Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Jul;174(14):4614–4621. doi: 10.1128/jb.174.14.4614-4621.1992

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.

B R Clarke 1, C Whitfield 1
PMCID: PMC206256  PMID: 1378055

Abstract

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.

Full text

PDF
4614

Images in this article

4616Image
on p.4616
4617Image
on p.4617
4618Image
on p.4618
4618Image
on p.4618
4619Image
on p.4619

Selected References

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

  1. Arakawa Y., Ohta M., Wacharotayankun R., Mori M., Kido N., Ito H., Komatsu T., Sugiyama T., Kato N. Biosynthesis of Klebsiella K2 capsular polysaccharide in Escherichia coli HB101 requires the functions of rmpA and the chromosomal cps gene cluster of the virulent strain Klebsiella pneumoniae Chedid (O1:K2). Infect Immun. 1991 Jun;59(6):2043–2050. doi: 10.1128/iai.59.6.2043-2050.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Barcak G. J., Wolf R. E., Jr Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli. J Bacteriol. 1988 Jan;170(1):365–371. doi: 10.1128/jb.170.1.365-371.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Batchelor R. A., Haraguchi G. E., Hull R. A., Hull S. I. Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli. J Bacteriol. 1991 Sep;173(18):5699–5704. doi: 10.1128/jb.173.18.5699-5704.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beger D. W., Heuzenroeder M. W., Manning P. A. Demonstration of clonal variation amongst O-antigen serotype variants of Escherichia coli O2 and O18 using DNA probes to the rfb region of the E. coli strain B41 (O101:K99/F41). FEMS Microbiol Lett. 1989 Feb;57(3):317–322. doi: 10.1016/0378-1097(89)90321-2. [DOI] [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. Darveau R. P., Hancock R. E. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol. 1983 Aug;155(2):831–838. doi: 10.1128/jb.155.2.831-838.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  10. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dolph P. J., Majerczak D. R., Coplin D. L. Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii. J Bacteriol. 1988 Feb;170(2):865–871. doi: 10.1128/jb.170.2.865-871.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Heuzenroeder M. W., Beger D. W., Thomas C. J., Manning P. A. Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/F41) and the genetic relationship to other O101 rfb loci. Mol Microbiol. 1989 Mar;3(3):295–302. doi: 10.1111/j.1365-2958.1989.tb00174.x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Johnson K. G., Perry M. B. Improved techniques for the preparation of bacterial lipopolysaccharides. Can J Microbiol. 1976 Jan;22(1):29–34. doi: 10.1139/m76-004. [DOI] [PubMed] [Google Scholar]
  18. Kessler A. C., Brown P. K., Romana L. K., Reeves P. R. Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6-dideoxyhexose abequose. J Gen Microbiol. 1991 Dec;137(12):2689–2695. doi: 10.1099/00221287-137-12-2689. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  21. Kol O., Wieruszeski J. M., Strecker G., Montreuil J., Fournet B., Zalisz R., Smets P. Structure of the O-specific polysaccharide chain from Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. Carbohydr Res. 1991 Sep 18;217:117–125. doi: 10.1016/0008-6215(91)84122-u. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Liu D., Verma N. K., Romana L. K., Reeves P. R. Relationships among the rfb regions of Salmonella serovars A, B, and D. J Bacteriol. 1991 Aug;173(15):4814–4819. doi: 10.1128/jb.173.15.4814-4819.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marolda C. L., Welsh J., Dafoe L., Valvano M. A. Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187. J Bacteriol. 1990 Jul;172(7):3590–3599. doi: 10.1128/jb.172.7.3590-3599.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McCallum K. L., Schoenhals G., Laakso D., Clarke B., Whitfield C. A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing. Infect Immun. 1989 Dec;57(12):3816–3822. doi: 10.1128/iai.57.12.3816-3822.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McCallum K. L., Whitfield C. The rcsA gene of Klebsiella pneumoniae O1:K20 is involved in expression of the serotype-specific K (capsular) antigen. Infect Immun. 1991 Feb;59(2):494–502. doi: 10.1128/iai.59.2.494-502.1991. [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 U., Mayer H. Genetic location of genes encoding enterobacterial common antigen. J Bacteriol. 1985 Aug;163(2):756–762. doi: 10.1128/jb.163.2.756-762.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. NAIDE Y., NIKAIDO H., MAEKELAE P. H., WILKINSON R. G., STOCKER B. A. SEMIROUGH STRAINS OF SALMONELLA. Proc Natl Acad Sci U S A. 1965 Jan;53:147–153. doi: 10.1073/pnas.53.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Nyman K., Plosila M., Howden L., Mäkelä P. H. Genetic determination of lipopolysaccharide: locus of O-specific unit polymerase in group E of salmonella. Zentralbl Bakteriol Orig A. 1979 Apr;243(2-3):355–362. [PubMed] [Google Scholar]
  32. Oxley D., Wilkinson S. G. Structures of neutral glycans isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O16 and O20. Carbohydr Res. 1989 Oct 31;193:241–248. doi: 10.1016/0008-6215(89)85122-5. [DOI] [PubMed] [Google Scholar]
  33. Richards J. C., Leitch R. A. Elucidation of the structure of the Pasteurella haemolytica serotype T10 lipopolysaccharide O-antigen by n.m.r. spectroscopy. Carbohydr Res. 1989 Mar 15;186(2):275–286. doi: 10.1016/0008-6215(89)84041-8. [DOI] [PubMed] [Google Scholar]
  34. Sanderson K. E., MacAlister T., Costerton J. W., Cheng K. J. Permeability of lipopolysaccharide-deficient (rough) mutants of Salmonella typhimurium to antibiotics, lysozyme, and other agents. Can J Microbiol. 1974 Aug;20(8):1135–1145. doi: 10.1139/m74-176. [DOI] [PubMed] [Google Scholar]
  35. Sanderson K. E., Roth J. R. Linkage map of Salmonella typhimurium, edition VII. Microbiol Rev. 1988 Dec;52(4):485–532. doi: 10.1128/mr.52.4.485-532.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schnaitman C. A., Austin E. A. Efficient incorporation of galactose into lipopolysaccharide by Escherichia coli K-12 strains with polar galE mutations. J Bacteriol. 1990 Sep;172(9):5511–5513. doi: 10.1128/jb.172.9.5511-5513.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  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. Valvano M. A., Marolda C. L. Relatedness of O-specific lipopolysaccharide side chain genes from strains of Shigella boydii type 12 belonging to two clonal groups and from Escherichia coli O7:K1. Infect Immun. 1991 Nov;59(11):3917–3923. doi: 10.1128/iai.59.11.3917-3923.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Whitfield C., Richards J. C., Perry M. B., Clarke B. R., MacLean L. L. Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1. J Bacteriol. 1991 Feb;173(4):1420–1431. doi: 10.1128/jb.173.4.1420-1431.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wilkinson R. G., Gemski P., Jr, Stocker B. A. Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol. 1972 May;70(3):527–554. doi: 10.1099/00221287-70-3-527. [DOI] [PubMed] [Google Scholar]

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

RESOURCES