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. 1997 Sep;179(18):5783–5788. doi: 10.1128/jb.179.18.5783-5788.1997

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli.

C Ulmke 1, J W Lengeler 1, K Schmid 1
PMCID: PMC179467  PMID: 9294435

Abstract

The conjugative plasmid pRSD2 carries a raf operon that encodes a peripheral raffinose metabolic pathway in enterobacteria. In addition to the previously known raf genes, we identified another gene, rafY, which in Escherichia coli codes for an outer membrane protein (molecular mass, 53 kDa) similar in function to the known glycoporins LamB (maltoporin) and ScrY (sucrose porin). Sequence comparisons with LamB and ScrY revealed no significant similarities; however, both lamB and scrY mutants are functionally complemented by RafY. Expressed from the tac promoter, RafY significantly increases the uptake rates for maltose, sucrose, and raffinose at low substrate concentrations; in particular it shifts the apparent K(m) for raffinose transport from 2 mM to 130 microM. Moreover, RafY permits diffusion of the tetrasaccharide stachyose and of maltodextrins up to maltoheptaose through the outer membrane of E. coli. A comparison of all three glycoporins in regard to their substrate selectivity revealed that both ScrY and RafY have a broad substrate range which includes alpha-galactosides while LamB seems to be restricted to malto-oligosaccharides. It supports growth only on maltodextrins but not, like the others, on raffinose and stachyose.

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Selected References

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  1. Aslanidis C., Schmid K., Schmitt R. Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli. J Bacteriol. 1989 Dec;171(12):6753–6763. doi: 10.1128/jb.171.12.6753-6763.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aslanidis C., Schmitt R. Regulatory elements of the raffinose operon: nucleotide sequences of operator and repressor genes. J Bacteriol. 1990 Apr;172(4):2178–2180. doi: 10.1128/jb.172.4.2178-2180.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benz R., Francis G., Nakae T., Ferenci T. Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site. Biochim Biophys Acta. 1992 Mar 2;1104(2):299–307. doi: 10.1016/0005-2736(92)90044-m. [DOI] [PubMed] [Google Scholar]
  4. Bockmann J., Heuel H., Lengeler J. W. Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132. Mol Gen Genet. 1992 Oct;235(1):22–32. doi: 10.1007/BF00286177. [DOI] [PubMed] [Google Scholar]
  5. Brass J. M., Bauer K., Ehmann U., Boos W. Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli. J Bacteriol. 1985 Feb;161(2):720–726. doi: 10.1128/jb.161.2.720-726.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burkardt H. J., Mattes R., Schmid K., Schmitt R. Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli. Mol Gen Genet. 1978 Oct 25;166(1):75–84. doi: 10.1007/BF00379731. [DOI] [PubMed] [Google Scholar]
  7. Death A., Notley L., Ferenci T. Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress. J Bacteriol. 1993 Mar;175(5):1475–1483. doi: 10.1128/jb.175.5.1475-1483.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hardesty C., Ferran C., DiRienzo J. M. Plasmid-mediated sucrose metabolism in Escherichia coli: characterization of scrY, the structural gene for a phosphoenolpyruvate-dependent sucrose phosphotransferase system outer membrane porin. J Bacteriol. 1991 Jan;173(2):449–456. doi: 10.1128/jb.173.2.449-456.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Klein W., Boos W. Induction of the lambda receptor is essential for effective uptake of trehalose in Escherichia coli. J Bacteriol. 1993 Mar;175(6):1682–1686. doi: 10.1128/jb.175.6.1682-1686.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  11. Luckey M., Nikaido H. Specificity of diffusion channels produced by lambda phage receptor protein of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jan;77(1):167–171. doi: 10.1073/pnas.77.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nikaido H. Porins and specific diffusion channels in bacterial outer membranes. J Biol Chem. 1994 Feb 11;269(6):3905–3908. [PubMed] [Google Scholar]
  13. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  14. Puppe W., Siebers A., Altendorf K. The phosphorylation site of the Kdp-ATPase of Escherichia coli: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit. Mol Microbiol. 1992 Dec;6(23):3511–3520. doi: 10.1111/j.1365-2958.1992.tb01786.x. [DOI] [PubMed] [Google Scholar]
  15. Randall-Hazelbauer L., Schwartz M. Isolation of the bacteriophage lambda receptor from Escherichia coli. J Bacteriol. 1973 Dec;116(3):1436–1446. doi: 10.1128/jb.116.3.1436-1446.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sahin-Tóth M., Frillingos S., Lengeler J. W., Kaback H. R. Active transport by the CscB permease in Escherichia coli K-12. Biochem Biophys Res Commun. 1995 Mar 28;208(3):1116–1123. doi: 10.1006/bbrc.1995.1449. [DOI] [PubMed] [Google Scholar]
  17. Schirmer T., Keller T. A., Wang Y. F., Rosenbusch J. P. Structural basis for sugar translocation through maltoporin channels at 3.1 A resolution. Science. 1995 Jan 27;267(5197):512–514. doi: 10.1126/science.7824948. [DOI] [PubMed] [Google Scholar]
  18. Schmid K., Ebner R., Altenbuchner J., Schmitt R., Lengeler J. W. Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400. Mol Microbiol. 1988 Jan;2(1):1–8. doi: 10.1111/j.1365-2958.1988.tb00001.x. [DOI] [PubMed] [Google Scholar]
  19. Schmid K., Ebner R., Jahreis K., Lengeler J. W., Titgemeyer F. A sugar-specific porin, ScrY, is involved in sucrose uptake in enteric bacteria. Mol Microbiol. 1991 Apr;5(4):941–950. doi: 10.1111/j.1365-2958.1991.tb00769.x. [DOI] [PubMed] [Google Scholar]
  20. Schmid K., Ritschewald S., Schmitt R. Relationships among raffinose plasmids determined by the immunochemical cross-reaction of their alpha-galactosidases. J Gen Microbiol. 1979 Oct;114(2):477–481. doi: 10.1099/00221287-114-2-477. [DOI] [PubMed] [Google Scholar]
  21. Schmid K., Schupfner M., Schmitt R. Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12. J Bacteriol. 1982 Jul;151(1):68–76. doi: 10.1128/jb.151.1.68-76.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schülein K., Schmid K., Benzl R. The sugar-specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate-specific porins. Mol Microbiol. 1991 Sep;5(9):2233–2241. doi: 10.1111/j.1365-2958.1991.tb02153.x. [DOI] [PubMed] [Google Scholar]
  23. Struyvé M., Moons M., Tommassen J. Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J Mol Biol. 1991 Mar 5;218(1):141–148. doi: 10.1016/0022-2836(91)90880-f. [DOI] [PubMed] [Google Scholar]
  24. Szmelcman S., Schwartz M., Silhavy T. J., Boos W. Maltose transport in Escherichia coli K12. A comparison of transport kinetics in wild-type and lambda-resistant mutants as measured by fluorescence quenching. Eur J Biochem. 1976 May 17;65(1):13–19. doi: 10.1111/j.1432-1033.1976.tb10383.x. [DOI] [PubMed] [Google Scholar]
  25. Tanaka S., Lerner S. A., Lin E. C. Replacement of a phosphoenolpyruvate-dependent phosphotransferase by a nicotinamide adenine dinucleotide-linked dehydrogenase for the utilization of mannitol. J Bacteriol. 1967 Feb;93(2):642–648. doi: 10.1128/jb.93.2.642-648.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ubben D., Schmitt R. A transposable promoter and transposable promoter probes derived from Tn1721. Gene. 1987;53(1):127–134. doi: 10.1016/0378-1119(87)90100-4. [DOI] [PubMed] [Google Scholar]
  27. Welte W., Nestel U., Wacker T., Diederichs K. Structure and function of the porin channel. Kidney Int. 1995 Oct;48(4):930–940. doi: 10.1038/ki.1995.374. [DOI] [PubMed] [Google Scholar]
  28. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. de Vries G. E., Raymond C. K., Ludwig R. A. Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6080–6084. doi: 10.1073/pnas.81.19.6080. [DOI] [PMC free article] [PubMed] [Google Scholar]

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