in the GABA pathway, GSST, was mapped by interrupted mating, using one of the GSST-less mutants, DB742, isolated in this work. The mutated locus, gabT, is situated at about min 73 of the E. coli chromosome, close to the gltC gene. Genetic evidence concerning the sensitivity of the enzymes of the GABA pathway to catabolite repression under different physiological conditions suggests that the two structural genes of the GABA regulon do not constitute one operon.

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