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. 1990 Jan;172(1):424–430. doi: 10.1128/jb.172.1.424-430.1990

Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product.

G Sweet 1, C Gandor 1, R Voegele 1, N Wittekindt 1, J Beuerle 1, V Truniger 1, E C Lin 1, W Boos 1
PMCID: PMC208448  PMID: 2152911

Abstract

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.

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Selected References

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  1. Albin R., Silverman P. M. Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome. Mol Gen Genet. 1984;197(2):261–271. doi: 10.1007/BF00330972. [DOI] [PubMed] [Google Scholar]
  2. Berman-Kurtz M., Lin E. C., Richey D. P. Promoter-like mutant with increased expression of the glycerol kinase operon of Escherichia coli. J Bacteriol. 1971 Jun;106(3):724–731. doi: 10.1128/jb.106.3.724-731.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Bremer E., Silhavy T. J., Weisemann J. M., Weinstock G. M. Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step. J Bacteriol. 1984 Jun;158(3):1084–1093. doi: 10.1128/jb.158.3.1084-1093.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cozzarelli N. R., Freedberg W. B., Lin E. C. Genetic control of L-alpha-glycerophosphate system in Escherichia coli. J Mol Biol. 1968 Feb 14;31(3):371–387. doi: 10.1016/0022-2836(68)90415-4. [DOI] [PubMed] [Google Scholar]
  8. Cozzarelli N. R., Lin E. C. Chromosomal location of the structural gene for glycerol kinase in Escherichia coli. J Bacteriol. 1966 May;91(5):1763–1766. doi: 10.1128/jb.91.5.1763-1766.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Freedberg W. B., Lin E. C. Three kinds of controls affecting the expression of the glp regulon in Escherichia coli. J Bacteriol. 1973 Sep;115(3):816–823. doi: 10.1128/jb.115.3.816-823.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Freundlieb S., Boos W. Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. J Biol Chem. 1986 Feb 25;261(6):2946–2953. [PubMed] [Google Scholar]
  11. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  12. HAYASHI S., LIN E. C. CAPTURE OF GLYCEROL BY CELLS OF ESCHERICHIA COLI. Biochim Biophys Acta. 1965 Mar 29;94:479–487. doi: 10.1016/0926-6585(65)90056-7. [DOI] [PubMed] [Google Scholar]
  13. Heller K. B., Lin E. C., Wilson T. H. Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli. J Bacteriol. 1980 Oct;144(1):274–278. doi: 10.1128/jb.144.1.274-278.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Icho T., Bulawa C. E., Raetz C. R. Molecular cloning and sequencing of the gene for CDP-diglyceride hydrolase of Escherichia coli. J Biol Chem. 1985 Oct 5;260(22):12092–12098. [PubMed] [Google Scholar]
  15. KOCH J. P., HAYASHI S., LIN E. C. THE CONTROL OF DISSIMILATION OF GLYCEROL AND L-ALPHA-GLYCEROPHOSPHATE IN ESCHERICHIA COLI. J Biol Chem. 1964 Sep;239:3106–3108. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lin E. C. Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol. 1976;30:535–578. doi: 10.1146/annurev.mi.30.100176.002535. [DOI] [PubMed] [Google Scholar]
  18. Lopilato J., Bortner S., Beckwith J. Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet. 1986 Nov;205(2):285–290. doi: 10.1007/BF00430440. [DOI] [PubMed] [Google Scholar]
  19. Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Minton N. P. Improved plasmid vectors for the isolation of translational lac gene fusions. Gene. 1984 Nov;31(1-3):269–273. doi: 10.1016/0378-1119(84)90220-8. [DOI] [PubMed] [Google Scholar]
  22. Pettigrew D. W., Ma D. P., Conrad C. A., Johnson J. R. Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme. J Biol Chem. 1988 Jan 5;263(1):135–139. [PubMed] [Google Scholar]
  23. Pichersky E., Gottlieb L. D., Hess J. F. Nucleotide sequence of the triose phosphate isomerase gene of Escherichia coli. Mol Gen Genet. 1984;195(1-2):314–320. doi: 10.1007/BF00332765. [DOI] [PubMed] [Google Scholar]
  24. Postma P. W., Lengeler J. W. Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol Rev. 1985 Sep;49(3):232–269. doi: 10.1128/mr.49.3.232-269.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Power J. The L-rhamnose genetic system in Escherichia coli K-12. Genetics. 1967 Mar;55(3):557–568. doi: 10.1093/genetics/55.3.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  27. Richey D. P., Lin E. C. Importance of facilitated diffusion for effective utilization of glycerol by Escherichia coli. J Bacteriol. 1972 Nov;112(2):784–790. doi: 10.1128/jb.112.2.784-790.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ross D. G., Swan J., Kleckner N. Nearly precise excision: a new type of DNA alteration associated with the translocatable element Tn10. Cell. 1979 Apr;16(4):733–738. doi: 10.1016/0092-8674(79)90089-8. [DOI] [PubMed] [Google Scholar]
  29. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanno Y., Wilson T. H., Lin E. C. Control of permeation to glycerol in cells of Escherichia coli. Biochem Biophys Res Commun. 1968 Jul 26;32(2):344–349. doi: 10.1016/0006-291x(68)90392-6. [DOI] [PubMed] [Google Scholar]
  31. Schweizer H., Boos W., Larson T. J. Repressor for the sn-glycerol-3-phosphate regulon of Escherichia coli K-12: cloning of the glpR gene and identification of its product. J Bacteriol. 1985 Feb;161(2):563–566. doi: 10.1128/jb.161.2.563-566.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simons R. W., Hoopes B. C., McClure W. R., Kleckner N. Three promoters near the termini of IS10: pIN, pOUT, and pIII. Cell. 1983 Sep;34(2):673–682. doi: 10.1016/0092-8674(83)90400-2. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  35. Zhu Y., Lin E. C. L-1,2-propanediol exits more rapidly than L-lactaldehyde from Escherichia coli. J Bacteriol. 1989 Feb;171(2):862–867. doi: 10.1128/jb.171.2.862-867.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Jong W. W., Zweers A., Cohen L. H. Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem Biophys Res Commun. 1978 May 30;82(2):532–539. doi: 10.1016/0006-291x(78)90907-5. [DOI] [PubMed] [Google Scholar]

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