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Simple water extracts of all Escherichia coli O antigen test strains 01–0150 and K antigen test strains K1–K91 were examined in immunoelectrophoresis test. The precipitation arcs corresponding to the O antigen specificity and to the thermostable polysaccharide K antigen were easy to identify. All strains gave an O antigen precipitation arc found either on the anodic or the kathodic side of the application basin, and close to this. Only a limited number of strains contained a special thermostable K polysaccharide, always negatively charged. According to the movement of the O antigen and to presence or non-presence of a polysaccharide K antigen, the extracts and thus the strains could be divided into a few groups which fitted well with our present knowledge about pathogenicity in different E. coli strains. Serotypes found frequently in normal faeces and in extra intestinal disease had kathodic O antigens and a special negatively charged K antigen. The so-called enteropathogenic types (from infantile diarrhoea) had a kathodic O antigen and no special K antigen. Types from dysentery-like disease had a negatively charged O antigen but no special thermostable K antigen. Thus E. coli strains which may invade the tissues when conditions allow have a negatively charged surface antigen, either O antigen lipo-polysaccharide or K antigen polysaccharide or both. Acidic components, most often hexuronic acids or neuramic acid, were found in side chains from most of the strains with an anodic O antigen.

The chemical structure of the K4-specific capsular polysaccharide (K4 antigen) of Escherichia coli O5: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)-beta-D-glucuronyl-(1,4)-beta-D-N-acetylgalactosaminyl(1- to which beta-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.

Bacteriophage Ω8 is propagated in Escherichia coli E56b (08: K27-:H-), a non-capsulated strain. Another non-capsulated strain, E. coli 2398 (08:K?-:H-), is killed by bacteriophage Ω8 without phage propagation. This strain was formerly believed to be E. coli 093:K?-:H-, cross-reacting with strain E56b. We have established chemical and serological identity of the 08-specific lipopolysaccharides of the two strains. The 08-specific lipopolysaccharides of both strains inhibited the infection of Escherichia coli E56b with bacteriophage Ω8 equally well. The adsorption rate constants of Ω8 were identical for the two strains of E. coli 08. Evidence was obtained with 32P-labelled bacteriophage Ω8 for penetration of viral DNA into both bacterial strains. In host strain E56b, phage particle synthesis occurred normally. In strain 2398 the viral DNA was not degraded but its expression was blocked. The killing effect of Ω8 on E. coli strain 2398 is supposed to be due to damage of the cytoplasmic membrane, which could not be reversed under the influence of viral information. This was indicated by a blockage of cellular respiration, β-galactoside transport and RNA as well as protein synthesis.

The Escherichia coli K5 capsular polysaccharide has the same (GlcUA-->GlcNAc)n structure as the nonsulfated heparan sulfate/heparin precursor polysaccharide. A monoclonal antibody (mAb 865) against the K5 polysaccharide has been described (Peters, H., Jurs, M., Jann, B., Jann, K., Timmis, K. N., and Bitter-Sauermann, D. (1985) Infect. Immun. 50, 459-466). In this report, we demonstrate the binding of anti-K5 mAb 865 to N-acetylated sequences in heparan sulfates and heparan sulfate proteoglycans but not to heparin. This is shown by direct binding and fluid phase inhibition of mAb 865 in an enzyme-linked immunosorbent assay. In this system we found that the binding of the mAb decreased with increasing sulfate content of the polysaccharide. By testing chemically modified K5 and heparin polysaccharides, we found that each of the modifications that occur during heparan sulfate (HS) synthesis (N-sulfation, C-5 epimerization, and O-sulfation) prevents recognition by mAb 865. Samples of heparan sulfate from human aorta (HS-II) were selectively degraded so as to allow the separate isolation of N-sulfated and N-acetylated block structures. N-Sulfated oligosaccharides (obtained after N-deacetylation by hydrazinolysis followed by nitrous acid deamination at pH 3.9) were not recognized by mAb 865, in contrast to N-acetylated oligosaccharides (obtained after nitrous acid deamination at pH 1.5), although the reactivity was lower than for intact HS-II. Analysis of the latter's pH 1.5 deamination products by gel filtration indicated that a minimal size of 18 saccharide units was necessary for antibody binding. These results lead us to propose bivalent antibody-heparan sulfate interaction, in which both F(ab) domains of the mAb interact with their epitopes, both of which are present in a single large (>/=18 saccharide units) N-acetylated domain and additionally with single epitopes present in two N-acetylated sequences (each

The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined. This region, essential for the biosynthesis of the K5 polysaccharide, contained four genes, termed kfiA-D. The G + C ratio was 33.4%, which was lower than the typical G + C ratio for E. coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster. Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping. Promoter activity was confirmed by promoter-probe analysis. The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the KfiC gene resulted in increased K5 transferase activity. The predicted amino acid sequence of KfiD had homology to a number of NAD-dependent dehydrogenase enzymes and was demonstrated to be a UDP-glucose dehydrogenase that catalyses the information of UDP-glucuronic acid from UDP-glucose.

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 rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.

In Escherichia coli with group II capsules, the synthesis of capsular polysaccharide and its cellular expression are encoded by the kps gene cluster, which is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of E. coli with the group II capsular K5 polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, D, U, C and S genes. In this communication we describe the overexpression of the kpsD and kpsU genes as well as the isolation of the KpsU protein from the recombinant bacteria by chloroform treatment. The purified KpsU protein exhibited CMP-Kdo-synthetase activity. The N-terminal sequence and two internal peptide sequences of the isolated protein are in agreement with that previously predicted from the DNA sequence of the kpsU gene. The kinetic data of the CMP-Kdo-synthetase participating in K5 capsule expression (K-CMP-Kdo-synthetase) differ from those described for the CMP-Kdo-synthetase, participating in lipopolysaccharide synthesis (L-CMP-Kdo-synthetase).

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]

Expression of the Escherichia coli K5 antigen was used as a model system to study the role of known regulators of gene expression on production of group II capsules in E. coli. Only mutations in the rfaH gene had an effect on production of the K5 antigen, abolishing the expression of any detectable capsule at 37 degrees C. None of the mutations studied induced capsule expression at 18 degrees C. A sequence, termed JUMPstart, found in group II capsule gene clusters and upstream of a number of polysaccharide biosynthesis genes in enteric bacteria is homologous to sequences found in RfaH regulated operons. This may indicate a common mode of regulation of these polysaccharide biosynthesis genes by RfaH.

Two distinct forms of the O6 antigen (LPS) from E. coli were analysed using 1 H and 13 C NMR spectroscopoy. Their structures were found to be In the O6-specific polysaccharide from E. coli O6:K2 and O6:K13, X is β- 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 β- d -Glc p NAc.

The E. coli K5 capsular polysaccharide is composed of 4)GlcpA(beta 1-4)GlcpNAc(alpha 1-disaccharide units. A partially N-deacetylated/N-sulfated heptasaccharide, derived from this polymer and having a nonreducing terminal GlcNAc unit, was used as acceptor for a mastocytoma microsomal GlcA-transferase involved in heparin biosynthesis. An octasaccharide with nonreducing-terminal GlcA similarly served as acceptor for the microsomal GlcNAc-transferase. Analysis of the labeled octa- and nona-saccharides formed by transfer of monosaccharide units from UDP-[14C]GlcA and UDP-[3H]GlcNAc, respectively, showed that both glycosyltransferases could utilize partially N-sulfated acceptors. The GlcA-transferase showed a marked preference for a terminal GlcNAc-GlcA-GlcNSO3-sequence, particularly when this sequence was followed by an additional N-sulfated disaccharide unit. Enzymes catalyzing the same GlcA and GlcNAc transfer reactions were solubilized from E. coli K5 membranes. The K5 capsular polysaccharide, like the heparin/heparan sulfate precursor polysaccharide, thus probably grows by stepwise, alternating addition of the two constituent monosaccharide units, from the corresponding UDP-sugars, to the nonreducing ends of the chains. Moreover, the bacterial glycosyltransferases utilized the same partially N-sulfated oligosaccharide substrates as the mammalian enzymes, and with similar preference for N-sulfate groups in certain positions.

Summary: Subcloning, transposon insertion, and deletion analysis revealed that the Escherichia coli 09 rfb region is about 12 kb in size. The region encodes at least seven polypeptides of 89, 74, 55, 50, 44, 41 and 39·5 kDa. Southern hybridization analysis of rfb regions of E. coli 08 and 09, and Klebsiella 03 and 05 serotypes (all of these O polysaccharides are mannose homopolymers and the structures of the repeating unit of E. coli 09 and Klebsiella 03 are identical) showed that a central region specific for E. coli 09 and Klebsiella 03 is flanked by two regions common to all four. Complementation experiments using strains with known defects and specific tests for the enzymic activity showed that the 50 and 55 kDa polypeptides, encoded by the common region, are phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), respectively. Nucleotide sequencing of the region revealed the presence of two genes, rfbK and rfbM, analogous to the corresponding genes of Salmonella typhimurium. In E. coli 09, rfbK and rfbM encode proteins of 460 amino acids (50809 Da) and 471 amino acids (52 789 Da). The amino acid sequence of GMP was conserved in RfbMs of E. coli 07 and Salmonella groups B, C1 and C2, CpsB of S. typhimurium, AlgA of Pseudomonas aeruginosa, and XanB of Xanthomonas campestris. The phylogenetic trees of PMM and GMP were different in topology and in the evolutionary distances from ancestors.

Two distinct forms of the O4 antigen (LPS) from E. coli were analysed by 1 H and 3 C 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 In the O4-specific polysaccharided from E. coli O4:K3, O4:K6, and O4:K12, X is α- d -Glcp. In the O4 specific polysaccharide from E. coli O4:K52, the rhamnose residue is not substituted (X = H).

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 chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

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 1 H and 13 C NMR spectroscopy showed that the O56 polysaccharide has the primary structure

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.

Escherichia coli may cause intestinal or extraintestinal infections. Generally, extraintestinal E. coli are encapsulated. The capsules are important virulence determinants, which enable the pathogenic bacteria to evade or counteract the unspecific host defense during the early (preimmune) phase of infection. They interfere with the action of complement and phagocytes. This effect is generally transient and overcome by capsule-specific antibodies in the immune phase of the host defense. In some cases, capsules are not or only poorly immunogenic, as a result of structural relationship or identity with host material. Strains with such capsules (e.g., K1 or K5) are very virulent. Bacterial capsules consist of acidic polysaccharides, which are made up from oligosaccharide repeating units. The capsules of E. coli are divided into two groups, which differ in chemistry, biochemistry, and genetic organization. All capsular polysaccharides are chromosomally determined: those of group I close to his and those of group II close to serA. The biosynthesis and surface expression have been extensively studied with representatives of group II capsular polysaccharides. It could be shown that their biosynthesis is directed from a gene block that determines the synthesis of the polysaccharide, its translocation across the cytoplasmic membrane, as well as its surface expression in a coordinate process. The chemical nature of group II capsular polysaccharides, as well as the mechanism(s) of their biosynthesis and expression, is presented. Key words: Escherichia coli, capsular polysaccharides, structure, genetics, biology.

The O-specific polysaccharide moiety of the O1A1 antigen (lipopolysaccharide) from E. coli 01:K1 consists of l-rhamnose, N-acetyl-d-glucosamine and N-acetyl-d-mannosamine in the molar ratio of 3:1:1. By using fragmentation procedures, methylation analysis, and NMR spectroscopy, the O1A1 polysaccharide was found to have the structure

Summary From the capsular K40 polysaccharide of E. coli O8: K40: H9, a fraction was obtained by gel permeation chromatography which in SDS-PAGE exhibited a ladder-like pattern characteristic of lipopolysaccharides. In Western blots, this fraction reacted with a K40-specific antiserum but not with an O8-specific antiserum. It contained, in addition to the constituents of the K40 polysaccharide (glucuronic acid, glucosamine and serine), glucose, galactose, heptose, and KDO. Mild acid hydrolysis of this fraction liberated a lipid moiety which by chemical analysis was characterized as lipid A. From these results, we conclude that the capsular polysaccharide of E. coli O8: K40: H9 is in part bound to core lipid A. The significance of this finding is discussed.

Colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli was dissociated into one type of subunit (15 kDa). The dissociation was achieved either by heating CFA/I in sodium dodecyl sulfate at 100 degrees C or by heating it for 20 min in water. Heating in water to 100 degrees C yielded only in the 15-kDa subunit, but heating to 85 degree C yielded small amounts of oligomers in addition. The monomeric subunits obtained after heating in water are stable, as demonstrated by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heating prior to the electrophoretic run. These subunits inhibited CFA/I-induced hemagglutination, indicating that they had maintained their receptor-binding properties. When the hybridoma technique was used, two types of monoclonal anti-CFA/I antibodies were obtained. Antibodies obtained by immunization with the purified subunits were more reactive with subunits than with fimbriae, as shown by enzyme-linked immunosorbent assay. These antibodies strongly inhibited CFA/I-induced hemagglutination. When examined by immunoelectron microscopy, these antibodies seemed to label the fimbrial tips. A similar labeling pattern was obtained with gold particles modified with the receptor ganglioside GM2. Antibodies obtained by immunization with fimbriae reacted in enzyme-linked immunosorbent assays equally well with fimbriae and subunits. They inhibited CFA/I-induced hemagglutination only slightly. Immunoelectron microscopy revealed that these antibodies labeled the fimbriae densely and regularly over their entire lengths. In a coagglutination experiment with Staphylococcus aureus and monoclonal antibodies, the subunits retained their receptor-binding properties. From these results, we conclude that CFA/I fimbriae consist entirely of one type of adhesive subunit, of which only the one at the tip is accessible to the receptor.

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.

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 K11 antigen of Escherichia coli contains glucose, fructose, and phosphate in the molar ratios 2:1:1, and a backbone of -4)-beta-D-glucopyranosyl-(1----4)-alpha-D-glucopyranosyl phosphate-(1----to which beta-D-fructofuranose is linked at position 3 of the beta-D-glucopyranosyl residue. The fructose, which is the immunodominant sugar of the K11 antigen, is released from the polysaccharide under mild acidic conditions (70 degrees, pH 5.0).

Publisher Summary Capsular polysaccharides (CPS) are acidic polysaccharides made up of repeating oligosaccharide units, which may be either linear or branched. The primary structures can be formulated by the respective repeating unit and the joining linkages. The CPSs are sometimes substituted with O-formyl- or O-acetyl-groups (in ester linkage) or with pyruvate (in ketosidic linkage). Amino acids are found linked either to the hydroxyl groups of sugar rings (esters) or to the carboxyl group of hexuronic acids (amides). In general, the CPSs are distinctly longer, up to about 100 kDa per chain, than the polysaccharide moieties (O-PS) of the cell wall (outer membrane) O-antigens of Gram-negative bacteria (LPS, up to about 10–20kDa). In some Gram-negative bacteria, the LPS seems to occur in two physically distinct forms with the same primary structure. The lack of an outer membrane, and hence the lack of a periplasmic space, simplifies the surface presentation of CPS. In general, bacterial CPS induces an immune response that results in the production of protective anticapsular antibodies in the exposed host. With whole bacteria or when coupled to a carrier protein as immunogen, CPS induces a T-cell-dependent immune response that results in the production of IgG antibodies. These can be boosted by subsequent injections of the immunogen. In contrast, pure soluble CPS induces a T-cell-independent response that results in IgM antibodies that cannot be boosted. CPSs may also exert activities that are not primarily involved in immunological processes. The CPS of Bacteroides fragilis, which forms a strong complex of two distinct polysaccharides, induces intestinal abscesses.

CMP-Kdo synthetases from Gram-negative bacteria activate Kdo for incorporation into lipo- and capsule-polysaccharides. Here we report the crystal structure of the capsule-specific 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 beta-sheet with surrounding helices. The chain fold is novel; it is remotely related to a double Rossmann fold. A large pocket at the carboxyl terminal ends of the central. beta-strands most likely accommodates the catalytic center. A putative phosphate binding site at the loop between the first beta-strand and the following helix is indicated by a bound iridium hexachloride anion.

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.

The structures of the 6-deoxytalose-containing O-specific polysaccharides from the O45 antigen, an O45-related antigen (O45rel), and the O66 antigen (lipopolysaccharides, LPSs) of Escherichia coli were elucidated by chemical characterization and by one- and two-dimensional 1 H and 13 C NMR spectroscopy. The O45 and O45-related polysaccharides have the following general structure: For the O45 antigen, X is α- d -Fuc p NAc and for the O45-related antigen, X is β- d -Glc p NAc . The structure of the O66 polysaccharide is

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.

The polysaccharide moiety of the O83 antigen (lipopolysaccharide, LPS) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-glucuronic acid in the molar ratios 1:2:1:1. Methylation analysis of the polysaccharide and derived oligosaccharides as well as one- and two-dimensional 1H and 13C NMR spectroscopy of the polysaccharide at pD 1 and 6 showed that the O83 polysaccharide has the primary structure-->6)-alpha-D-Glc p-(1-->4)-beta-D-Glc pA-(1-->6)-beta-D-Gal p-(1-->4)-beta-D- Gal p-(1-->4)-beta-D-Glc pNAc-(1-->.

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]

The genes directing the expression of group II capsules in Escherichia coli are organized into three regions. The central region 2 is type specific and thought to determine the synthesis of the respective polysaccharide, whilst the flanking regions 1 and 3 are common to all group II gene clusters and direct the surface expression of the capsular polysaccharide. In this communication we analyze the involvement of region 1 and 3 genes in the synthesis of the capsular KS polysaccharide. Recombinant E. coli strains harboring all KS specific region 2 genes and having various combinations of region 1 and 3 gene were studied using immunoelectron microscopy. Membranes from these bacteria were incubated with UDP[14C]GlcA and UDPG1cNAc in the absence or presence of KS polysaccharide as an exogenous acceptor. It was found that recombinant strains with only gene region 2 did not produce the K5 polysaccharide. Membranes of such strains did not synthesize the polymer and did not elongate K5 polysaccharide added as an exogenous acceptor. An involvement of genes from region 1 (notably kps C and kpsS) and from region 3 (notably kpsT) in the K5 polysaccharide synthesis was apparent and is discussed.

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].

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 wild-type O4 LPS [Haraguchi, G. E., Zahringer, 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 α-l-rhamnopyranose for β-d-galactofuranose. The relevance of these results for lipopolysaccharide cloning and lipopolysaccharide biosynthesis are discussed.

The adhesion of pathogenic bacteria to the epithelial surfaces of a host is a prelude to infection, followed by multiplication of the bacteria and their penetration into the host’s cells or their damaging of the epithelium by the action of their toxins. Adhesion is mediated by interaction between bacterial recognition proteins (adhesins, hemagglutinins, lectins) and receptors on the host ce11.1,2 Most receptors are complex carbohydrates but components of the extracellular matrix and other proteins have also been identified as such2,3 (see Korhonen et al., in this volume). Adhesion can be measured by counting the adhering cells under standard conditions; however, since most adhesive bacteria agglutinate erythrocytes, adhesion can also be monitored easily by hemagglutination. Both adhesion and hemagglutination are inhibited by receptor analogs (glycoproteins, glycolipids, oligosaccharides) and this can be used to determine adhesion specificity. Thus, one way of characterizing bacterial adhesins is via the specificity of their receptors.

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.