Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B

Abstract

Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gitS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with gn apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gitP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li'. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, mnonensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 ,IM in the gltP system and 3.5 ,IM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gitS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.

The Escherichia coli mutant requiring D-glutamic acid is the result of mutations in two distinct genetic loci

“…A second possibility was suggested by the fact that E. coli is known to be relatively poor at transporting D-glutamic acid intracellularly (1,6,32). This raised the possibility that E. coli WM335 was in fact a double mutant, having a defect in the dga locus and an additional mutation in the transport locus that influenced the uptake of exogeneous D-glutamate.…”

“…This raised the possibility that E. coli WM335 was in fact a double mutant, having a defect in the dga locus and an additional mutation in the transport locus that influenced the uptake of exogeneous D-glutamate. The most likely candidate of the three known D-glutamate transport systems was gUtS (1,6,12). When the gltS gene of E. coli WM335 was linked to a transposon and transduced into E. coli K-12, it was then possible to subsequently introduce the dga locus from WM335 and obtain a D-glutamate-requiring E. coli K-12.…”

“…The gltS gene from E. coli WM335 was cloned by using SalI-digested genomic DNA and ligation into pUC18. The clone containing the gltS gene was selected in K-12 by growth on M9 minimal medium with glutamate as the sole carbon and nitrogen source (6). A candidate plasmid with a 7.1-kb insert was found to contain the gltS region (pRDD021, Fig.…”

Secondary solute transport in bacteria

“…Finally, glutamate uptake by E. coli has been characterized as glutamate-H+-Na + symport and detailed kinetic analyses of ligand binding and transport have been performed [63,64]. More recently, it has been shown that the observed glutamate-H+-Na ÷ symport is the result of two activities (transport systems), i.e., GltS-mediated glutamate-Na ÷ symport [65] and GltP-mediated glutamate-H + symport [50]. A similar situation holds for proline transport in E. coli and S. typhimurium where the transport activities have been resolved to PutP-mediated proline-Na ÷ and ProPmediated proline-H ÷ symport activities [60,66,67].…”

“…Based on these findings and observations that replacement of either Cys-281 or Cys-344 causes complete resistance to NEM [280], whereas the wild-type PutP protein is inhibited in a substrate protectable manner, a model has been proposed in which Gly-22 (helix I), Cys-141 (helix III), Arg-257, Cys-281 (helix VII) and Cys-344 (helix VIII) are in close contact and form at least part of the cation-binding site [189]. On the basis of some similarity between Na+-solute symporters, a consensus sequence motif (--Gly---Ala .... Leu---GlyArg), corresponding with GlY328-Ala366-Leu371-GlY375Arg376 in PutP, has been implicated in sodium binding [65]. A similar motif has also been detected in the sodium-proton-glutamate symport systems of B. stearothermophilus and B. caldotenax [101], the proton-dependent glutamate uptake system (GItP) of E. coli [50], but, for instance, not in the MelB protein of E. coli [310] and the alanine carrier protein of thermophilic bacterium PS3 [311].…”