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A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated glP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing glP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5-and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and (B-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na+-independent, binding-protein-independent glutamate-aspartate transport system. Escherichia coli K-12 strains generally do not grow well on L-glutamate as a sole source of carbon and energy, probably because the transport systems for glutamate are repressed in wild-type strains (11). Mutants with derepressed levels of glutamate uptake were isolated by selecting for sensitivity to toxic analogs of glutamate (6,15). Studies with these and other strains identified five systems for dicarboxylic amino acid transport in E. coli DW2 (15): (i) a binding-protein-independent, Na+-dependent, glutamatespecific system; (ii) a binding-protein-dependent, Na+-independent system for transport of glutamate and aspartate; (iii) a binding-protein-independent, Na+-independent glutamateaspartate system; (iv) a binding-protein-independent, aspartate-specific system; and (v) a dicarboxylic acid transport system that carries aspartate in addition to malate, fumarate, and succinate. Cloning of the genes encoding the various components of the glutamate-aspartate transport systems would provide a method for studying regulation of expression of the genes as well as the nature and function of the gene products.In a recent paper (4), the cloning of two E. coli B genes for glutamate and aspartate transport was described. One gene (gltS) specified a glutamate-specific, Na+-dependent carrier and corresponds to the E. coli K-12 gItS gene mapped at 82 min on the E. coli chromosome (11). The other gene (gltP) specified a Na+-independent, glutamate-aspartate carrier. In this paper, we describe the cloning, expression, sequencing, and function of a gene for glutamate-aspartate transport. Since there are significant similarities between the properties of the gltP gene cloned by Deguchi et al. (4) and the gene d...

The active transport of glutamine by Escherichia coli occurs via a single osmotic shock-sensitive transport system which is known to be dependent upon a periplasmic binding protein specific for glutamine. We obtained a mutant that had elevated levels of glutamine transport and overproduced the glutamine binding protein. From this strain many point mutants and deletion-carrying strains defective in glutamine transport were isolated by a variety of techniques. The genetic locus coding for the glutamine transport system, glnP, and the regulatory mutation which causes overproduction of the transport system were both shown to map at 17.7 min on the E. coli chromosome, and it was demonstrated that the glnP locus contains the structural gene for the glutamine binding protein. Evidence was also obtained that the glutamine transport system, by an unknown mechanism, plays a direct role in the catabolism of glutamate and, hence, of glutamine and proline as well.

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 ,uM L-glutamine in the presence of chloramphenicol (100 ,ug/ml). The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min. The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 ,uM) with respect to the [14C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs y-L-glutamyl hydroxamate (Ki = 360 ,uM) and y-L-glutamyl hydrazide (Ki = 800 ,uM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 ,uM), was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition. A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells. These findings indicate that ammonium transport in E. coli is regulated by the internal glutamine pool via feedback inhibition.

We treated the glutamine binding protein with diethyl pyrocarbonate (DEPC) and N-bromosuccinimide (NBS) to modify respectively the sole histidine and tryptophan residues and examined the effect of these modifications on the ability of the binding protein to bind glutamine as well as the ability to restore glutamine transport in membrane vesicles of Escherichia coli. Under the conditions used, both DEPC and NBS markedly inhibited the ability to restore glutamine transport in vesicles without any significant effect on glutamine binding. Moreover, saturating quantities of glutamine had no protective effect on the inactivation of the binding protein by DEPC or NBS. Fluorometric measurement and amino acid analysis indicate that the inactivation of the binding protein in restoring vesicle transport by NBS can be attributed to the oxidation of a single tryptophan residue. Similar analysis and the inability of hydroxylamine to reverse the effect of DEPC indicate that the effects of DEPC can probably be attributed to alterations of the sole histidine and/or one or more lysine residues of the binding protein. We conclude that the glutamine binding protein possesses at least two largely nonoverlapping functional domains, one responsible for glutamine binding and the other for the interaction with the other components of the glutamine transport system.