Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 May;179(10):3232–3238. doi: 10.1128/jb.179.10.3232-3238.1997

Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens.

J Drummelsmith 1, P A Amor 1, C Whitfield 1
PMCID: PMC179101  PMID: 9150218

Abstract

Individual Escherichia coli strains produce several cell surface polysaccharides. In E. coli E69, the his region of the chromosome contains the rfb (serotype O9 lipopolysaccharide O-antigen biosynthesis) and cps (serotype K30 group IA capsular polysaccharide biosynthesis) loci. Polymorphisms in this region of the Escherichia coli chromosome reflect extensive antigenic diversity in the species. Previously, we reported a duplication of the manC-manB genes, encoding enzymes involved in GDP-mannose formation, upstream of rfb in strain E69 (P. Jayaratne et al., J. Bacteriol. 176:3126-3139, 1994). Here we show that one of the manC-manB copies is flanked by IS1 elements, providing a potential mechanism for the gene duplication. Adjacent to manB1 on the IS1-flanked segment is a further open reading frame (ugd), encoding uridine-5'-diphosphoglucose dehydrogenase. The Ugd enzyme is responsible for the production of UDP-glucuronic acid, a precursor required for K30 antigen synthesis. Construction of a chromosomal ugd::Gm(r) insertion mutation demonstrated the essential role for Ugd in the biosynthesis of the K30 antigen and confirmed that there is no additional functional ugd copy in strain E69. PCR amplification and Southern hybridization were used to examine the distribution of IS1 elements and ugd genes in the vicinity of rfb in other E. coli strains, producing different group IA K antigens. The relative order of genes and, where present, IS1 elements was established in these strains. The regions adjacent to rfb in these strains are highly variable in both size and gene order, but in all cases where a ugd homolog was present, it was found near rfb. The presence of IS1 elements in the rfb regions of several of these strains provides a potential mechanism for recombination and deletion events which could contribute to the antigenic diversity seen in surface polysaccharides.

Full Text

The Full Text of this article is available as a PDF (289.8 KB).

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. 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]
  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. Bik E. M., Bunschoten A. E., Gouw R. D., Mooi F. R. Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis. EMBO J. 1995 Jan 16;14(2):209–216. doi: 10.1002/j.1460-2075.1995.tb06993.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Binotto J., MacLachlan P. R., Sanderson K. E. Electrotransformation in Salmonella typhimurium LT2. Can J Microbiol. 1991 Jun;37(6):474–477. doi: 10.1139/m91-078. [DOI] [PubMed] [Google Scholar]
  6. Burrows L. L., Charter D. F., Lam J. S. Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster. Mol Microbiol. 1996 Nov;22(3):481–495. doi: 10.1046/j.1365-2958.1996.1351503.x. [DOI] [PubMed] [Google Scholar]
  7. Chu S., Noonan B., Cavaignac S., Trust T. J. Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5754–5758. doi: 10.1073/pnas.92.12.5754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Comstock L. E., Johnson J. A., Michalski J. M., Morris J. G., Jr, Kaper J. B. Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1. Mol Microbiol. 1996 Feb;19(4):815–826. doi: 10.1046/j.1365-2958.1996.407928.x. [DOI] [PubMed] [Google Scholar]
  9. Dodgson C., Amor P., Whitfield C. Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens. J Bacteriol. 1996 Apr;178(7):1895–1902. doi: 10.1128/jb.178.7.1895-1902.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dougherty B. A., van de Rijn I. Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity. J Biol Chem. 1993 Apr 5;268(10):7118–7124. [PubMed] [Google Scholar]
  11. Drake C. R., Boulnois G. J., Roberts I. S. The Escherichia coli serA-linked capsule locus and its flanking sequences are polymorphic, genetic evidence for the existence of more than two groups of capsule gene clusters. J Gen Microbiol. 1993 Aug;139(8):1707–1714. doi: 10.1099/00221287-139-8-1707. [DOI] [PubMed] [Google Scholar]
  12. Franco A. V., Liu D., Reeves P. R. A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains. J Bacteriol. 1996 Apr;178(7):1903–1907. doi: 10.1128/jb.178.7.1903-1907.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Homonylo M. K., Wilmot S. J., Lam J. S., MacDonald L. A., Whitfield C. Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface. Can J Microbiol. 1988 Oct;34(10):1159–1165. doi: 10.1139/m88-204. [DOI] [PubMed] [Google Scholar]
  16. Jann K., Dengler T., Jann B. Core-lipid A on the K40 polysaccharide of Escherichia coli O8:K40:H9, a representative of group I capsular polysaccharides. Zentralbl Bakteriol. 1992 Jan;276(2):196–204. doi: 10.1016/s0934-8840(11)80006-x. [DOI] [PubMed] [Google Scholar]
  17. Jann K., Jann B. The K antigens of Escherichia coli. Prog Allergy. 1983;33:53–79. doi: 10.1159/000407421. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kido N., Torgov V. I., Sugiyama T., Uchiya K., Sugihara H., Komatsu T., Kato N., Jann K. 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. J Bacteriol. 1995 Apr;177(8):2178–2187. doi: 10.1128/jb.177.8.2178-2187.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kroll J. S. The genetics of encapsulation in Haemophilus influenzae. J Infect Dis. 1992 Jun;165 (Suppl 1):S93–S96. doi: 10.1093/infdis/165-supplement_1-s93. [DOI] [PubMed] [Google Scholar]
  21. Laakso D. H., Homonylo M. K., Wilmot S. J., Whitfield C. Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20, in Escherichia coli K12. Can J Microbiol. 1988 Aug;34(8):987–992. doi: 10.1139/m88-173. [DOI] [PubMed] [Google Scholar]
  22. MacLachlan P. R., Keenleyside W. J., Dodgson C., Whitfield C. Formation of the K30 (group I) capsule in Escherichia coli O9:K30 does not require attachment to lipopolysaccharide lipid A-core. J Bacteriol. 1993 Dec;175(23):7515–7522. doi: 10.1128/jb.175.23.7515-7522.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Machida Y., Machida C., Ohtsubo E. Insertion element IS1 encodes two structural genes required for its transposition. J Mol Biol. 1984 Aug 5;177(2):229–245. doi: 10.1016/0022-2836(84)90454-6. [DOI] [PubMed] [Google Scholar]
  24. McCallum K. L., Laakso D. H., Whitfield C. Use of a bacteriophage-encoded glycanase enzyme in the generation of lipopolysaccharide O side chain deficient mutants of Escherichia coli O9:K30 and Klebsiella O1:K20: role of O and K antigens in resistance to complement-mediated serum killing. Can J Microbiol. 1989 Nov;35(11):994–999. doi: 10.1139/m89-166. [DOI] [PubMed] [Google Scholar]
  25. Meng A. Simplified downward alkaline transfer of DNA. Biotechniques. 1994 Jul;17(1):72–73. [PubMed] [Google Scholar]
  26. Morona R., Mavris M., Fallarino A., Manning P. A. Characterization of the rfc region of Shigella flexneri. J Bacteriol. 1994 Feb;176(3):733–747. doi: 10.1128/jb.176.3.733-747.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morona R., van den Bosch L., Manning P. A. Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri. J Bacteriol. 1995 Feb;177(4):1059–1068. doi: 10.1128/jb.177.4.1059-1068.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mutharia L. W., Raymond B. T., Dekievit T. R., Stevenson R. M. Antibody specificities of polyclonal rabbit and rainbow trout antisera against Vibrio ordalii and serotype 0:2 strains of Vibrio anguillarum. Can J Microbiol. 1993 May;39(5):492–499. doi: 10.1139/m93-070. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Reeves P. R., Hobbs M., Valvano M. A., Skurnik M., Whitfield C., Coplin D., Kido N., Klena J., Maskell D., Raetz C. R. Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol. 1996 Dec;4(12):495–503. doi: 10.1016/s0966-842x(97)82912-5. [DOI] [PubMed] [Google Scholar]
  32. Schmidt G., Jann B., Jann K., Orskov I., Orskov F. Genetic determinants of the synthesis of the polysaccharide capsular antigen K27(A) of Escherichia coli. J Gen Microbiol. 1977 Jun;100(2):355–361. doi: 10.1099/00221287-100-2-355. [DOI] [PubMed] [Google Scholar]
  33. Schweizer H. D. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques. 1993 Nov;15(5):831–834. [PubMed] [Google Scholar]
  34. Sekine Y., Nagasawa H., Ohtsubo E. Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1. Mol Gen Genet. 1992 Nov;235(2-3):317–324. doi: 10.1007/BF00279376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Sieberth V., Rigg G. P., Roberts I. S., Jann K. Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes. J Bacteriol. 1995 Aug;177(15):4562–4565. doi: 10.1128/jb.177.15.4562-4565.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stevenson G., Neal B., Liu D., Hobbs M., Packer N. H., Batley M., Redmond J. W., Lindquist L., Reeves P. Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J Bacteriol. 1994 Jul;176(13):4144–4156. doi: 10.1128/jb.176.13.4144-4156.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  39. Umeda M., Ohtsubo E. Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome. Gene. 1991 Feb 1;98(1):1–5. doi: 10.1016/0378-1119(91)90096-t. [DOI] [PubMed] [Google Scholar]
  40. Whitfield C., Amor P. A., Köplin R. Modulation of the surface architecture of gram-negative bacteria by the action of surface polymer:lipid A-core ligase and by determinants of polymer chain length. Mol Microbiol. 1997 Feb;23(4):629–638. doi: 10.1046/j.1365-2958.1997.2571614.x. [DOI] [PubMed] [Google Scholar]
  41. Whitfield C., Schoenhals G., Graham L. Mutants of Escherichia coli O9:K30 with altered synthesis and expression of the capsular K30 antigen. J Gen Microbiol. 1989 Oct;135(10):2589–2599. doi: 10.1099/00221287-135-10-2589. [DOI] [PubMed] [Google Scholar]
  42. Xiang S. H., Hobbs M., Reeves P. R. Molecular analysis of the rfb gene cluster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains. J Bacteriol. 1994 Jul;176(14):4357–4365. doi: 10.1128/jb.176.14.4357-4365.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES