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The use of gene fusions to study bacterial transport proteins

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Summary

The transport of solutes by bacteria has been studied for about thirty years. Early experiments on amino acid entry and galactoside accumulation provided concrete evidence that bacteria possessed specific transport systems and that these were subject to regulation. Since then a large number of transport systems have been discovered and studied extensively. Many of these use entirely different strategies for capturing or accumulating substrates. This diversity reflects variation in the availability of nutrients and ions in the different environments tolerated and inhabited by microorganisms. Examination of a few bacterial transport systems provides an opportunity to gain insight into a wide range of topics in the area of membrane transport. These include: the identification of carrier proteins and their arrangement in the membrane, the regulation of transport protein synthesis by environmental factors, and the localization of transport proteins to their extracytoplasmic destinations.

It has been possible to construct a number of bacterial strains in which the gene (lacZ) which codes for the cytoplasmic enzyme β-galactosidase is fused to genes which code for transport proteins. The following article is intended to illustrate how these gene fusions have been used to study the regulation and structure of transport proteins inEscherichia coli.

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References

  1. Accola, R.S., Celada, F. 1976. Antibody mediated activation of a deletion mutant β-galactosidase defective in the α-region.FEBS Lett. 67:299–302

    Google Scholar 

  2. Bassford, P., Beckwith, J.R., Berman, M., Brickman, E., Casadaban, M., Guarente, L., St. Girons, I., Sarthy, A., Schwartz, M., Shuman, H., Silhavy, T. 1978. Genetic fusions of thelac operon: A new approach to the study of biological processes.In: The Operon. J.H. Miller and W. Reznikoff, editors. pp. 245–261. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  3. Bassford, P.J., Kadner, R.J. 1978. Role of the outer membrane in active transport.In: Bacterial Transport. B.P. Rosen, editor. pp. 443–462. M. Dekker, New York

    Google Scholar 

  4. Bavoil, P., Hofnung, M., Nikaido, H. 1980. Identification of a cytoplasmic-membrane associated component of the maltose transport system ofEscerichia coli.J. Biol. Chem. 255:8366–8369

    Google Scholar 

  5. Bayer, M.E. 1968. Areas of adhesion between wall and membrane ofEscherichia coli.J. Gen Microbiol. 53:595–404

    Google Scholar 

  6. Bedouelle, H., Bassford, P.J., Jr., Fowler, A.V., Zabin, I., Beckwith, J. 1980. Mutations which alter the function of the signal sequence of the maltose binding protein ofEscerichia coli.Nature (London) 285:78–81

    Google Scholar 

  7. Berger, E.A., Heppel, L.A. 1974. Different mechanisms of energy coupling for the shock-sensitive and shock-resistant amino acid permeases ofEscherichia coli.J. Biol. Chem. 249:7747–7755

    Google Scholar 

  8. Boos, W. 1974. Bacterial Transport.Annu. Rev. Biochem. 43:123–146

    Google Scholar 

  9. Braun, V., Sieglin, U. 1970. The covalent murein-lipoprotein structure of theEscherichia coli cell wall. The attachment site of the lipoprotein on the murein.Eur. J. Biochem 13:336–346

    Google Scholar 

  10. Brickman, E., Silhavy, T.J., Bassford, P.J., Shuman, H.A., Beckwith, J.R. 1979. Sites within genelacZ ofEscherichia coli for formation of active hybrid β-galactosidase molecules.J. Bacteriol. 139:13–18

    Google Scholar 

  11. Casadaban, M. 1976. Transposition and fusion of thelac operon to selected promoters inE. coli using bacteriophage λ and Mu.J. Mol. Biol. 104:541–555

    Google Scholar 

  12. Datta, D.B., Kramer, C., Henning, U. 1976. Diploidy for a structural gene specifying a major protein of the outer cell envelope membrane fromEscherichia coli K-12.J. Bacteriol. 128:834–841

    Google Scholar 

  13. Debarbouille, M., Shuman, H.A., Silhavy, T.J., Schwartz, M. 1978. Dominant constitutive mutations inmalT, the positive regulator gene of the maltose regulon inEscherichia coli.J. Mol. Biol. 124:359–371

    Google Scholar 

  14. Dietzel, I., Kolb, V., Boos, W. 1978. Pole cap formation inEscherichia coli following induction of the maltose binding protein.Arch. Miktobiolo. 118:207–218

    Google Scholar 

  15. Emr, S.D., Hall, M.N., Silhavy, T.J. 1980. A mechanism of localization: The signal hypothesis and bacteria.J. Cell Biol. 86:701–711

    Google Scholar 

  16. Emr, S.D., Silhavy, T.J. 1980. Mutations affecting localization of anEscherichia coli outer membrana protein, the bacteriophage lambda receptor. J. Mol. Biol.141:63–90

    Google Scholar 

  17. Epstein, W., Laimins, L., Hesse, J. 1979. A phosphorylated intermediate of the Kdp system, an ATP-driven K− transport system ofE. coli. Proc. XIth Intl. Congress of Biochemistry, Toronto, p. 449

  18. Epstein, W., Laimins, L. 1980. Potassium transport inEscherichia coli: Diverse systems with common control by osmotic forces.Trends Biochem. Sci. 5:21–23

    Google Scholar 

  19. Epstein, W., Schultz, S.G. 1965. Cation transport inEscherichia coli V. Regulation of cation content.J. Gen. Physiol. 49:221–234

    Google Scholar 

  20. Epstein, W., Whitelaw, V., Hesse, J. 1978. A K+ Transport ATPase inEscherichia coli.J. Biol. Chem. 253:6666–6668

    Google Scholar 

  21. Fowler, A.V., Zabin, I. 1977. The amino acid sequence of β-galactosidase ofEscherichia coli.Proc. Natl. Acad. Sci. USA 74:1507–1511

    Google Scholar 

  22. Fox, C.F., Kennedy, E.P. 1965. Specific labelling and partial purification of the M protein, A component of the β-galactosidase transport systems ofE. coli.Proc. Natl. Acad. Sci. USA 54:891–899

    Google Scholar 

  23. Hall, M.N., Silhavy, T.J. 1979. Transcriptional regulation ofEscherichia coli K-12 major outer membrane protein 1b.J. Bacteriol. 140:342–350

    Google Scholar 

  24. Hall, M.N., Silhavy, T.J. 1980. TheompB locus and the regulation of the major outer membrane porin proteins ofEscherichia coli K-12.J. Mol. Biol. (in press)

  25. Hatfield, D., Hofnung, M., Schwartz, M. 1969. Genetic analysis of the maltose A region inEscherchia coli.J. Bacteriol. 98:559–567

    Google Scholar 

  26. Hong, J.S., Hunt, A.G., Masters, P.S., Lieberman, M.A. 1979. Requirement of acetyl phosphate for the binding protein dependent transport systems inEscherichia coli.Proc. Natl. Acad. Sci. USA 76:1213–1217

    Google Scholar 

  27. Kawaji, H., Mizuno, T., Mizushima, S. 1979. Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins 0–8 and 0–9 ofEscherichia coli K-12.J. Bacteriol. 140:843–847

    Google Scholar 

  28. Kellermann, O., Szmelcman, S. 1974. Active transport of maltose inEscherichia coli K-12: Involvement of a “periplasmic” maltose binding protein.Eur. J. Biochem. 47:139–149

    Google Scholar 

  29. Laimins, L., Rhoades, D.B., Altendorf, K., Epstein, W. 1978. Identification of the structural proteins of an ATP-driven potassium transport system inEscherichia coli.Proc. Natl. Acad. Sci. USA 75:3216–3219

    Google Scholar 

  30. Laimins, L., Rhoades, D.B., Epstein, W. 1980. Osmotic control of kdp operon expression inE. coli. Proc. Natl. Acad. Sci. USA (in press)

  31. Lugtenberg, B., Peters, R., Bernheimer, H., Berendsen, W. 1976. Influence of cultural conditions and mutations on the composition of the outer membrane proteins ofEscherichia coli.Mol. Gen. Genet. 147:251–262

    Google Scholar 

  32. Miller, J., reznikoff, W. 1979. The Operon. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  33. Mitchell, P. 1961.In: Biological Structure and Function. T. W. Goodwin and O. Lindberg, editors. Vol. 2, pp. 581–603 Academic Press, New York

    Google Scholar 

  34. Moreno, F., Fowler, A. V., Hall, M., Silhavy, T.J., Zabin, I., Schwartz, M. 1980. A signal sequence is not sufficient for protein export.Nature (London) 286:356–359

    Google Scholar 

  35. Müller-Hill, B., Kania, J. 1974.Lac repressor can be fused to β-galactosidase,Nature (London) 249:561

    Google Scholar 

  36. Nakae, T. 1975. Outer membrane ofSalmonella typhimurium: Reconstitution of sucrose-permeable membrane vesicles.Biochem. Biophys. Res. Commun. 64:1224–1230

    Google Scholar 

  37. Nakae, T. 1976. Outer membrane ofS. typhimurium. Isolation of a complex that produces transmembrane channels.J. Biol. Chem. 251:2176–2178

    Google Scholar 

  38. Neu, H.C., Heppel, L. 1965. The release of enzymes fromEscherichia coli by osmotic shock and during the formation of spheroplasts.J. Biol. Chem. 240:3685–3692

    Google Scholar 

  39. Osborn, M.J., Wu, H.C.P. 1980. Proteins of the outer membrane of gram negative bacteria.Annu. Rev. Microbiol. (in press)

  40. Randall-Hazelbauer, L.L., Schwartz, M. 1973. Isolation of the bacteriophage lambda receptor fromEscherichia coli K-12.J. Bacteriol. 116:1436–1446

    Google Scholar 

  41. Rhoades, D.B., Epstein, W. 1977. Energy coupling to net K+ transport inEscherichia coli K-12.J. Biol. Chem. 252:1394–1401

    Google Scholar 

  42. Rhoades, D.B., Laimins, L., Epstein, W. 1978. Functional organization of thekdp genes ofEscherichia coli K-12.J. Bacteriol. 135:445–452

    Google Scholar 

  43. Rhoades, D.B., Waters, F.B., Epstein, W. 1976. Cation transport inEscherichia coli. VIII. Potassium transport mutants.J. Gen. Physiol. 67:325–341

    Google Scholar 

  44. Sarma, V., Reeves, P. 1977. Genetic locus (ompB) affecting a major outer-membrane protein inEscherichia coli K-12.J. Bacteriol. 132:23–27

    Google Scholar 

  45. Sarthy, A., Fowler, A.V., Zabin, I., Beckwith, J. 1979. The use of gene fusions to determine a partial signal sequence of alkaline phosphatase.J. Bacteriol. 139:932–939

    Google Scholar 

  46. Schnaitman, C. 1971. Solubilization of the cytoplasmic membrane ofEscherichia coli by Triton X-100.J. Bacteriol. 108:545–552

    Google Scholar 

  47. Shuman, H.A., Beckwith, J.R. 1979. Mutants ofEscherichia coli K-12 that allow transport of maltosevia the β-galactoside transport system.J. Bacteriol. 137:365–373

    Google Scholar 

  48. Shuman, H.A., Silhavy, T.J. 1981. Identification of themalK gene product: A peripheral membrane component of theE. coli maltose transport system.J. Biol. Chem. 256:560–562

    Google Scholar 

  49. Shuman, H.A., Silhavy, T.J., Beckwith, J. 1980. Labeling proteins with β-galactosidase by gene fusion: Identification of a cytoplasmic membrane component of theEscherichia coli maltose transport system.J. Biol. Chem. 255:168–174

    Google Scholar 

  50. Silhavy, T.J., Bassford, P.J., Jr., Beckwith, J. 1979. A genetic approach to the study of protein localization inEscherichia coli.In: Bacterial Outer Membranes: Biogenesis and Function. M. Inouye, editor. John Wiley and Sons, New York

    Google Scholar 

  51. Silhavy, T.J., Casadaban, M., Shuman, H.A., Beckwith, J.R. 1976. Conversion of β-galactosidase to a membrane-bound state by gene fusion.Proc. Natl. Acad. Sci. USA 73:3423–3427

    Google Scholar 

  52. Smit, J., Nikaido, H. 1978. Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on sites of insertion of insertion of porins and growth of cell surface inSalmonella typhimurium.J. Bacteriol. 135:687–702

    Google Scholar 

  53. Szmelcman, S., Schwartz, M., Silhavy, M., Boos, W. 1976. Maltose transport inEscherichia coli K-12. A comparison of transport kinetics in wild type and λ-resistant mutants with the dissociation constants of the maltose binding protein as measured by fluorescence quenching.Eur. J. Biochem. 65:13–19

    Google Scholar 

  54. Van Alphen, W., Lugtenberg, B. 1977. Influence of osmolarity of the growth medium on the outer membrane protein pattern ofEscherichia coli.J. Bacteriol. 131:623–630

    Google Scholar 

  55. Verhoef, C., Lugtenberg, B., Boxtel, R. van, Graaff, P. de, Verheij, H. 1979. Genetics and biochemistry of the peptidoglycan-associated proteinsb andc ofEscherichia coli K-12.Mol. Gen. Genet. 169:137–146

    Google Scholar 

  56. Wandersman, C., Moreno, F., Schwartz, M. 1980. Pleiotropic mutants ofExcherichia coli K-12 resistant to phage TP1, a phage which can use either of two outer membrane proteins as its receptor.J. Bacteriol. 143:1374–1383

    Google Scholar 

  57. Wanner, B.L., Sarthy, A., Beckwith, J. 1979.Escherichia coli pleiotropic mutant that reduces amounts of several periplamic and outer membrane proteins.J. Bacteriol. 140:229–239

    Google Scholar 

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  1. Department of Biochemistry and Molecular Biology, Harvard University, 02138, Cambridge, Massachusetts

    Howard A. Shuman

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Shuman, H.A. The use of gene fusions to study bacterial transport proteins. J. Membrain Biol. 61, 1–11 (1981). https://doi.org/10.1007/BF01870747

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