Yeheskel S. Halpern

Glutamate-binding protein and its relation to glutamate transport in escherichia coli K-12

Abstract A highly specific, energy-dependent active transport system of glutamate has been found in Escherichia coli K-12. Mutants capable of utilizing glutamate as the sole carbon and energy source exhibit several-fold higher rates of glutamate uptake than the wild-type parent. Spheroplasts prepared from one such mutant, SC7, retain only 10–30% of the glutamate uptake capacity of intact cells. The capacity of spheroplasts for glutamate uptake can be fully restored by the addition of concentrated supernatant fluid obtained in the preparation of spheroplasts. This supernatant contains a protein fraction capable of binding L-glutamate with a K m of 6.7 × 10 −6 M. L-glutamate-γ-methyl ester, a competitive inhibitor of glutamate uptake also inhibits competitively the binding of glutamate to the protein. L-alanine, a non-competitive inhibitor of glutamate uptake, inhibits its binding by the protein in a non-competitive fashion.

The metabolic pathway of glutamate in escherichia coli K-12

Abstract Mutants unable to grow on glutamate as the sole source of carbon and energy, isolated from glutamate-utilizing Escherichia coli K-12 strains, are described. One mutant had a very low aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1, formerly known as glutamate-oxaloacetate transaminase) activity (10% of wild-type activity). Another mutant lacked aspartate ammonialysae ( l -aspartate ammonia-lysae, EC 4.3.1.1, formerly known as aspartase) activity completely. These two mutants were unable to grow in a glutamate-minimal medium either at 30° or at 42°. A third mutant was selected for its inability to grow on glutamate at 42° but it could utilize glutamate for growth at 30°. This mutant was shown to have a thermolabile aspartate ammonia-lyase. All three mutants exhibited normal (wild-type) levels of glutamate dehydrogenase ( l -glutamate:NADP oxido-reductase (deaminating), EC 1.4.1.4) activity. On the basis of these data and earlier findings from this and other laboratories it is concluded that the major pathway of glutamate metabolism in E. coli is via trans-amination with oxaloacetate to give α-ketoglutarate and aspartate, and subsequent deamination of the aspartate to fumarate.

Sodium-dependent glutamate transport in membrane vesicles of Escherichia coli K-12

Mutants of E. coli K-l 2 which utilize glutamate as a major carbon source transport glutamate more effectively than wild type strains which are unable to grow on this amino acid [l-4] . Glutamate transport in one such mutant, strain CS7, has been shown to require sodium which increases the apparent affinity of the transport system for glutamate, but does not affect its capacity [5]. It has also been reported that E. coli K-12 exhibits carrier-mediated, first-order efflux of glutamate; however, the efflux rate is not altered by mutations which increase the rate of glutamate uptake [6-81. Recently, a specific glutamate-binding protein has been isolated from strain CS7, and purified to homogeneity [9,10]. The K, for Lglutamate binding to this protein and the Ki values for certain glutamate analogues are similar to the appropriate kinetic values obtained for glutamate transport in intact cells. Moreover, mutants with increased glutamate transport activity have almost twice as much glutamate-binding protein as the wild-type parent [9,10]. Although these findings implicate the glutamate-binding protein in transport, membrane vesicles prepared from glutamateutilizing mutants transport glutamate more than 1 Otimes better than wild-type preparations, but have no detectable binding protein [ 1 l] . The present study demonstrates that glutamate transport in membrane vesicles of strain CS7 also requires sodium, while no such requirement is apparent for glutamate binding to purified binding protein.

Effect of glucose and other carbon compounds on the transport of α-methylglucoside in Escherichia coli K12

Abstract The effect of glucose, succinate, sorbitol, glycerol and arabinose on the rate of uptake of α-[ 14 C]methylglucoside by Escherichia coli K12 was studied in comparison with the effect of these substances on the rate of α-methylglucoside exit from cells preloaded with the radioactive compound. Glucose was a very potent inhibitor of uptake and greatly accelerated exit, its effect on the former reaction exceeding four-fold that on the latter. Each of the other compounds tested was much less active than glucose and affected both uptake and exit of methylglucoside to a similar extent. The inhibition of uptake and the acceleration of exit by glucose were not affected by sodium azide. On the other hand, azide practically abolished the effect of succinate and glycerol, both on the uptake and exit reactions and reduced to 50% the effect of sorbitol. These results provide further support for the conclusion reached by Hoffee et al. that α-methylglucoside is transported by a glucose permease and that the inhibitory effect of metabolizable compounds on its uptake is due to energy supply for the exit reaction. Another possible mechanism for acceleration of exit in the presence of hexitols and sugars is also discussed.

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Further studies of glutamate transport in Escherichia coli. some features of the exit process

Abstract The rate of loss of radioactive glutamate by preloaded Escherichia coli K-12 cells after dilution into glutamate-free medium was studied. The exit process is a first order reaction strongly dependent on temperature. The Q10 of the reaction in the glutamate-utilizing mutant, CS1, was 2.4. NaN3 accelerated glutamate exit by a factor of about 2.5; the Q10 of exit in the presence of NaN3 was approx. 1.8. Non-radioactive L -glutamate, DL -glutamate-γ-methyl ester, L -alanine and L -aspartate, all of which inhibit glutamate uptake in this organism, accelerated the rate of loss of the amino acid by preloaded cells. Glycine, which does not inhibit uptake, also had no effect on the exit reaction. Growth on glutamate did not enhance the exit reaction; preloaded glutamate-grown cells lost glutamate to the medium at a somewhat slower (by about 20%) rate than did cells grown on succinate. E. coli K-12 strain CS101, unable to grow on glutamate as the source of carbon because of poor uptake of this amino acid, also showed a rapid exit process; the latter was even slightly faster than in the glutamate-utilizing mutant (by some 20%). The mechanism of glutamate exit and its relationship to the uptake process are discussed.

Comparison of some physicochemical and catalytic properties of glutamate decarboxylase from various Escherichia coli K-12 sources

Abstract A wide variation was found in the specific activity of l -glutamate decarboxylase (EC 4.1.1.15) among genetically related Escherichia coli K-12 strains grown under different conditions. Results are presented showing that the K m values for two substrates, l -glutamate and l -glutamate-γ-methyl ester, the K i values for five competitive inhibitors of the l -glutamate decarboxylase reaction, and the absorption spectrum, heat stability and electrophoretic mobility of l -glutamate decarboxylase, were practically identical when tested with four different preparations from strains CS101 and CS7B grown on glucose or succinate at different temperatures. It is concluded that l -glutamate decarboxylase obtained from different strains grown under different conditions represents the same molecular species, and that the wide variation in specific activity observed is due to differences in the amount of enzyme formed by the different cultures

Effect of glucose on the utilization of succinate and the activity of tricarboxylic acid-cycle enzymes in Escherichia coli

Abstract When cultures of Escherichia coli growing exponentially in a glucose-saltys medium were transferred to a similar medium containing succinate as the sole source of carbon, a lag in growth of about 4 h was observed. The rate of O2 uptake by non-proliferating suspensions of glucose-grown cells in the presence of succinate was about 9% of that observed with succinate-grown cells. The rate of O2 uptake by cell-free extracts of glucose-grown cells in the presence of succinate (and other tricaboxylic acid cycle intermediates) and the activity of tricaboxylic acid-cylce enzymes in these extracts were 2–4-fold lower than the rate of O2 uptake and the coresponding enzymatic activities in extracts of succinate-grown cells. The intracellular concentration of “succinate” following incubation with [14C]succinate was 3–5 fold higher in succinate- grown than in glucose-grown cells. It is concluded that the lag in growth observed upon transfer of glucose-grown E. coli to a succinate medium is a combined effect of the repression by glucose of tricarboxylic acid-cycle enzymes and the active transport of succinate into the cell.

Induction and repression of glutamic acid decarboxylase in Escherichia coli

Abstract The effects of the carbon source and temperature on the formation of glutamic decarboxylase by Escherichia coli strains H and W and by various mutants derived from these strains have been studied. Decarboxylase formation by strain H was not appreciably affected by the carbon source used or by temperature. Strain H/Gl1, when grown at 37°, showed inducer requirement in the presence of glucose and other sugars and repression by succinate and pyruvate which could not be overcome by glutamate. At 30° this mutant behaved like the parent strain H. The other strains tested showed different patterns of physiological control of decarboxylase synthesis. It is suggested that glutamate induction and succinate repression might represent different regulatory mechanisms of a temperature-sensitive control system.

Mutations affecting the regulation of γ-aminobutyrate utilization inEscherichia coli K-12

Four genes,gabCPDT, are involved in the utilization of γ-aminobutyrate (GABA) byEscherichia coli K-12. Thegab gene cluster maps nearrecA andsrl, at 57.5 min.gabP, gabD andgabT specify the synthesis of GABA transport carrier, succinic semialdehyde dehydrogenase (SSDH), and glutamate-succinic semialdehyde transaminase (GSST), respectively;gabC controls the synthesis of all three proteins. GABA-nonutilizing mutants carrying deletions insrl extended into thegab cluster have been isolated. The mutants completely lost the capacity for GABA transport, while preserving full activity of GSST and SSDH, suggesting thatgabC is not a promoter-operator locus or a gene coding for an activator protein. A mutation ingabD (M-16) that abolished SSDH activity had the following additional properties: It exerted a bipolar effect on the neighboring genes, greatly reducing the activities of GSST and SSDH; the polar effect ongabP but not ongabT was fully suppressed by the knownrho mutation suA78; at least three classes of GABA-utilizing revertants of M-16 were obtained: (i) revertants with allgab activities restored to the parental levels; (ii) revertants with SSDH activity still missing, but with the other activities fully repaired; (iii) revertants with no SSDH activity, with GSST partly recovered, but with transport fully repaired. It is suggested that thegab cluster is transcribed bidirectionally from a promoter in thegabD region and that the mutation in strain M-16 may be due to DNA insertion in that region.