Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Jun;172(6):3214–3220. doi: 10.1128/jb.172.6.3214-3220.1990

Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

B Wallace 1, Y J Yang 1, J S Hong 1, D Lum 1
PMCID: PMC209127  PMID: 1971622

Abstract

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.

Full text

PDF
3214

Images in this article

3216Image
on p.3216
3217Image
on p.3217

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  2. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Deguchi Y., Yamato I., Anraku Y. Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B. J Bacteriol. 1989 Mar;171(3):1314–1319. doi: 10.1128/jb.171.3.1314-1319.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Goldrick D., Yu G. Q., Jiang S. Q., Hong J. S. Nucleotide sequence and transcription start point of the phosphoglycerate transporter gene of Salmonella typhimurium. J Bacteriol. 1988 Aug;170(8):3421–3426. doi: 10.1128/jb.170.8.3421-3426.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. Marcus M., Halpern Y. S. Genetic analysis of the glutamate permease in Escherichia coli K-12. J Bacteriol. 1969 Mar;97(3):1118–1128. doi: 10.1128/jb.97.3.1118-1128.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Masters P. S., Hong J. S. Genetics of the glutamine transport system in Escherichia coli. J Bacteriol. 1981 Sep;147(3):805–819. doi: 10.1128/jb.147.3.805-819.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Nohno T., Saito T., Hong J. S. Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ). Mol Gen Genet. 1986 Nov;205(2):260–269. doi: 10.1007/BF00430437. [DOI] [PubMed] [Google Scholar]
  10. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Schellenberg G. D., Furlong C. E. Resolution of the multiplicity of the glutamate and aspartate transport systems of Escherichia coli. J Biol Chem. 1977 Dec 25;252(24):9055–9064. [PubMed] [Google Scholar]
  12. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Willis R. C., Furlong C. E. Interactions of a glutamate-aspartate binding protein with the glutamate transport system of Escherichia coli. J Biol Chem. 1975 Apr 10;250(7):2581–2586. [PubMed] [Google Scholar]
  14. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES