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J Bacteriol. Jul 1994; 176(13): 4133–4143.
PMCID: PMC205613

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.


We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.

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  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Barcak GJ, Wolf RE., Jr Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli. J Bacteriol. 1988 Jan;170(1):365–371. [PMC free article] [PubMed]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • Carlin NI, Wehler T, Lindberg AA. Shigella flexneri O-antigen epitopes: chemical and immunochemical analyses reveal that epitopes of type III and group 6 antigens are identical. Infect Immun. 1986 Jul;53(1):110–115. [PMC free article] [PubMed]
  • Clark CA, Beltrame J, Manning PA. 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. [PubMed]
  • Claverie JM, Daulmerie C. Smoothing profiles with sliding windows: better to wear a hat! Comput Appl Biosci. 1991 Jan;7(1):113–115. [PubMed]
  • Cohen SN, Chang AC, Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. [PMC free article] [PubMed]
  • Collins LV, Hackett J. Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium. J Bacteriol. 1991 Apr;173(8):2521–2529. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Eisenberg D, Schwarz E, Komaromy M, Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. [PubMed]
  • Gemski P, Jr, Koeltzow DE, Formal SB. Phage conversion of Shigella flexneri group antigens. Infect Immun. 1975 Apr;11(4):685–691. [PMC free article] [PubMed]
  • Gray CH, Tatum EL. X-Ray Induced Growth Factor Requirements in Bacteria. Proc Natl Acad Sci U S A. 1944 Dec 15;30(12):404–410. [PMC free article] [PubMed]
  • Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. [PubMed]
  • Jiang XM, Neal B, Santiago F, Lee SJ, Romana LK, Reeves PR. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. [PubMed]
  • Klena JD, Schnaitman CA. 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. [PubMed]
  • Kogan G, Haraguchi G, Hull SI, Hull RA, Shashkov AS, 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. [PubMed]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed]
  • Larsen RD, Rajan VP, Ruff MM, Kukowska-Latallo J, Cummings RD, Lowe JB. Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8227–8231. [PMC free article] [PubMed]
  • Lindberg AA, Wollin R, Gemski P, Wohlhieter JA. Interaction between bacteriophage Sf6 and Shigella flexner. J Virol. 1978 Jul;27(1):38–44. [PMC free article] [PubMed]
  • Liu D, Haase AM, Lindqvist L, Lindberg AA, Reeves PR. 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. [PMC free article] [PubMed]
  • Liu D, Reeves PR. Escherichia coli K12 regains its O antigen. Microbiology. 1994 Jan;140(Pt 1):49–57. [PubMed]
  • Macpherson DF, Manning PA, Morona R. Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb locus of Shigella flexneri. Mol Microbiol. 1994 Jan;11(2):281–292. [PubMed]
  • Marolda CL, Valvano MA. 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. [PMC free article] [PubMed]
  • Marolda CL, Welsh J, Dafoe L, Valvano MA. Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187. J Bacteriol. 1990 Jul;172(7):3590–3599. [PMC free article] [PubMed]
  • Meier-Dieter U, Barr K, Starman R, Hatch L, Rick PD. 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]
  • Miller RD, Dykhuizen DE, Hartl DL. Fitness effects of a deletion mutation increasing transcription of the 6-phosphogluconate dehydrogenase gene in Escherichia coli. Mol Biol Evol. 1988 Nov;5(6):691–703. [PubMed]
  • Nasoff MS, Baker HV, 2nd, Wolf RE., Jr DNA sequence of the Escherichia coli gene, gnd, for 6-phosphogluconate dehydrogenase. Gene. 1984 Mar;27(3):253–264. [PubMed]
  • Parent R, Roy PH. The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat. J Bacteriol. 1992 May;174(9):2891–2897. [PMC free article] [PubMed]
  • Petrovskaya VG, Licheva TA. A provisional chromosome map of Shigella and the regions related to pathogenicity. Acta Microbiol Acad Sci Hung. 1982;29(1):41–53. [PubMed]
  • Peyru G, Fraenkel DG. Genetic mapping of loci for glucose-6-phosphate dehydrogenase, gluconate-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrase in Escherichia coli. J Bacteriol. 1968 Apr;95(4):1272–1278. [PMC free article] [PubMed]
  • Mohana Rao JK, Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Simmons DA, Romanowska E. Structure and biology of Shigella flexneri O antigens. J Med Microbiol. 1987 Jun;23(4):289–302. [PubMed]
  • Stevenson G, Neal B, Liu D, Hobbs M, Packer NH, Batley M, Redmond JW, 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. [PMC free article] [PubMed]
  • Tabata S, Higashitani A, Takanami M, Akiyama K, Kohara Y, Nishimura Y, Nishimura A, Yasuda S, Hirota Y. Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome. J Bacteriol. 1989 Feb;171(2):1214–1218. [PMC free article] [PubMed]
  • Vaara M. Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme. FEMS Microbiol Lett. 1992 Oct 15;76(3):249–254. [PubMed]
  • Valvano MA. Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli. Can J Microbiol. 1992 Jul;38(7):711–719. [PubMed]
  • Valvano MA, Crosa JH. Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1. Infect Immun. 1984 Oct;46(1):159–167. [PMC free article] [PubMed]
  • Valvano MA, Crosa JH. Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1. Infect Immun. 1989 Mar;57(3):937–943. [PMC free article] [PubMed]
  • Valvano MA, Silver RP, Crosa JH. Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1. Infect Immun. 1986 Apr;52(1):192–199. [PMC free article] [PubMed]
  • Verma NK, Brandt JM, Verma DJ, Lindberg AA. 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. [PubMed]
  • Whitfield C, Valvano MA. Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria. Adv Microb Physiol. 1993;35:135–246. [PubMed]
  • Wierenga RK, Terpstra P, Hol WG. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. [PubMed]
  • 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. [PubMed]
  • Yao Z, Liu H, Valvano MA. 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. [PMC free article] [PubMed]

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