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 (gltT(Bs)) and Bacillus caldotenax (gltT(Bc)) 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 gltT(Bs) and gltT(Bc), genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltT(Bs) and GltT(Bc)). 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 GltT(Bs) and GltT(Bc) 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

Adaptation of microorganisms and their transport systems to high temperatures

Growth of Bacteria and Archaea has been observed at temperatures up to 95 and 110 degrees C, respectively. These thermophiles are adapted to environments of high temperature by changes in the membrane lipid composition, higher thermostabilities of the (membrane) proteins, higher turnover rates of the energy transducing enzymes, and/or the (exclusive) use of sodium-ions rather than protons as coupling ion in energy transduction. The proton permeability of the cytoplasmic membrane of bacteria and archaea was observed to increase with the temperature. This increased proton permeability limits the maximum temperature of growth of bacteria. Higher growth temperatures can be reached by an increased proton pumping activity by using the less permeable sodium ions as coupling ions or by changing the lipid composition of the cytoplasmic membrane. The Na+/H+/glutamate transport proteins of the thermophiles Bacillus stearothermophilus (GltT(Bc)) and Bacillus caldotenax (GltT(Bc)) were studied extensively. These transportproteins have unique features. Transport of L-glutamate occurs in symport with 1 Na+ and 1 H+ when the transport proteins are expressed in their natural environment. The sodium ion dependency of the GltT transporters of these Bacillus strains was found to increase with temperature. However, when the GltT proteins are expressed in the mesophile Escherichia coli, electrogenic symport of L-glutamate occurs with greater than or equal to 2 H+. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, and that the conformation influences the coupling ion selectivity. The Na+/H+/glutamate transport proteins of B. stearothermophilus (GltT(Bc)) and B. caldotenax (GltT(Bc)) are homologous to transport systems of glutamate and structurally related compounds from mesophilic organisms. Both sodium, as well as proton coupled transporters, belong to this family of carboxylate transporters (FCT). (C) 1997 Elsevier Science Inc