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

Pazzani, Carlo; Rosenow, Carsten; Boulnois, Graham J.; Bronner, D; Jann, Klaus; Roberts, Ian S.

doi:10.1128/jb.175.18.5978-5983.1993

Cited by 76 publications

(84 citation statements)

References 41 publications

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“…This activity increase is caused by a second CKS determined by one of the temperature regulated capsule genes, kpsU [15]. The capsulespecific second enzyme was termed K-CKS in order to distinguish it from the isozyme L-CKS engaged in lipopolysaccharide synthesis.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…The capsulespecific second enzyme was termed K-CKS in order to distinguish it from the isozyme L-CKS engaged in lipopolysaccharide synthesis. K-CKS has recently been isolated and characterized [13][14][15][16]. It contains 245 amino acid residues among *Corresponding author.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…Primer design was based on the DNA sequence [15]. Digestion with EcoRI and HindllI yielded a 750 bp amplification product which was inserted into the expression vector pUAKyI [21].…”

Section: Expression and Purificationmentioning

confidence: 99%

“…The chain tracing was converted to a model using the program O [27] together with the DNA-derived amino acid sequence of Pazzani et al [15]. One subunit was fitted, and the other one was generated by NCS.…”

Section: Model Building and Refinementmentioning

confidence: 99%

“…All these enzymes catalyze the generation of CTP-activated sugar nucleotides. The presence of K-CKS in addition to L-CKS (lipopolysaccharide biogenesis) presumably allows for an independent regulation of group II capsule expression [15]. These capsules constitute important virulence determinants of pathogenic E. coli strains causing extraintestinal infections.…”

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confidence: 99%

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The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli

1996

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CMP-Kdo synthetases from Gram-negative bacteria activate Kdo for incorporation into lipo-and capsule-polysaccharities. Here we report the crystal structure of the capsulespecific synthetase from E. coli at 2.3 A resolution. The enzyme is a dimer of 2 x 245 amino acid residues assuming C2 symmetry. It contains a central predominantly parallel [~-sheet with surrounding helices. The chain fold is novel; it is remotely related to a double Rossmaun fold. A large pocket at the carboxyl terminal ends of the central ~-strands most likely accommodates the catalytic center. A putative phosphate binding site at the loop between the first ~strand and the following helix is indicated by a bound iridium hexachloride anion.Key words: Capsular polysaccharide; CMP-Kdo synthetase (Escherichia coli); X-ray analysis; Crystal structure; Saccharide activation which 44% are identical with L-CKS. The kinetic data of the two isozymes differ distinctly [16].The rapid spread of antibiotic resistance among bacterial strains and the fact that Kdo is absent from mammalian cells [17] make K-CKS an attractive target molecule for drug design as an anti-infective measure. Cloning of the kpsU gene from the K5 antigen gene cluster [16] permitted structural analysis of this enzyme. Here, we describe the overexpression, purification, crystallization and X-ray structure determination of K-CKS. To our knowledge this is the first actually presented structure of a sugar-activating enzyme. A short note announcing an L-CKS crystal structure has been given earlier [18]. The AMP-transferring enzyme kanamycin nucleotidyltransferase [19] and the sugar phosphate-transferring enzyme galactose-l-phosphate uridylyltransferase [20] use similar substrates, but they do not activate sugars and they differ structurally from K-CKS.

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Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…Primer design was based on the DNA sequence [15]. Digestion with EcoRI and HindllI yielded a 750 bp amplification product which was inserted into the expression vector pUAKyI [21].…”

Section: Expression and Purificationmentioning

confidence: 99%

Section: Model Building and Refinementmentioning

confidence: 99%

mentioning

confidence: 99%

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The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli

1996

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Extracellular Polysaccharides

Sutherland¹

1996

Biotechnology

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The extracellular polysaccharides of seventeen strains (four species) of Rhizobiurn were studied chromatographically. The seven strains from lucerne (R. meliloti) produced polysaccharides which contained glucose but consistently lacked glucuronic acid. The remaining ten strains, which were isolated from pea (R. leguminosarum), clover (R. triifolii) and bean (R. phaseoli), produced a polysac-charide in which both glucose and glucuronic acid were demonstrated. These latter strains could be grouped into three types according to their glucose : glucuronic acid ratio, but such groups did not correspond with the species. In 1931 Hopkins, Peterson & Fred identified glucuronic acid in the extracel-lular polysaccharide of strains of Rhizobium meliloti, R. leguminosarum and R. trifolii. Schluchterer Br; Stacey (1945) found that the polysaccharide from a clover strain consisted of a glucose-glucuronic acid chain. Contrary to these findings, De Leizaola & Dedonder (1955) found rhamnose, glucose, galactose, mannose, xylose and arabinose in preparations from four species of Rhizobium (pea, soya bean, clover, lupin), but reported complete absence of glucuronic acid. Kleczkowski & Kleczkowski (1952) reported having found fructose in a clover strain, as well as glucose and glucuronic acid in this and in a strain of R. leguminosarum. By using antisera developed against the parent bacterium, these workers obtained precipitation of antibody with the polysaccharide prepared from R. leguminosarum. With R. trifolii the polysaccharide did not cause precipitation in the homologous antiserum. In the present investigation, polysaccharides from a selection of Rhizobium strains representing four species have been examined : (a) chromatographically, with a view to investigating the report of De Leizaola & Dedonder (1955); (b) quantitatively, by methods of spectrophotometric analysis, to determine their glucose : glucuronic acid ratio. METHODS Organisms and methods of culture. The following organisms were selected for study: Rhizobium trvolii (clover strains) SU36, SUSS, SU64, SU 91, SU298/ 533, NA30, NZ 19; R. leguminosarum (pea strains) SU302, SU331; R. phaseoli (bean strains) NA 1 ; R. nzeliloti .(lucerne strains) SU 27, SU 47, SU 62, SU 74, SU76, SUlOl, SU277. (SU refers to strains from the Sydney University collection ; NAY New South Wales Department of Agriculture; NZ, the Department of Scientific and Industrial Research, New Zealand.) The organisms were inoculated into the medium formulated by Norris (1958) which contains (g./l.) mannitol, 10; disodium hydrogen phosphate, 0.45 ; magnesium sulphate heptahydrate, 0.1 ; potassium nitrate, 0.6 ; ferric chloride, 0.01; thiamin and biotin, trace. For each strain, four 1.1. flasks 31 G. Microb. XXI

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Extracellular Polysaccharides

Sutherland¹

2001

Biotechnology Set

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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

Cited by 76 publications

References 41 publications

The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli

The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli

Extracellular Polysaccharides

Extracellular Polysaccharides

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