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The capsular K5 polysaccharide of Escherichia coli is the receptor of the capsule-specific coliphage K5, which harbors an enzyme that degrades the capsular K5 polysaccharide to a number of oligosaccharides. Analysis of the degradation products using gel permeation chromatography, the periodate-thiobarbituric acid and bicinchoninic acid reactions, and nuclear magnetic resonance spectroscopy showed that the major reaction products are hexa-, octa-, and decasaccharides with 4,5-unsaturated glucuronic acid (delta4,5GlcA) at their nonreducing end. Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase.

Structures for the N-acetylneuraminic acid (Neu5Ac)-containing O56 and O24 polysaccharides of Escherichia coli have been reported previously. During these studies unusual chemical shifts had been observed for the NMR signals for H-3eq and C-3 of the Neu5Ac residues of both polysaccharides. In further pursuing this phenomenon, we have reinvestigated the O56 and O24 polysaccharides as well as derived oligosaccharides by one- and two-dimensional NMR spectroscopy. The results showed that structures of both polysaccharides (PSs) had to be modified and formulated as [formula: see text] 2D ROESY spectra revealed a strong NOE between H-3eq of Neu5Ac and the protons of the side-chain sugar (H-3 and H-5 of alpha-D-Gal p in the O56 PS and H-3 of alpha-D-Glc p in the O24 PS) and also between H-3ax of Neu5Ac and H-3 of beta-D-Glc p in the main chain. This indicated a close spatial association of the seven-linked alpha-Neu5Ac and the side-chain residues alpha-D-Gal p (O56 PS) and alpha-D-Glc p (O25 PS), respectively. The strong long-range spatial contacts caused the unusual chemical shifts of H-3eq and C-3 of Neu5Ac.

The polysaccharide moiety of the O16 antigen (lipopolysaccharide) consists of D-glucopyranose, D-galactofuranose, L-rhamnopyranose, and 2-acetamido-2-deoxy-D-glucopyranose in the molar ratios 1:1:1:1. It is O-acetylated with one acetyl group per repeating unit. One- and two-dimensional NMR spectroscopy of the polysaccharide before and after O-deacetylation showed that the O16 polysaccharide has the structure [formula: see text]

Two distinct forms of the O6 antigen (LPS) from E. coli were analysed using 1H and 13C NMR spectroscopy. Their structures were found to be [formula: see text] In the O6-specific polysaccharide from E. coli O6:K2 and O6:K13, X is beta-D-Glc p, as had previously been shown for the O6 polysaccharide from E. coli O6:K15; in the O6 specific polysaccharide from E. coli O6:K54, X is beta-D-Glc pNAc.

The polysaccharide moiety of the O22-antigen (lipopolysaccharide, LPS) consists of 2-acetamido-2-deoxy-D-galactose, D-glucuronic acid, D-glucose, and D-galactose in the molar ratios 2:1:1:1. Methylation analysis as well as 1D and 2D NMR spectroscopy showed that the O22 polysaccharide has the primary structure [formula: see text]

Two distinct forms of the O4 antigen (LPS) from E. coli were analysed by 1H and 13C NMR spectroscopy. Both consisted of D-glucose, L-rhamnose, 2-acetamido-2,6-dideoxy-L-galactose (L-FucNAc), and 2-acetamido-2-deoxy-D-glucose. Their structures were found to be [formula: see text]. In the O4-specific polysaccharide from E. coli O4:K3, O4:K6, and O4:K12, X is alpha-D-Glcp. In the O4 specific polysaccharide from E. coli O4:K52, the rhamnose residue is not substituted (X = H).

The polysaccharide moiety of the O23 antigen (lipopolysaccharide) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose in the molar ratios 2:1:2:1. Methylation analysis of the polysaccharide as well as one- and two-dimensional 1H and 13C NMR spectroscopy of the polysaccharide and a trisaccharide obtained by Smith degradation showed that the O23 polysaccharide has the primary structure [formula: see text].

A computer evaluation of 13C-NMR data for the title polysaccharides based on the monosaccharide and methylation analysis data led to the structure of the repeating unit of the O1A polysaccharide as well as to several probable structures of the O1C polysaccharide, of which the correct one was inferred by means of a single NOE experiment. The analysis of the spectrum of the O1B polysaccharide was unsuccessful, due to the presence in its structure of the fragment alpha-L-Rha-(1-->2)-alpha-D-Gal-(1-->3)-D-GlcNAc with the terminal (1-->2)-linkage, whose spectral data could not be calculated by additive schemes using only glycosylation effects. However in reevaluation of the O1B spectral data by taking into account the deviations from additivities of the chemical shifts values in spectra of the related trisaccharides, to reveal the most probable structure of the O1B's repeating unit. [formula: see text]

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.

The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

The K10 antigen from Escherichia coli O11:K10:H10 consists of equimolar amounts of rhamnose and 4,6-dideoxy-4-malonylaminoglucose [Qui4NMal; 4-(2-carboxyacetamido)-4,6-dideoxyglucose]. Methylation analysis and 1 and 2D NMR spectroscopy showed that the K10 capsular polysaccharide has the structure [formula: see text]

In previous studies it had been shown that lipopolysaccharide from O4-specific recombinant Escherichia coli, had serological reactivities and a chemical composition that differed from wildtype O4 LPS [Haraguchi, G.E., Zähringer, U., Jann, B., Jann, K., Hull, R.A. & Hull, S.I. (1991) Microb. Pathog. 10, 351-361]. Here we present the structural elucidation of the O-specific moieties from lipopolysaccharides of some of the recombinant strains obtained in previous studies. Compositional analysis, methylation, chemical reactions and NMR spectroscopy showed that, during genetic manipulations (recombination, cosmid cloning, plasmid subcloning), a gradual structural change in the O-specific polysaccharides was observed in the recombinant strains. These changes comprised of an alteration in the position of glucose (side chain) substitution, a change in the anomeric configuration of the main-chain N-acetylglucosamine and an exchange of alpha-L-rhamnopyranose for beta-D-galactofuranose. The relevance of these results for lipopolysaccharide cloning and lipopolysaccharide biosynthesis are discussed.

The O56 polysaccharide moiety of the O56 antigen (LPS) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and N-acetylneuraminic acid in the molar ratios 1:1:1:1. Methylation analysis, periodate oxidation, mild acid hydrolysis, as well as 1H and 13C NMR spectroscopy showed that the O56 polysaccharide has the primary structure [formula: see text]

The primary structure of an emulsion-stabilizing exopolysaccharide from the adhesive, hydrophobic Rhodococcus strain No. 33 was elucidated by NMR spectroscopy, methylation analyses, periodate oxidation and oligosaccharide analyses. The polysaccharide PS-33 consisted of rhamnose, galactose, glucose and glucuronic acid in molar ratios of 2:1:1:1. The main chain contained 3-substituted alpha-D-glucuronic acid linked to the 3-position at alpha-L-rhamnose, in addition to 3-substituted residues of beta-D-galactose and alpha-D-glucose. The alpha-L-rhamnose of the side chain was linked to position 4 of the galactose. In addition, the polysaccharide was O-acetylated, corresponding to one acetyl group per repeating unit. From the results two structural possibilities could be suggested. As the polysaccharide carries hydrophobic groups (methyl of rhamnose/O-acetyl), it is very likely that these are of general significance for the emulsifying activity of polysaccharides. It also seems to be possible that this polysaccharide is at least partially responsible for the hydrophobic cell surface properties of the Rhodococcus strain No. 33 and it may be involved in hydrophobic interactions when adhering to hydrophobic interfaces.

The O-specific moieties of the O1B antigen (lipopolysaccharide) from Escherichia coli O1B:K1 and the O1C antigen from E. coli O1C:K- both consist of L-rhamnose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in a molar ratio of 2:1:1:1. By using fragmentation procedures, methylation analysis, and one- and two-dimensional nuclear magnetic resonance spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In the O1B polysaccharide X is 2, and in the O1C polysaccharide X is 3. With the recently published structure of the O1A polysaccharides (B. Jann, A. S. Shashkov, D. S. Gupta, S. M. Panasenko, and K. Jann, Carbohydr. Polym. 18:51-57 1992), three related O1 antigens are now known. Their common (O1-specific) epitope is suggested to be the side-chain N-acetyl-D-mannosamine residue.

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 [formula: see text] In O18A-PS and O18A1-PS x = 2, whereas in O18B-PS and in O18B11-PS x = 3. In all four polysaccharides alpha-D-Galp (residue D) is substituted at O-3. This substituent L (residue E) is beta-D-GlcpNAc-(1 in O18A-PS and O18A1-PS and it is alpha-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 alpha-D-GlcpNAc residue A is substituted with alpha-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.

The 0104 antigen (lipopolysaccharide, LPS) of Escherichia coli has an acidic O specific polysaccharide. From the aqueous phase of a phenol water extraction of E. coli O104: K-, a fraction was obtained by ultracentrifugation and Cetavlon precipitation of the supernatant, which was enriched in long-chain LPS. Compositional analysis, NMR spectroscopy, periodate oxidation and methylation analysis showed that the polysaccharide chain of O104 LPS II consisted of galactose, N-acetylgalactosamine and neuraminic acid and acetate in the molar ratio of 2:1:1:1 and contained 3-beta Gal, 3-beta GalNAc, 4-alpha Gal, and 4-alpha(9-OAc-NeuNAc) in linear sequence. The same results were obtained with the capsular K9 polysaccharide from E. coli O9:K9, as presented here and reported previously (Dutton et al. (1987) Carbohydr. Res. 170, 193-206).

The capsular polysaccharide (K98 antigen) of E. coli O7:K98:H6 contains rhamnose, glucuronic acid, and acetate in the molar ratios 3:1:0.6. Methylation analysis, oligosaccharide analysis, and 1D- and 2D-n.m.r. spectroscopy revealed the polysaccharide to be a glucuronic acid-substituted rhamnan with the structure [formula; see text] Of the 3-linked rhamnose residues, approximately 60% are O-acetylated at position 2.

Biosynthesis of the capsular K5 polysaccharide of Escherichia coli, which has the structure 4)-beta GlcA-1,4-alpha GlcNAc-(1, was studied with membrane preparations from an E. coli K5 wild-type strain and from a recombinant K-12 strain expressing the K5 capsule. Polymerization occurs at the inner face of the cytoplasmic membrane without the participation of lipid-linked oligosaccharides. The serological K5 specificity of the in vitro product was determined with a K5-specific monoclonal antibody in an antigen-binding assay. The K5 polysaccharide, as obtained from the membranes after an in vitro incubation, has 2-keto-3-deoxyoctulosonic acid as the reducing sugar, which indicates that the polysaccharide grows by chain elongation at the nonreducing end.

The Escherichia coli O4 serotype is among those commonly isolated from urinary tract infections. In order to study the genetics of the O-antigen, the O4 biosynthesis genes from a uropathogenic E. coli have previously been cloned into E. coli K-12. A subclone, GH58, has been identified which reacts with antisera against the O4 serotype. In contrast to the wild-type parental strain, lipopolysaccharide (LPS) from this clone is devoid of rhamnose and does not cross-react with O18 antisera. The recombinant plasmid from GH58, pGH58, was used to transform the rfb deletion strain HU1190. The resultant strain agglutinates in O4 antisera, but produces unpolymerized LPS. Escherichia coli K-12 strains HB101 and RC712 containing pGH58 produce polymerized LPS, indicating that the genetic background of the host can influence the LPS encoded by recombinant molecules. A cosmid, pGH84, has been identified which encompasses the entire pGH58 gene sequences and includes an additional 34 kilobases of DNA. HU1190 containing this cosmid agglutinates in O4 antisera and produces a polymerized LPS. By constructing several deletion subclones of pGH84, we have localized the genes necessary for polymerized LPS to a 5.5 kb ClaI-BamHI fragment. P1 transductants that make polymerized and unpolymerized O4 LPS have also been identified.

Escherichia coli 21535 (O21:K4:H4 with nonfimbrial adhesin NFA-6) and 21511 (O7:K98:H6 with nonfimbrial adhesin NFA-4) were analyzed by immunoelectron microscopy with a K98-specific antiserum and K4- and NFA-4-specific and NFA-6-reactive monoclonal antibodies. The bacteria were analyzed in ultrathin sections after stabilization of the capsules with specific antibodies by embedding in Epon 812 as well as in Lowicryl K4M. With the Lowicryl-embedded samples, the polysaccharide K antigens were labeled by the immunogold technique. It was found that with both strains all bacteria expressed the polysaccharide capsule, while in each case about 20% expressed the protein capsule in addition. Thus, in both invasive E. coli strains, bacteria are present which express composite capsules with the adhesin (recognition protein) at the cell-distal outer region and the K antigen (acidic polysaccharide) at the cell-proximal inner region. These findings are discussed with respect to the participation of the capsular compartments in unspecific host defense.

The temperature-regulated expression of capsular group II polysaccharides of Escherichia coli (B. Jann and K. Jann, (1990) Curr. Top. Microbiol. Immunol. 150: 19-42) depends on an elevated concentration of CMP-KDO, as evidenced by an increased activity of CMP-KDO synthetase. The increase in activity of CMP-KDO synthetase is observed only in cytoplasmic fractions of bacteria which had been grown at 37 degrees C but not after growth at 18 degrees C. The activity of CMP-KDO synthetase thus parallels the activity of the (membrane-associated) system synthesizing capsules of group II in E. coli. No such dependence of capsule expression on CMP-KDO was observed with E. coli with capsules of group I. A number of E. coli strains with capsular polysaccharides, which on the basis of genetic determination and chemical characteristics are considered as group II capsules, show no temperature regulation of their capsules and do not depend on an elevated CMP-KDO concentration for capsule expression. The capsular polysaccharides of these E. coli strains, which possibly represent a new group of E. coli capsules are tentatively classified as group I/II.

The K16-antigen from E. coli Rk 21510 (O7:K16:H-) is shown to consist of the repeating unit ----2)-beta-D-Ribf-(1----3)-beta-D-Ribf-(1----5)-alpha-Kd op-(2---- of which approximately 33% is O-acetylated at position 3 of the 2-linked ribose.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.

We have examined the alterations in lipopolysaccharide during aggregation and early development in Myxococcus xanthus. The lipopolysaccharide was isolated and characterized from cells developing on agar during glycerol induction and vegetative growth. A methylated amino sugar was identified as 6-O-methylgalactosamine by gas-liquid chromatography-mass spectrometry. This novel sugar was enriched in cells developing on agar.

The structures of the capsular K18, K22, and K100 antigens of E. coli O23:K18:H15, O23:K22:H15, and O75:K100:H5, respectively, were elucidated by determination of composition, 1H-, 13C-, and 31P-n.m.r. spectroscopy, periodate oxidation, alkaline hydrolysis followed by incubation with alkaline phosphatase, and methylation analysis of the polymers and their neutral fragmentation products. The polymers are poly(ribosyl-ribitol phosphates) related to the capsular antigen of H. influenzae (Hib). The K22 antigen has the repeating unit -P-2)-beta-Rib-(1----2)-RibOH-(5-, and the K18 antigen has the same polymer chain with partial 3-O-acetylation of the ribose moiety. The K100 antigen consists of repeating units of -P-3)-beta-Rib-(1----2)-RibOH-(5- and seems to have a secondary structure different from that of the other antigens. Together with the Hib capsular antigens, the structure of which was reported as -P-3)-beta-Rib-(1----1)-RibOH-(5-, these capsular antigens represent a structurally related group of capsular polymers.

The chemical structure of the K14-antigenic polysaccharide (K14 antigen) of Escherichia coli 06:K14:H31 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and study of the oligosaccharides obtained by partial hydrolysis. The polysaccharide consists of [O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1 leads to 5)-O-(3-deoxy-beta-D-manno-octulopyranosylonic acid)-(2 leads to 6)] repeating units, approximately 60% of the octonic acid units being O-acetylated and approximately 10% O-propionylated at O-8. The sequence of acetylated and propionylated residues is not known. The serologically-specific part of the K14 antigen residues in the polysaccharide part.

The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and methylation analysis. The K95 polysaccharide, which contains furanosidic 3-deoxy-D-manno-2-octulosonic acid (KDOf) residues, consists of----3)-beta-D-Rib-(1----8)-KDOf-(2----repeating units, has a molecular weight of approximately 25,000 (approximately 65 repeating units), and is randomly O-acetylated (1 acetyl group per repeating unit at unknown positions).

Most of the his+ hybrids from crosses between the Escherichia coli donor Hfr45(O8:K27) and different E. coli O9 recipients expressed the donor O8 antigen specificity and produced the capsular antigen K27. Therefore these hybrids must have inherited the his-linked donor rfb region determining the synthesis of O8- specific polysaccharides as well as his-linked genes involved in K27 antigen synthesis. In the living state these hybrids were inagglutinable in O8 antiserum like the donor cells. However, when E. coli K12 and O8:K42- were used as recipients most of the his+ hybrids were agglutinable in O8 and K27 antisera. The amounts of K27 antigen present in these hybrids, designated as K27i (intermediate) forms, were sufficient to evoke the production of K27 antibodies in rabbits, but insufficient to inhibit O-agglutination of the respective cells. The additional transfer of the trp region of E. coli O8:K27 into such K27i forms frequently resulted in O-inagglutinable K27+ hybrids. This is attributed to the introduction of trp-linked genes which apparently play a role in the synthesis of K27 capsular antigen. Tus it is concluded that at least two gene loci, one close to his and the other close to trp, are required for the synthesis of the complete capsular antigen K27.

Compositional analysis of the intact and carboxyl-reduced capsular polysaccharide of Acinetobacter calcoaceticus BD4 (PS-4) showed it to consist of L-rhamnose, D-glucose, D-glucuronic and D-mannose in molar ratios of 4:1:1:1. 13C-nuclear magnetic resonance spectroscopy, methylation analysis, oligosaccharide analysis and base-catalyzed beta-elimination were used to elucidate the primary structure. Oligosaccharides were obtained by enzymatic depolymerization with a specific bacteriophage-induced depolymerase and by partial acid hydrolysis. Form the results it is concluded that PS-4 consists of repeating units of the heptasaccharide (Formula: see text). The bacteriophage-induced depolymerase was found to be an endo-beta-D-glucosidase that hydrolyzed the bond beta-D-Glc-(1----3)-L-Rha to generate a heptasaccharide in 40% yield.

The chemical structure of the K52 antigenic capsular polysaccharide (K52 antigen) of Escherichia coli O4:K52: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 alpha-galactose units interlinked between C1 and C3 by phosphodiester bridges. This poly(alpha-galactosyl-phosphate) is substituted at C2 of each galactose unit by beta-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 K52 polysaccharide has an average molecular mass of 34 kDa, thus consisting of approximately 65 fructosyl-galactosyl-phosphate repeating units.

Crossed immunoelectrophoresis has shown that the Escherichia coli polysaccharide K2 antigen exists in two forms, K2ab and K2a. In confirmation of this finding, chemical structural analysis showed that K2ab, in addition to the galactose, glycerol and phosphate groups common to K2ab and K2a, contained 0 acetyl groups. The contrast between the earlier and the present K2 designation is discussed and the superiority of the CIE technique for this type of analysis is underlined.

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1----6)-O -(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)] repeating units. All of the glucuronic acid residues are substituted amidically with L-serine.

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 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), galactose (Gal), galactosamine (GalN) and 4-O-(1'-carboxyethyl)-D-glucopyranose (glucolactilic acid, GlcLA) 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 (see article). 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.

The lipopolysaccharide from Escherichia coli O9:K30- was isolated in about 2% yield with aqueous 45% phenol at 65 degrees C, followed by ultracentrifugation. 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 leads to 2 and 1 leads to 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 alpha-mannosidase from jack bean meal, as well as a specific rotation of [alpha]25D = +89 degrees indicated that all mannosyl linkages have the alpha-configuration. Smith degradation resulted in the liberation of mannosyl (1 leads to 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 alpha-mannosyl(1 leads to 3)-alpha-mannosyl-(1 leads to 2)-alpha-mannosyl-(1 leads to 2)-alpha-mannosyl-(1 leads to 2)-mannose, which are joined in the polysaccharide through alpha-(1 leads to 3)-mannosyl linkages.

Crossreactions of 24 K polysaccharides and 4 O polysaccharides of E. coli in antisera to 27 pneumococcal types, 3 anti-Salmonella sera, and anti-Klebsiella Kl serum are discussed in relation to structural features of the polysaccharides insofar as these are known. Predictions based on the crossprecipitations are also ventured for several instances in which structures are as yet undetermined.

The structure of the capsular K74 antigen of E. coli H702c (O44:K74:H18) was elucidated by determination of the composition, 1H- and 13C-n.m.r. and c.d. spectroscopy, periodate oxidation, and methylation analysis of the polysaccharide and of a trisaccharide obtained by mild acid hydrolysis. The K74 antigen has the repeating unit----3)-beta-D-Ribf-(1----2)-beta-D-Ribf-(1----6)-beta-++ +KDOf-(2----. Of the repeating units, approximately 65% are O-acetylated, most probably at C-2 of the 3-linked ribose.

The lipopolysaccharides of ampicillin-resistant cell-wall-defective mutants of Escherichia coli 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.

The lipopolysaccharide of Escherichia coli BB and a number of R-phage selected (e.g. T3, T4) cell-wall-defective mutants were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. This revealed the sugar sequence in the hexose-heptose region of the core. The linkage of heptose (Hep) to 2-keto-3-deoxyoctonate (KDO) was established as ... Hep 1,5 leads to KDO ... by methylation analysis. The substituted derivative of KDO was identified by gas chromatography and mass spectrometry. The KDO region contains three KDO units. Its structure was elaborated by (a) selective removal and identification of 7-phosphoryl ethanolamine-KDO (KDO-PN), (b) periodate oxidation and thiobarbituric acid reaction in conjunction with mild hydrolysis, (c) a modified methylation analysis. Phosphate substitution of E. coli BB core was studied by beta-elimination and using the information obtained with KDO-PN. The structures of the cell wall lipopolysaccharides from E. coli BB and cell-wall-defective mutants are given.

The major surface antigens of Escherichia coli are the cell wall lipopolysaccharides (LPS; O antigens) and the capsular polysaccharides (PS; K antigens). These polysaccharides are synthesized at the cytoplasmic membrane of the bacteria; the LPS are transported to the outer membrane, where they reside, whereas the PS are secreted into capsules. The LPS consist of lipid A covalently linked to the core oligosaccharide, which itself is covalently linked to the O-specific polysaccharide. The latter, which determines the O specificity of the bacteria may be neutral or contain negative charges (carboxyl groups or phosphate). The relatedness of E. coli to other genera (e.g., Klebsiella or Shigella) frequently is borne out by structural identity. This intergeneric relation is paralleled by similar pathogenic properties of the bacteria in question. The capsular antigens of E. coli are acidic polysaccharides, which can be divided into groups (I and II) on the basis of molecular size, nature of the acidic component, coexpression with O antigens, and temperature regulation of their biosynthesis. The major acidic components are hexuronic acid (mainly of Klebsiella-like group I) as well as 2-keto-3-deoxy-D-mannooctulonic acid, N-acetylneuraminic acid, or phosphate (mainly in Neisseria- and Haemophilus-like group II). Relatedness of encapsulated E. coli to encapsulated bacteria of other genera (Neisseria, Haemophilus, Klebsiella) is based on structural identity or similarity of the respective capsules. Identity not only refers to structure and serology of these capsules but also to the pathogenicity of the respective bacteria (e.g., E. coli K1 and Neisseria meningitidis b). Bacterial pathogenicity may be caused by the host's inability to raise an immune response to bacterial capsules (E. coli K1 and K5) because of the identity of the capsular polysaccharides and the host carbohydrates. This can be described as camouflage used by the bacteria as a strategem for bacterial virulence.