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. 1995 Jul;177(14):3992–3997. doi: 10.1128/jb.177.14.3992-3997.1995

Cloning and analysis of gene clusters for production of the Escherichia coli K10 and K54 antigens: identification of a new group of serA-linked capsule gene clusters.

R Pearce 1, I S Roberts 1
PMCID: PMC177129  PMID: 7608072

Abstract

The polysaccharide capsules of Escherichia coli have been classified into three groups: I, II, and I/II. The third group, I/II, has been poorly studied and possesses characteristics of both group I and group II capsules. In this report, we describe the cloning of the K10 and K54 capsule gene clusters, two representatives of group I/II capsules. Probes taken from DNA flanking regions 1 and 3 of the group II capsule clusters hybridized to these group I/II clones, confirming that the group I/II capsule genes are flanked by the same DNA and are therefore located in the same serA-linked region of the chromosome as group II capsule gene clusters. Southern blotting showed that homologous sequences were present in both the K10 and K54 capsule gene clusters and in other group I/II strains. No homology was detected between these sequences and the chromosomal DNA of either a group I or a group II strain. Likewise, no homology was detected to the chromosomal DNA of either a K11 or K19 strain, both of which had previously been classified as group I/II strains. In the K10 and K54 capsule gene clusters, these conserved sequences flanked a serotype-specific region in a manner analogous to group II capsule gene organization. Complementation of mutations in the kpsE, kpsD, and kpsC genes in region 1 of the K5 capsule gene cluster by subclones of the K10 and K54 capsule gene clusters indicated that certain stages in the export of group II and I/II capsules may be conserved. In the light of the findings presented here, we suggest that the group I/II capsule gene clusters are sufficiently different from group II capsule gene clusters to justify their renaming as group III.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boulnois G. J., Jann K. Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity. Mol Microbiol. 1989 Dec;3(12):1819–1823. doi: 10.1111/j.1365-2958.1989.tb00168.x. [DOI] [PubMed] [Google Scholar]
  3. Boulnois G. J., Roberts I. S. Genetics of capsular polysaccharide production in bacteria. Curr Top Microbiol Immunol. 1990;150:1–18. doi: 10.1007/978-3-642-74694-9_1. [DOI] [PubMed] [Google Scholar]
  4. Boulnois G. J., Roberts I. S., Hodge R., Hardy K. R., Jann K. B., Timmis K. N. Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production. Mol Gen Genet. 1987 Jun;208(1-2):242–246. doi: 10.1007/BF00330449. [DOI] [PubMed] [Google Scholar]
  5. Boulnois G., Drake R., Pearce R., Roberts I. Genome diversity at the serA-linked capsule locus in Escherichia coli. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):121–124. doi: 10.1111/j.1574-6968.1992.tb14029.x. [DOI] [PubMed] [Google Scholar]
  6. Bronner D., Sieberth V., Pazzani C., Roberts I. S., Boulnois G. J., Jann B., Jann K. Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster. J Bacteriol. 1993 Sep;175(18):5984–5992. doi: 10.1128/jb.175.18.5984-5992.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  8. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cieslewicz M. J., Steenbergen S. M., Vimr E. R. Cloning, sequencing, expression, and complementation analysis of the Escherichia coli K1 kps region 1 gene, kpsE, and identification of an upstream open reading frame encoding a protein with homology to GutQ. J Bacteriol. 1993 Dec;175(24):8018–8023. doi: 10.1128/jb.175.24.8018-8023.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [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. Drake C. R., Roberts I. S., Jann B., Jann K., Boulnois G. J. Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide, a fructose-substituted chondroitin. FEMS Microbiol Lett. 1990 Jan 1;54(1-3):227–230. doi: 10.1016/0378-1097(90)90287-z. [DOI] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Finke A., Jann B., Jann K. CMP-KDO-synthetase activity in Escherichia coli expressing capsular polysaccharides. FEMS Microbiol Lett. 1990 May;57(1-2):129–133. doi: 10.1016/0378-1097(90)90426-q. [DOI] [PubMed] [Google Scholar]
  15. Finke A., Roberts I., Boulnois G., Pzzani C., Jann K. Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis. J Bacteriol. 1989 Jun;171(6):3074–3079. doi: 10.1128/jb.171.6.3074-3079.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jann B., Jann K. Structure and biosynthesis of the capsular antigens of Escherichia coli. Curr Top Microbiol Immunol. 1990;150:19–42. doi: 10.1007/978-3-642-74694-9_2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Jann K., Jann B. Capsules of Escherichia coli, expression and biological significance. Can J Microbiol. 1992 Jul;38(7):705–710. doi: 10.1139/m92-116. [DOI] [PubMed] [Google Scholar]
  19. Kröncke K. D., Boulnois G., Roberts I., Bitter-Suermann D., Golecki J. R., Jann B., Jann K. Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli. J Bacteriol. 1990 Feb;172(2):1085–1091. doi: 10.1128/jb.172.2.1085-1091.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Moxon E. R., Kroll J. S. The role of bacterial polysaccharide capsules as virulence factors. Curr Top Microbiol Immunol. 1990;150:65–85. doi: 10.1007/978-3-642-74694-9_4. [DOI] [PubMed] [Google Scholar]
  22. Orskov F., Sharma V., Orskov I. Influence of growth temperature on the development of Escherichia coli polysaccharide K antigens. J Gen Microbiol. 1984 Oct;130(10):2681–2684. doi: 10.1099/00221287-130-10-2681. [DOI] [PubMed] [Google Scholar]
  23. Orskov I., Nyman K. Genetic mapping of the antigenic determinants of two polysaccharide K antigens, K10 and K54, in Escherichia coli. J Bacteriol. 1974 Oct;120(1):43–51. doi: 10.1128/jb.120.1.43-51.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Orskov I., Sharma V., Orskov F. Genetic mapping of the K1 and K4 antigens (L) of Escherichia coli. Non-allelism of K(L) antigens with K antigens of O8:K27(A), O8:K8(L) and O9:K57(B). Acta Pathol Microbiol Scand B. 1976 Jun;84(3):125–131. [PubMed] [Google Scholar]
  26. 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]
  27. Pazzani C., Rosenow C., Boulnois G. J., Bronner D., Jann K., Roberts I. S. Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide. J Bacteriol. 1993 Sep;175(18):5978–5983. doi: 10.1128/jb.175.18.5978-5983.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roberts I. S., Mountford R., Hodge R., Jann K. B., Boulnois G. J. Common organization of gene clusters for production of different capsular polysaccharides (K antigens) in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1305–1310. doi: 10.1128/jb.170.3.1305-1310.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roberts I., Mountford R., High N., Bitter-Suermann D., Jann K., Timmis K., Boulnois G. Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli. J Bacteriol. 1986 Dec;168(3):1228–1233. doi: 10.1128/jb.168.3.1228-1233.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  31. Sieberth V., Jann B., Jann K. Structure of the K10 capsular antigen from Escherichia coli O11:K10:H10, a polysaccharide containing 4,6-dideoxy-4-malonylamino-D-glucose. Carbohydr Res. 1993 Aug 17;246:219–228. doi: 10.1016/0008-6215(93)84034-4. [DOI] [PubMed] [Google Scholar]
  32. Silver R. P., Vann W. F., Aaronson W. Genetic and molecular analyses of Escherichia coli K1 antigen genes. J Bacteriol. 1984 Feb;157(2):568–575. doi: 10.1128/jb.157.2.568-575.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Steenbergen S. M., Vimr E. R. Mechanism of polysialic acid chain elongation in Escherichia coli K1. Mol Microbiol. 1990 Apr;4(4):603–611. doi: 10.1111/j.1365-2958.1990.tb00629.x. [DOI] [PubMed] [Google Scholar]
  36. Vimr E. R., Aaronson W., Silver R. P. Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1. J Bacteriol. 1989 Feb;171(2):1106–1117. doi: 10.1128/jb.171.2.1106-1117.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vimr E. R. Map position and genomic organization of the kps cluster for polysialic acid synthesis in Escherichia coli K1. J Bacteriol. 1991 Feb;173(3):1335–1338. doi: 10.1128/jb.173.3.1335-1338.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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