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The capsule-specific coliphage K5 of Escherichia coli contains an enzyme that degrades the capsular K5 polysaccharide into many oligosaccharides through beta-elimination. The polysaccharides are capsule phage receptors and the enzyme is a K5 polysaccharide lyase. The major components of the reaction are hexa-, octa- and decasaccharides. A 4,5-unsaturated glucuronic acid is present at the products nonreducing end.

A genetic analysis of subcloning, transposon insertion and detection revealed the Escherichia coli O9 rfb region to be approximately 12 kilobases in size, with the region encoding seven polypeptides. The 50 and 55 kDa polypeptides were phosphomannomutase and GDP-mannose pyrophosphorylase, respectively.

A group II K antigen capsular layer in Escherichia coli that expresses colanic acid is formed and analyzed. The presence of rcsB regulator sequences in E.coli K1 and K5 confirms that group II K antigens are not regulated by the rcs system. Colanic acid biosynthesis gene cluster is translated and regulated by the Rcs system. Colonic acid is an extracellular polysaccharide.

The region 1 mutants were characterized and examined by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity and chemical studies on K5 polysaccharides. Several mutations present in region 1 induced polysaccharide transposition and also inhibited the polymerization ability of the membranes. KDO and phosphatidic acid replacement were observed in extracellular and periplasmic polysaccharide, but not in cytoplasmic polysaccharide.

The O-specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L-rhamnose (Rha), N-acetyl-D-glucosamine, D-galactose, and D-glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one-and two-dimensional NMR spectroscopy, the structures of these polysaccharides were found to be [This symbol cannot be presented in ASCII format] In O18A-PS and O18A1-PS x = 2, whereas in O18B-PS and in O18B11-PS x = 3.In all four polysaccharides α-D-Galp (residue D) is substituted at O-3. This substituent L (residue E) is β-D-GlcpNAc-(1 in O18A-PS and O18A1-PS and it is α-D-Glcp-(1 in O18B-PS and O18B1-PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1-PS and O18B1-PS the α-D-GlcpNAc residue A is substituted with α-Glcp-(1 (residue F), which is linked to O-6 in O18A1-PS and to O-4 in O18B1-PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications. [ABSTRACT FROM AUTHOR]

The chemical structure of the K4-specific capsular polysaccharide (K4 antigen) of Escherichia coli 05: K4: H4 was elucidated by composition, carboxyl reduction periodate oxidation methylation nuclear-magnetic-resonance spectroscopy and enzymatic cleavage. The polysaccharide consists of a backbone with the structure → 3)-β-Dglucuronyl-(1,4)-β-D-N-acetylgalactosaminyl(1- to which β-fructofuranose is linked at C-3 of glucuronic acid. Mild acid hydrolysis liberated fructose and converted the K4 antigen into a polysaccharide which has the same structure as chondroitin. The defructosylated polysaccharide was a substrate for hyaluronidase and chondroitinase. The serological reactivity of the K4 polysaccharide was markedly reduced after defructosylation. [ABSTRACT FROM AUTHOR]

The chemical structure of the K52 antigenic capsular polysaccharide (K 52 antigen) of Escherichia coli O4:K 52: H- was elucidated by composition, nuclear magnetic resonance spectroscopy, methylation, periodate oxidation before and after graded acid hydrolysis and by oligosaccharide analysis. The polysaccharide consists of a backbone of α-galactose units interlinked between C1 and C3 by phosphodiester bridges. This poly(α-galactosyl-phosphate) is substituted at C2 of each galactose unit by β-fructofuranose residues. About 80% of the galactose units are O-acetylated at C4 and about 10% of the fructose units are both O-acetylated and O-propionylated at C1. The K 52 polysaccharide has an average molecular mass of 34 kDa, thus consisting of approximately 65 fructosyl-galactosyl-phosphate repeating units. [ABSTRACT FROM AUTHOR]

The chemical structure of the outer (hexose) regions of the core olugosaccharide from Escherichia coli 09 with the complete R1 core, and from a R1-derived rfe mutant were analyzed using compositional analysis, methylation and gas chromatography/mass spectrometry. It was found that, in contrast to the branched outer region of the R1 core, the outer region of the core from rfe mutant lacked terminal glucose and was linear. These results are in agreement with recent findings on the biosynthesis of the 09 antigen. They suggest a cotransfer with the 09-specific mannan to a 'pre-core' lacking terminal glucose, as the assembly (translocation) step in the 09 antigen synthesis. Thus it is suggested that the initation of O-chain synthesis (by the formation of an acceptor glucolipid) and the termination of core synthesis are closely correlated. In conjuction with previous biochemical data, the analytical results presented here indicate a novel core synthesis. [ABSTRACT FROM AUTHOR]

The O-specific polysaccharide moiety (04 polysaccharide) of the 04 antigen (lipopolysaccharide) from Escherichia coli 04:K12:H- was isolated in pure form by degradation of the lipopolysaccharide and chromatography on Sephadex G-50. The primary structure of the 04 polysaccharide was elucidated by composition, Smith degradation, nuclear magnetic resonance spectroscopy, methylation and oligosaccharide analysis. Oligosaccharides were obtained by deaminating fragmentation of partially and completely de-N-acetylated polysaccharides. The polysaccharide consists of repeating units of the pentasaccharide [This symbol cannot be presented in ASCII format] which are joined in the polymer through α-1,4-linkages. The 04 polysaccharide has a mean molecular mass of 13800 Da and consists of the core oligosaccharide and about 14 pentasaccharide repeating units. [ABSTRACT FROM AUTHOR]

The lipopolysaccharide from Escherichia coli O9: K30- was isolated in about 2% yield with aqueous 45 % phenol at 65 %C, followed by ulracentrifugation. The polysaccharide moiety was obtained by graded hydrolysis and gel permeation chromatography. It consisted of a mannan which carried on its reducing end the core oligosaccharide of the R1 type. The mannan contained 1→2 and 1→3 linkages in a ratio of 3:2, as determined by methylation analysis and mass spectrometry. On periodate oxidation, 58 % of the mannose residues were destroyed. Degradation of oligosaccharide mixtures with α-mannosidase from jack bean meal, as well as a specific rotation of [α]D25 = + 89° indicated that all mannosyl linkages have the α-configuration. Smith degradation resulted in the liberation of mannosyl(1→3)-mannose (bound to glyceraldehyde), as established by methylation analysis. From these results we conclude that the O9 polysaccharide of E. coli has a pentasaccharide repeating unit of α-mannosyl (1 → 3)-α-mannosyl-( 1→ 2)-α-mannosyI-( 1→2)-α-mannosyl-( 1 → 2)-mannose, which are joined in the polysaccharide through α-(1 → 3)-mannosyl linkages. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide preparations from R (rough) Escherichia coli O8-, SR (semirough) Salmonella typhimurium and S (smooth) stains E. coli O8 and Citrobacter 396 were disintegrated with sodium dodecylsulfate and subjected to polyacrylamide gel electrophoresis in the presence of 1% sodium dodecylsulfate. The results obtained were compared with those obtained from the same lipopolysaccharaide preparations by degradation analysis. In dodecylsulfate gel electrophoresis the lipopolysaccharide preparation from the E. coli R mutant and the S. typhimurium SR mutant showed one band each (R- and SR- band, respectively) with different electrophoretic, mobilities. The lipopolysaccharide preparation from the E. coli O8-strain exhibited two bands, one of which had the same electrophoretic mobility as the R-band and the other was identified as S-band. The lipopolysaccharide preparation from the Citrobacter 396 S-strain exhibited four bands: one R-band, one SR-band and two S-bands. The results showed that wild-type S strains contain more than one type of lipopolysaccharide. They differ in the length of their O-specific polysaccharide chains. The lipid A content of the different lipopolysaccharide was expressed in their electrophoretic mobilities. [ABSTRACT FROM AUTHOR]

Acapsular (K-) mutants selected from Escherichia coli O14:K7 proved to be rough (R) forms, indicating the absences, of O-specific polysaccharides in the wild type K+ strain. This suggestion was confirmed by chemical analysis and degradation studies of the lipopo]ysaccharides from K+ and K-strains. In order to determine the chromosomal site of the S/R mutation in E. coli O14:K7, recombination experiments were performed. An acapsular R form (F2513) of E. coli O14:K7 was crossed with the E. coli O8 donor Hfr59. Most of the recombinants selected for the donor his+ allele showed O8-specificity in consequence of the transfer of the his-linked donor rfb cluster, governing the synthesis of O8-specific polysaccharides. This indicates that the rough phenotype of E. coli O14 is caused by mutational defects in the rib genes and that. this R strain produces a complete lipopolysaccharide core structure. As shown by serological tests and phage typing the E. coli O14 lipopolysaccharide core is different from other known complete core types (R1-R3 and K12). Partial cross reactivity between the lipopolysaccharides of E. coli O14 and E. coli RI mutants indicates a certain degree of structural similarity of the respective cores. Glucose, galactose, heptose and 2-keto-3-deo×yoctonate were identified as constituents of both core oligosaccharides. Quantitative analysis, however, revealed differences in the molar ratios of galactose : glucose : heptose which in the core of E. coli O14 is 3:2:3 and in the R1 core is 2:3:3. The results described here showed that E. coli O14:K7 lacks O-specific polysaccharides and represents an encapsulated rough (R) strain with a complete lipopolysaccharide core designated as R4. [ABSTRACT FROM AUTHOR]

The lipopolysaccharides of two wild-type Escherichia coli K-12 strains, two core-deficient mutants and one SR recombinant with Salmonella typhimurium specificity were analyzed. The respective oligosaccharides were dephosphorylated and methylated. Chemical analysis of the oligosaccharides and mass spectrometric analysis of their methylated derivatives indicated tire presence of core structures with different degrees of completion. In different strains of E. coli K-12 the complete core is substituted at the non-reducing end with N-acetylglucosamine or with another substituent. There are indications that the latter may be N-acetylmannosaminuronic acid. In the SR recombinant the complete (N-acetylglucosamine-free) K-12 core is substituted with one S-specific oligosaccharide of S. typhimurium. The attachment site for all these substituents is the 6-position of the non-reducing core-terminal glucose. The heterogeneity of the K-12 core preparations and mode and nature of their substitution are discussed. [ABSTRACT FROM AUTHOR]

The lipopolysaccharides of ampicillin-resistant cell-wall-defective mutants of Escherichia coil K-12 were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the core oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. From core-defective mutants substructures of the K-12 core were obtained. Analysis of the lipopolysaccharide preparations from wild-type K-12 indicated the presence of several core structures with different degrees of completion. The lipopolysaccharide preparation was degraded and the oligosaccharide mixture was partially resolved by gel filtration chromatography. Methylation, gas chromatography and mass spectrometry of the oligosaccharides permitted the tentative formulation of the K-12 core structure. Alternative interpretations for this heterogeneity are discussed. [ABSTRACT FROM AUTHOR]

From Escherichia coli 0124 two lipopolysaccharide preparations were obtained with phenol/water extraction and cetavlon precipitation. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and chemical analysis showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. In passive haemagglutination and gel precipitation the two lipopolysaccharide preparations from E. coli 0124 and the corresponding preparations from Shigella dysenteriae type 3 reacted alike. The O-specific polysaccharide moiety was characterized with proton magnetic resonance spectroscopy, optical rotation and paper electrophoresis. The constituents were determined by gas chromatography and ion-exchange chromatography. The polysaccharide contained glucose (Glc), gatactose (Gal), galactosamine (GalN) and 4-O-(1′-carboxyethyl)-D-glucopyranose (glucolactilic acid, GIcLA) in the molar ratios of 1 : 2 : 1 : 1. Glucolactilic acid, which has a structure similar to muramic acid, was first found in Sh. dysenteriae. The polysaccharide from E. coli 0124 and oligosaccharides obtained from it by partial acid hydrolysis were subjected to methylation analysis using the method of combined gas chromatography—mass spectrometry. The results indicated that the pentasaccharide repeating unit of the polysaccharide is[This symbol cannot be presented in ASCII format] In the polysaccharide the repeating units are joined through galactofuranosidic linkages. This structure is identical with that of the somatic polysaccharide of Sh. dysenterae type 3. [ABSTRACT FROM AUTHOR]