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J Bacteriol. 1995 August; 177(15): 4562–4565.
PMCID: PMC177216

Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes.


Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthesis of the K5 polysaccharide. A DNA fragment containing kfiD was amplified by PCR and cloned into the gene fusion vector pGEX-2T to generate a GST-KfiD fusion protein. The fusion protein was isolated from the cytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced recombinant bacteria by affinity chromatography and cleaved with thrombin. The N-terminal amino acid sequence of the cleavage product KfiD' corresponded to the predicted amino acid sequence of KfiD with an N-terminal glycyl-seryl extension from the cleavage site of the fusion protein. Anti-KfiD antibodies obtained with KfiD' were used to isolate the intact KfiD protein from the cytoplasms of E. coli organisms overexpressing the kfiD gene. The fusion protein, its cleavage product (KfiD'), and overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein could thus be characterized as a UDPglucose dehydrogenase.

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Selected References

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  • Boulnois GJ, Roberts IS. Genetics of capsular polysaccharide production in bacteria. Curr Top Microbiol Immunol. 1990;150:1–18. [PubMed]
  • Boulnois GJ, Roberts IS, Hodge R, Hardy KR, Jann KB, Timmis KN. 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. [PubMed]
  • Bronner D, Sieberth V, Pazzani C, Roberts IS, Boulnois GJ, 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. [PMC free article] [PubMed]
  • Bronner D, Sieberth V, Pazzani C, Smith A, Boulnois G, Roberts I, Jann B, Jann K. Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli. FEMS Microbiol Lett. 1993 Nov 1;113(3):279–284. [PubMed]
  • Cross AS. The biologic significance of bacterial encapsulation. Curr Top Microbiol Immunol. 1990;150:87–95. [PubMed]
  • Dougherty BA, van de Rijn I. Molecular characterization of hasA from an operon required for hyaluronic acid synthesis in group A streptococci. J Biol Chem. 1994 Jan 7;269(1):169–175. [PubMed]
  • Dougherty BA, 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]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Fath MJ, Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. [PMC free article] [PubMed]
  • Gotschlich EC, Fraser BA, Nishimura O, Robbins JB, Liu TY. Lipid on capsular polysaccharides of gram-negative bacteria. J Biol Chem. 1981 Sep 10;256(17):8915–8921. [PubMed]
  • Higgins CF, Hyde SC, Mimmack MM, Gileadi U, Gill DR, Gallagher MP. Binding protein-dependent transport systems. J Bioenerg Biomembr. 1990 Aug;22(4):571–592. [PubMed]
  • Jann B, Jann K. Structure and biosynthesis of the capsular antigens of Escherichia coli. Curr Top Microbiol Immunol. 1990;150:19–42. [PubMed]
  • Jann K, Jann B. Capsules of Escherichia coli, expression and biological significance. Can J Microbiol. 1992 Jul;38(7):705–710. [PubMed]
  • Kröncke KD, Boulnois G, Roberts I, Bitter-Suermann D, Golecki JR, 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. [PMC free article] [PubMed]
  • Lipman DJ, Pearson WR. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. [PubMed]
  • Pavelka MS, Jr, Wright LF, Silver RP. 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. [PMC free article] [PubMed]
  • Pazzani C, Rosenow C, Boulnois GJ, Bronner D, Jann K, Roberts IS. 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. [PMC free article] [PubMed]
  • Reizer J, Reizer A, Saier MH., Jr A new subfamily of bacterial ABC-type transport systems catalyzing export of drugs and carbohydrates. Protein Sci. 1992 Oct;1(10):1326–1332. [PubMed]
  • Roberts IS, Mountford R, Hodge R, Jann KB, Boulnois GJ. Common organization of gene clusters for production of different capsular polysaccharides (K antigens) in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1305–1310. [PMC free article] [PubMed]
  • Schiller JG, Lamy F, Frazier R, Feingold DS. UDP-glucose dehydrogenase from Escherichia coli. Purification and subunit structure. Biochim Biophys Acta. 1976 Dec 22;453(2):418–425. [PubMed]
  • Silver RP, Vann WF, Aaronson W. Genetic and molecular analyses of Escherichia coli K1 antigen genes. J Bacteriol. 1984 Feb;157(2):568–575. [PMC free article] [PubMed]
  • Smith AN, Boulnois GJ, Roberts IS. 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. [PubMed]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Vann WF, Schmidt MA, Jann B, Jann K. The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin. Eur J Biochem. 1981 May 15;116(2):359–364. [PubMed]
  • Vimr ER, Aaronson W, Silver RP. Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1. J Bacteriol. 1989 Feb;171(2):1106–1117. [PMC free article] [PubMed]

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