Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.
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 gltP) 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 gltP 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 beta-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.
Wallace
B
B
School of Microbiology, University of New South Wales, Kensington, Australia.
Yang
Y J
YJ
Hong
J S
JS
Lum
D
D
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid