Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax.
The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.
Tolner
B
B
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Poolman
B
B
Konings
W N
WN
D000595:Amino Acid Sequence; D026922:Amino Acid Transport Systems, Acidic; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011522:Protons; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D027981:Symporters
D026922:Amino Acid Transport Systems, Acidic; D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003062:Codon; D005971:Glutamates; D011522:Protons; D027981:Symporters; C077711:gltT protein, Bacillus caldotenax; C077709:gltT protein, Bacillus stearothermophilus; D018698:Glutamic Acid; D012964:Sodium