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. 1997 Sep;179(17):5570–5573. doi: 10.1128/jb.179.17.5570-5573.1997

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

M F Varela 1, R J Brooker 1, T H Wilson 1
PMCID: PMC179430  PMID: 9287014

Abstract

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

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Selected References

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  1. Brooker R. J., Wilson T. H. Isolation and nucleotide sequencing of lactose carrier mutants that transport maltose. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3959–3963. doi: 10.1073/pnas.82.12.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collins J. C., Permuth S. F., Brooker R. J. Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose. J Biol Chem. 1989 Sep 5;264(25):14698–14703. [PubMed] [Google Scholar]
  5. Eelkema J. A., O'Donnell M. A., Brooker R. J. An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. II. Second site revertants of the thiodigalactoside-dependent proton leak by the Val177/Asn319 permease. J Biol Chem. 1991 Mar 5;266(7):4139–4144. [PubMed] [Google Scholar]
  6. Foster D. L., Boublik M., Kaback H. R. Structure of the lac carrier protein of Escherichia coli. J Biol Chem. 1983 Jan 10;258(1):31–34. [PubMed] [Google Scholar]
  7. Franco P. J., Eelkema J. A., Brooker R. J. Isolation and characterization of thiodigalactoside-resistant mutants of the lactose permease which possess an enhanced recognition for maltose. J Biol Chem. 1989 Sep 25;264(27):15988–15992. [PubMed] [Google Scholar]
  8. Frillingos S., Kaback H. R. Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification. Biochemistry. 1996 Apr 2;35(13):3950–3956. doi: 10.1021/bi952601m. [DOI] [PubMed] [Google Scholar]
  9. Goswitz V. C., Brooker R. J. Isolation of lactose permease mutants which recognize arabinose. Membr Biochem. 1993 Jan-Mar;10(1):61–70. doi: 10.3109/09687689309150253. [DOI] [PubMed] [Google Scholar]
  10. Hobson A. C., Gho D., Müller-Hill B. Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene. J Bacteriol. 1977 Sep;131(3):830–838. doi: 10.1128/jb.131.3.830-838.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jung K., Jung H., Kaback H. R. Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling. Biochemistry. 1994 Apr 5;33(13):3980–3985. doi: 10.1021/bi00179a026. [DOI] [PubMed] [Google Scholar]
  12. Kaback H. R., Frillingos S., Jung H., Jung K., Privé G. G., Ujwal M. L., Weitzman C., Wu J., Zen K. The lactose permease meets Frankenstein. J Exp Biol. 1994 Nov;196:183–195. doi: 10.1242/jeb.196.1.183. [DOI] [PubMed] [Google Scholar]
  13. Kaback H. R., Jung K., Jung H., Wu J., Privé G. G., Zen K. What's new with lactose permease. J Bioenerg Biomembr. 1993 Dec;25(6):627–636. doi: 10.1007/BF00770250. [DOI] [PubMed] [Google Scholar]
  14. King S. C., Hansen C. L., Wilson T. H. The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli. Biochim Biophys Acta. 1991 Feb 25;1062(2):177–186. doi: 10.1016/0005-2736(91)90390-t. [DOI] [PubMed] [Google Scholar]
  15. King S. C., Wilson T. H. Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier. Replacement of histidine 322 by tyrosine or phenylalanine. J Biol Chem. 1989 May 5;264(13):7390–7394. [PubMed] [Google Scholar]
  16. King S. C., Wilson T. H. Identification of valine 177 as a mutation altering specificity for transport of sugars by the Escherichia coli lactose carrier. Enhanced specificity for sucrose and maltose. J Biol Chem. 1990 Jun 15;265(17):9638–9644. [PubMed] [Google Scholar]
  17. Krämer R. Functional principles of solute transport systems: concepts and perspectives. Biochim Biophys Acta. 1994 Mar 29;1185(1):1–34. doi: 10.1016/0005-2728(94)90189-9. [DOI] [PubMed] [Google Scholar]
  18. Lee J. I., Hwang P. P., Hansen C., Wilson T. H. Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli. J Biol Chem. 1992 Oct 15;267(29):20758–20764. [PubMed] [Google Scholar]
  19. Lee J. I., Hwang P. P., Wilson T. H. Lysine 319 interacts with both glutamic acid 269 and aspartic acid 240 in the lactose carrier of Escherichia coli. J Biol Chem. 1993 Sep 25;268(27):20007–20015. [PubMed] [Google Scholar]
  20. Lee J. I., Varela M. F., Wilson T. H. Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli. Biochim Biophys Acta. 1996 Jan 12;1278(1):111–118. doi: 10.1016/0005-2736(95)00209-x. [DOI] [PubMed] [Google Scholar]
  21. Maloney P. C. Bacterial transporters. Curr Opin Cell Biol. 1994 Aug;6(4):571–582. doi: 10.1016/0955-0674(94)90079-5. [DOI] [PubMed] [Google Scholar]
  22. Markgraf M., Bocklage H., Müller-Hill B. A change of threonine 266 to isoleucine in the lac permease of Escherichia coli diminishes the transport of lactose and increases the transport of maltose. Mol Gen Genet. 1985;198(3):473–475. doi: 10.1007/BF00332941. [DOI] [PubMed] [Google Scholar]
  23. Müller-Hill B., Crapo L., Gilbert W. Mutants that make more lac repressor. Proc Natl Acad Sci U S A. 1968 Apr;59(4):1259–1264. doi: 10.1073/pnas.59.4.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Newman M. J., Foster D. L., Wilson T. H., Kaback H. R. Purification and reconstitution of functional lactose carrier from Escherichia coli. J Biol Chem. 1981 Nov 25;256(22):11804–11808. [PubMed] [Google Scholar]
  25. Olsen S. G., Greene K. M., Brooker R. J. Lactose permease mutants which transport (malto)-oligosaccharides. J Bacteriol. 1993 Oct;175(19):6269–6275. doi: 10.1128/jb.175.19.6269-6275.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Poolman B., Konings W. N. Secondary solute transport in bacteria. Biochim Biophys Acta. 1993 Nov 2;1183(1):5–39. doi: 10.1016/0005-2728(93)90003-x. [DOI] [PubMed] [Google Scholar]
  27. Shuman H. A., Beckwith J. Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system. J Bacteriol. 1979 Jan;137(1):365–373. doi: 10.1128/jb.137.1.365-373.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Teather R. M., Müller-Hill B., Abrutsch U., Aichele G., Overath P. Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector. Mol Gen Genet. 1978 Feb 27;159(3):239–248. doi: 10.1007/BF00268260. [DOI] [PubMed] [Google Scholar]
  29. Ujwal M. L., Jung H., Bibi E., Manoil C., Altenbach C., Hubbell W. L., Kaback H. R. Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy. Biochemistry. 1995 Nov 14;34(45):14909–14917. doi: 10.1021/bi00045a036. [DOI] [PubMed] [Google Scholar]
  30. Varela M. F., Wilson T. H. Molecular biology of the lactose carrier of Escherichia coli. Biochim Biophys Acta. 1996 Aug 7;1276(1):21–34. doi: 10.1016/0005-2728(96)00030-8. [DOI] [PubMed] [Google Scholar]
  31. Vogel H., Wright J. K., Jähnig F. The structure of the lactose permease derived from Raman spectroscopy and prediction methods. EMBO J. 1985 Dec 16;4(13A):3625–3631. doi: 10.1002/j.1460-2075.1985.tb04126.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wilson D. M., Wilson T. H. Cation specificity for sugar substrates of the melibiose carrier in Escherichia coli. Biochim Biophys Acta. 1987 Nov 13;904(2):191–200. doi: 10.1016/0005-2736(87)90368-3. [DOI] [PubMed] [Google Scholar]

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