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. 1994 Jul;176(13):4144–4156. doi: 10.1128/jb.176.13.4144-4156.1994

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.

G Stevenson 1, B Neal 1, D Liu 1, M Hobbs 1, N H Packer 1, M Batley 1, J W Redmond 1, L Lindquist 1, P Reeves 1
PMCID: PMC205614  PMID: 7517391

Abstract

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

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Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bastin D. A., Romana L. K., Reeves P. R. Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain. Mol Microbiol. 1991 Sep;5(9):2223–2231. doi: 10.1111/j.1365-2958.1991.tb02152.x. [DOI] [PubMed] [Google Scholar]
  6. Brahmbhatt H. N., Wyk P., Quigley N. B., Reeves P. R. Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the O antigen of Salmonella typhimurium LT2. J Bacteriol. 1988 Jan;170(1):98–102. doi: 10.1128/jb.170.1.98-102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown P. K., Romana L. K., Reeves P. R. Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D. Mol Microbiol. 1991 Aug;5(8):1873–1881. doi: 10.1111/j.1365-2958.1991.tb00811.x. [DOI] [PubMed] [Google Scholar]
  8. Brown P. K., Romana L. K., Reeves P. R. Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B. Mol Microbiol. 1992 May;6(10):1385–1394. doi: 10.1111/j.1365-2958.1992.tb00859.x. [DOI] [PubMed] [Google Scholar]
  9. Burland V., Plunkett G., 3rd, Daniels D. L., Blattner F. R. DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication. Genomics. 1993 Jun;16(3):551–561. doi: 10.1006/geno.1993.1230. [DOI] [PubMed] [Google Scholar]
  10. Churchward G., Belin D., Nagamine Y. A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984 Nov;31(1-3):165–171. doi: 10.1016/0378-1119(84)90207-5. [DOI] [PubMed] [Google Scholar]
  11. Clark C. A., Beltrame J., Manning P. A. The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6. Gene. 1991 Oct 30;107(1):43–52. doi: 10.1016/0378-1119(91)90295-m. [DOI] [PubMed] [Google Scholar]
  12. Clarke B. R., Whitfield C. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide. J Bacteriol. 1992 Jul;174(14):4614–4621. doi: 10.1128/jb.174.14.4614-4621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cossart P., Groisman E. A., Serre M. C., Casadaban M. J., Gicquel-Sanzey B. crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli. J Bacteriol. 1986 Aug;167(2):639–646. doi: 10.1128/jb.167.2.639-646.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fobes W. S., Gander J. E. Evidence for uridine 5'-( -D-galactopyranosyl pyrophosphate):NAD 2-hexosyl oxidoreductase in Penicillium charlesii. Biochem Biophys Res Commun. 1972 Oct 6;49(1):76–83. doi: 10.1016/0006-291x(72)90011-3. [DOI] [PubMed] [Google Scholar]
  16. Gleeson T. J., Staden R. An X windows and UNIX implementation of our sequence analysis package. Comput Appl Biosci. 1991 Jul;7(3):398–398. doi: 10.1093/bioinformatics/7.3.398. [DOI] [PubMed] [Google Scholar]
  17. Hammerschmidt S., Birkholz C., Zähringer U., Robertson B. D., van Putten J., Ebeling O., Frosch M. Contribution of genes from the capsule gene complex (cps) to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis. Mol Microbiol. 1994 Mar;11(5):885–896. doi: 10.1111/j.1365-2958.1994.tb00367.x. [DOI] [PubMed] [Google Scholar]
  18. Haraguchi G. E., Zähringer U., Jann B., Jann K., Hull R. A., Hull S. I. Genetic characterization of the O4 polysaccharide gene cluster from Escherichia coli. Microb Pathog. 1991 May;10(5):351–361. doi: 10.1016/0882-4010(91)90080-t. [DOI] [PubMed] [Google Scholar]
  19. Harris P. J., Henry R. J., Blakeney A. B., Stone B. A. An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr Res. 1984 Apr 2;127(1):59–73. doi: 10.1016/0008-6215(84)85106-x. [DOI] [PubMed] [Google Scholar]
  20. Jiang X. M., Neal B., Santiago F., Lee S. J., Romana L. K., Reeves P. R. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. doi: 10.1111/j.1365-2958.1991.tb00741.x. [DOI] [PubMed] [Google Scholar]
  21. Johnson M. T., Gander J. E. Purification and properties of a nucleoside diphosphosugar: NAD+ 2-hexosyl oxidoreductase. Biochim Biophys Acta. 1978 Mar 14;523(1):9–18. [PubMed] [Google Scholar]
  22. Kenne L., Lindberg B., Petersson K. Basic structure of the oligosaccharide repeating-unit of the Shigella flexneri O-antigens. Carbohydr Res. 1977 Jul;56(2):363–370. doi: 10.1016/s0008-6215(00)83357-1. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kogan G., Haraguchi G., Hull S. I., Hull R. A., Shashkov A. S., Jann B., Jann K. Structural analysis of O4-reactive polysaccharides from recombinant Escherichia coli. Changes in the O-specific polysaccharide induced by cloning of the rfb genes. Eur J Biochem. 1993 May 15;214(1):259–265. doi: 10.1111/j.1432-1033.1993.tb17919.x. [DOI] [PubMed] [Google Scholar]
  25. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  26. Lew H. C., Mäkelä P. H., Kuhn H. M., Mayer H., Nikaido H. Biosynthesis of enterobacterial common antigen requires dTDPglucose pyrophosphorylase determined by a Salmonella typhimurium rfb gene and a Salmonella montevideo rfe gene. J Bacteriol. 1986 Nov;168(2):715–721. doi: 10.1128/jb.168.2.715-721.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liu D., Reeves P. R. Escherichia coli K12 regains its O antigen. Microbiology. 1994 Jan;140(Pt 1):49–57. doi: 10.1099/13500872-140-1-49. [DOI] [PubMed] [Google Scholar]
  28. Marumo K., Lindqvist L., Verma N., Weintraub A., Reeves P. R., Lindberg A. A. Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC. Eur J Biochem. 1992 Mar 1;204(2):539–545. doi: 10.1111/j.1432-1033.1992.tb16665.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Mäkelä P. H., Schmidt G., Mayer H., Nikaido H., Whang H. Y., Neter E. Enterobacterial common antigen in rfb deletion mutants of Salmonella typhimurium. J Bacteriol. 1976 Sep;127(3):1141–1149. doi: 10.1128/jb.127.3.1141-1149.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Neuhard J., Thomassen E. Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene. J Bacteriol. 1976 May;126(2):999–1001. doi: 10.1128/jb.126.2.999-1001.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nikaido H., Levinthal M., Nikaido K., Nakane K. Extended deletions in the histidine-rough-B region of the Salmonella chromosome. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1825–1832. doi: 10.1073/pnas.57.6.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Orskov F., Orskov I., Evans D. J., Jr, Sack R. B., Sack D. A., Wadström T. Special Escherichia coli serotypes among enterotoxigenic strains from diarrhoea in adults and children. Med Microbiol Immunol. 1976 Jun 1;162(2):73–80. doi: 10.1007/BF02121318. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Rayssiguier C., Thaler D. S., Radman M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature. 1989 Nov 23;342(6248):396–401. doi: 10.1038/342396a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Rick P. D., Mayer H., Neumeyer B. A., Wolski S., Bitter-Suermann D. Biosynthesis of enterobacterial common antigen. J Bacteriol. 1985 May;162(2):494–503. doi: 10.1128/jb.162.2.494-503.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Sarvas M., Nikaido H. Biosynthesis of T1 antigen in Salmonella: origin of D-galactofuranose and D-ribofuranose residues. J Bacteriol. 1971 Mar;105(3):1063–1072. doi: 10.1128/jb.105.3.1063-1072.1971. [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. Simmons D. A., Romanowska E. Structure and biology of Shigella flexneri O antigens. J Med Microbiol. 1987 Jun;23(4):289–302. doi: 10.1099/00222615-23-4-289. [DOI] [PubMed] [Google Scholar]
  43. Sunshine M. G., Kelly B. Extent of host deletions associated with bacteriophage P2-mediated eduction. J Bacteriol. 1971 Nov;108(2):695–704. doi: 10.1128/jb.108.2.695-704.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Trejo A. G., Chittenden G. J., Buchanan J. G., Baddiley J. Uridine diphosphate alpha-D-galactofuranose, an intermediate in the biosynthesis of galactofuranosyl residues. Biochem J. 1970 Apr;117(3):637–639. doi: 10.1042/bj1170637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Verma N. K., Brandt J. M., Verma D. J., Lindberg A. A. Molecular characterization of the O-acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri. Mol Microbiol. 1991 Jan;5(1):71–75. doi: 10.1111/j.1365-2958.1991.tb01827.x. [DOI] [PubMed] [Google Scholar]
  46. Wang L., Romana L. K., Reeves P. R. Molecular analysis of a Salmonella enterica group E1 rfb gene cluster: O antigen and the genetic basis of the major polymorphism. Genetics. 1992 Mar;130(3):429–443. doi: 10.1093/genetics/130.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yao Z., Liu H., Valvano M. A. Acetylation of O-specific lipopolysaccharides from Shigella flexneri 3a and 2a occurs in Escherichia coli K-12 carrying cloned S. flexneri 3a and 2a rfb genes. J Bacteriol. 1992 Dec;174(23):7500–7508. doi: 10.1128/jb.174.23.7500-7508.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yao Z., Valvano M. A. Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a. J Bacteriol. 1994 Jul;176(13):4133–4143. doi: 10.1128/jb.176.13.4133-4143.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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