CHARACTERIZATION OF THE PROTON GLUTAMATE SYMPORT PROTEIN OF BACILLUS-SUBTILIS AND ITS FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI

Transport of acidic amino acids in Bacillus subtilis is an electrogeni c process in which L-glutamate or L-aspartate is symported with at lea st two protons. This is shown by studies of transport in membrane vesi cles in which a proton motive force is generated by oxidation of ascor bate-phenazine methosulfate or by artificial ion gradients. An inwards directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-gl utamate and L-aspartate. L-Glutamate transport is inhibited by beta-hy droxyaspartate and cysteic acid but not by alpha-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltP( Bsu)) was cloned by complementation of Escherichia coli JC5412 for gro wth on glutamate as the sole source of carbon, energy, and nitrogen, a nd its nucleotide sequence was determined. Putative promoter, terminat or, and ribosome binding site sequences were found in the flanking reg ions. UUG is most likely the start codon, gltP(Bsu) encodes a polypept ide of 414 amino acid residues and is homologous to several proteins t hat transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium- and proton co upled transporters belong to this family of dicarboxylate transporters . Hydropathy profiling and multiple alignment of the family of carboxy late transporters suggest that each of the proteins spans the cytoplas mic membrane 12 times with both the amino and carboxy termini on the i nside.