Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Apr;177(8):2178–2187. doi: 10.1128/jb.177.8.2178-2187.1995

Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.

N Kido 1, V I Torgov 1, T Sugiyama 1, K Uchiya 1, H Sugihara 1, T Komatsu 1, N Kato 1, K Jann 1
PMCID: PMC176863  PMID: 7536735

Abstract

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.

Full Text

The Full Text of this article is available as a PDF (306.5 KB).

Selected References

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

  1. Alexander D. C., Valvano M. A. Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. J Bacteriol. 1994 Nov;176(22):7079–7084. doi: 10.1128/jb.176.22.7079-7084.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barr K., Rick P. D. Biosynthesis of enterobacterial common antigen in Escherichia coli. In vitro synthesis of lipid-linked intermediates. J Biol Chem. 1987 May 25;262(15):7142–7150. [PubMed] [Google Scholar]
  3. Bastin D. A., Stevenson G., Brown P. K., Haase A., Reeves P. R. Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length. Mol Microbiol. 1993 Mar;7(5):725–734. doi: 10.1111/j.1365-2958.1993.tb01163.x. [DOI] [PubMed] [Google Scholar]
  4. Batchelor R. A., Alifano P., Biffali E., Hull S. I., Hull R. A. Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation. J Bacteriol. 1992 Aug;174(16):5228–5236. doi: 10.1128/jb.174.16.5228-5236.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bronner D., Clarke B. R., Whitfield C. Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1. Mol Microbiol. 1994 Nov;14(3):505–519. doi: 10.1111/j.1365-2958.1994.tb02185.x. [DOI] [PubMed] [Google Scholar]
  6. Bronner D., Sieberth V., Pazzani C., Smith A., Boulnois G., Roberts I., Jann B., Jann K. Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli. FEMS Microbiol Lett. 1993 Nov 1;113(3):279–284. doi: 10.1111/j.1574-6968.1993.tb06527.x. [DOI] [PubMed] [Google Scholar]
  7. Carlomagno M. S., Chiariotti L., Alifano P., Nappo A. G., Bruni C. B. Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons. J Mol Biol. 1988 Oct 5;203(3):585–606. doi: 10.1016/0022-2836(88)90194-5. [DOI] [PubMed] [Google Scholar]
  8. Curvall M., Lindberg B., Lönngren J., Nimmich W. Structural studies on the Klebsiella O group 3 lipopolysaccharide. Acta Chem Scand. 1973;27(7):2645–2649. doi: 10.3891/acta.chem.scand.27-2645. [DOI] [PubMed] [Google Scholar]
  9. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frosch M., Edwards U., Bousset K., Krausse B., Weisgerber C. Evidence for a common molecular origin of the capsule gene loci in gram-negative bacteria expressing group II capsular polysaccharides. Mol Microbiol. 1991 May;5(5):1251–1263. doi: 10.1111/j.1365-2958.1991.tb01899.x. [DOI] [PubMed] [Google Scholar]
  11. Hasegawa T., Ohta M., Kido N., Kato N., Miyamoto K., Koshiura R. Comparative studies on antitumor activity of Klebsiella O3 lipopolysaccharide and its polysaccharide fraction in mice. Jpn J Pharmacol. 1985 Aug;38(4):355–360. doi: 10.1254/jjp.38.355. [DOI] [PubMed] [Google Scholar]
  12. Hashimoto Y., Li N., Yokoyama H., Ezaki T. Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi. J Bacteriol. 1993 Jul;175(14):4456–4465. doi: 10.1128/jb.175.14.4456-4465.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Higgins C. F., Hyde S. C., Mimmack M. M., Gileadi U., Gill D. R., Gallagher M. P. Binding protein-dependent transport systems. J Bioenerg Biomembr. 1990 Aug;22(4):571–592. doi: 10.1007/BF00762962. [DOI] [PubMed] [Google Scholar]
  14. Holst O., Zähringer U., Brade H., Zamojski A. Structural analysis of the heptose/hexose region of the lipopolysaccharide from Escherichia coli K-12 strain W3100. Carbohydr Res. 1991 Aug 20;215(2):323–335. doi: 10.1016/0008-6215(91)84031-9. [DOI] [PubMed] [Google Scholar]
  15. Jann K., Goldemann G., Weisgerber C., Wolf-Ullisch C., Kanegasaki S. Biosynthesis of the O9 antigen of Escherichia coli. Initial reaction and overall mechanism. Eur J Biochem. 1982 Sep;127(1):157–164. doi: 10.1111/j.1432-1033.1982.tb06850.x. [DOI] [PubMed] [Google Scholar]
  16. Jann K., Kanegasaki S., Goldemann G., Mäkelä P. H. On the effect of rfe mutation on the biosynthesis of the 08 and 09 antigens of E. coli. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1185–1191. doi: 10.1016/0006-291x(79)90242-0. [DOI] [PubMed] [Google Scholar]
  17. Jann K., Pillat M., Weisgerber C., Shibaev V. N., Torgov V. I. Biosynthesis of the O9 antigen of Escherichia coli. Synthetic glycosyldiphosphomoraprenols as probes for requirement of mannose acceptors. Eur J Biochem. 1985 Sep 2;151(2):393–397. doi: 10.1111/j.1432-1033.1985.tb09114.x. [DOI] [PubMed] [Google Scholar]
  18. Jansson P. E., Lönngren J., Widmalm G., Leontein K., Slettengren K., Svenson S. B., Wrangsell G., Dell A., Tiller P. R. Structural studies of the O-antigen polysaccharides of Klebsiella O5 and Escherichia coli O8. Carbohydr Res. 1985 Dec 15;145(1):59–66. doi: 10.1016/s0008-6215(00)90412-9. [DOI] [PubMed] [Google Scholar]
  19. Jayaratne P., Bronner D., MacLachlan P. R., Dodgson C., Kido N., Whitfield C. Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group I K30 capsular polysaccharide. J Bacteriol. 1994 Jun;176(11):3126–3139. doi: 10.1128/jb.176.11.3126-3139.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kanegasaki S., Jann K. Demonstration by membrane reconstitution of a butanol-soluble intermediate in the biosynthesis of the O9 antigen of Escherichia coli. Eur J Biochem. 1979 Apr 2;95(2):287–293. doi: 10.1111/j.1432-1033.1979.tb12964.x. [DOI] [PubMed] [Google Scholar]
  21. Kido N., Ohta M., Iida K., Hasegawa T., Ito H., Arakawa Y., Komatsu T., Kato N. Partial deletion of the cloned rfb gene of Escherichia coli O9 results in synthesis of a new O-antigenic lipopolysaccharide. J Bacteriol. 1989 Jul;171(7):3629–3633. doi: 10.1128/jb.171.7.3629-3633.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klena J. D., Schnaitman C. A. Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Mol Microbiol. 1993 Jul;9(2):393–402. doi: 10.1111/j.1365-2958.1993.tb01700.x. [DOI] [PubMed] [Google Scholar]
  23. Kroll J. S., Loynds B., Brophy L. N., Moxon E. R. The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export. Mol Microbiol. 1990 Nov;4(11):1853–1862. doi: 10.1111/j.1365-2958.1990.tb02034.x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Liu D., Haase A. M., Lindqvist L., Lindberg A. A., Reeves P. R. Glycosyl transferases of O-antigen biosynthesis in Salmonella enterica: identification and characterization of transferase genes of groups B, C2, and E1. J Bacteriol. 1993 Jun;175(11):3408–3413. doi: 10.1128/jb.175.11.3408-3413.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marolda C. L., Valvano M. A. Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1). J Bacteriol. 1993 Jan;175(1):148–158. doi: 10.1128/jb.175.1.148-158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meier-Dieter U., Barr K., Starman R., Hatch L., Rick P. D. Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster. J Biol Chem. 1992 Jan 15;267(2):746–753. [PubMed] [Google Scholar]
  28. Meier-Dieter U., Starman R., Barr K., Mayer H., Rick P. D. Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis. J Biol Chem. 1990 Aug 15;265(23):13490–13497. [PubMed] [Google Scholar]
  29. Miyamoto K., Koshiura R., Hasegawa T., Kato N. Antitumor activity of Klebsiella 03 lipopolysaccharide in mice. Jpn J Pharmacol. 1984 Sep;36(1):51–57. doi: 10.1254/jjp.36.51. [DOI] [PubMed] [Google Scholar]
  30. Mulford C. A., Osborn M. J. An intermediate step in translocation of lipopolysaccharide to the outer membrane of Salmonella typhimurium. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1159–1163. doi: 10.1073/pnas.80.5.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mäkelä P. H., Jahkola M., Lüderitz O. A new gene cluster rfe concerned with the biosynthesis of Salmonella lipopolysaccharide. J Gen Microbiol. 1970 Jan;60(1):91–106. doi: 10.1099/00221287-60-1-91. [DOI] [PubMed] [Google Scholar]
  32. Mäkelä P. H. Participation of lipopolysaccharide genes in the determination of the entobacterial common antigen: analysis in Salmonella groups B and C1. J Bacteriol. 1974 Sep;119(3):765–770. doi: 10.1128/jb.119.3.765-770.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Orskov I., Orskov F., Jann B., Jann K. Serology, chemistry, and genetics of O and K antigens of Escherichia coli. Bacteriol Rev. 1977 Sep;41(3):667–710. doi: 10.1128/br.41.3.667-710.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pavelka M. S., Jr, Wright L. F., Silver R. P. Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1. J Bacteriol. 1991 Aug;173(15):4603–4610. doi: 10.1128/jb.173.15.4603-4610.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reeves P. Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet. 1993 Jan;9(1):17–22. doi: 10.1016/0168-9525(93)90067-R. [DOI] [PubMed] [Google Scholar]
  36. Reizer J., Reizer A., Saier M. H., Jr A new subfamily of bacterial ABC-type transport systems catalyzing export of drugs and carbohydrates. Protein Sci. 1992 Oct;1(10):1326–1332. doi: 10.1002/pro.5560011012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rick P. D., Hubbard G. L., Barr K. Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. J Bacteriol. 1994 May;176(10):2877–2884. doi: 10.1128/jb.176.10.2877-2884.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Schmidt G., Jann B., Jann K. Immunochemistry of R lipopolysaccharides of Escherichia coli. Different core regions in the lipopolysaccharides of O group 8. Eur J Biochem. 1969 Oct;10(3):501–510. doi: 10.1111/j.1432-1033.1969.tb00717.x. [DOI] [PubMed] [Google Scholar]
  40. Schnaitman C. A., Klena J. D. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev. 1993 Sep;57(3):655–682. doi: 10.1128/mr.57.3.655-682.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shibaev V. N. Biosynthesis of bacterial polysaccharide chains composed of repeating units. Adv Carbohydr Chem Biochem. 1986;44:277–339. doi: 10.1016/s0065-2318(08)60080-3. [DOI] [PubMed] [Google Scholar]
  42. Smith A. N., Boulnois G. J., Roberts I. S. Molecular analysis of the Escherichia coli K5 kps locus: identification and characterization of an inner-membrane capsular polysaccharide transport system. Mol Microbiol. 1990 Nov;4(11):1863–1869. doi: 10.1111/j.1365-2958.1990.tb02035.x. [DOI] [PubMed] [Google Scholar]
  43. Sugiyama T., Kido N., Komatsu T., Ohta M., Jann K., Jann B., Saeki A., Kato N. Genetic analysis of Escherichia coli O9 rfb: identification and DNA sequence of phosphomannomutase and GDP-mannose pyrophosphorylase genes. Microbiology. 1994 Jan;140(Pt 1):59–71. doi: 10.1099/13500872-140-1-59. [DOI] [PubMed] [Google Scholar]
  44. Sugiyama T., Kido N., Komatsu T., Ohta M., Kato N. Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium. J Bacteriol. 1991 Jan;173(1):55–58. doi: 10.1128/jb.173.1.55-58.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weisgerber C., Jann B., Jann K. Biosynthesis of the 09 antigen of Escherichia coli. Core structure of rfe mutant as indication of assembly mechanism. Eur J Biochem. 1984 May 2;140(3):553–556. doi: 10.1111/j.1432-1033.1984.tb08137.x. [DOI] [PubMed] [Google Scholar]
  46. Weisgerber C., Jann K. Glucosyldiphosphoundecaprenol, the mannose acceptor in the synthesis of the O9 antigen of Escherichia coli. Biosynthesis and characterization. Eur J Biochem. 1982 Sep;127(1):165–168. doi: 10.1111/j.1432-1033.1982.tb06851.x. [DOI] [PubMed] [Google Scholar]
  47. Whitfield C., Valvano M. A. Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria. Adv Microb Physiol. 1993;35:135–246. doi: 10.1016/s0065-2911(08)60099-5. [DOI] [PubMed] [Google Scholar]
  48. Zhang L., al-Hendy A., Toivanen P., Skurnik M. Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol Microbiol. 1993 Jul;9(2):309–321. doi: 10.1111/j.1365-2958.1993.tb01692.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES