Primary structure of the Escherichia coli serotype K30 capsular polysaccharide.

Methylation, 1H nuclear magnetic resonance, and bacteriophage degradation results indicate that the Escherichia coli serotype K30 capsular polysaccharide consists of leads to 2)-alpha-D-Manp-(1 leads to 3)-beta-D-Galp-(1 leads to chains carrying beta-D-GlcUAp-(1 leads to 3)-alpha-D-Galp-(1 leads to branches at position 3 of the mannoses.     

Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.     

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.     

Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Cloning, expression, and purification of the K5 capsular polysaccharide lyase (KflA) from coliphage K5A: evidence for two distinct K5 lyase enzymes

The Escherichia coli K5 capsular polysaccharide [-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.     

Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues

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).     

Genetic determinants of the synthesis of the polysaccharide capsular antigen K27(A) of Escherichia coli.

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.     

Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli

Abstract 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 kps S) and from region 3 (notably kps T) in the K5 polysaccharide synthesis was apparent and is discussed.     

Structure of the fructose-containing K52 capsular polysaccharide of uropathogenic Escherichia coli O4:K52:H-

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.     

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.     

Structure of the core regions in lipopolysaccharides from Escherichia coli K12 W2252-11U-, the Ter-15 mutant, and Ter-15 (F'-lac) and Ter-15 (F+) cells

From Escherichia coli K12 W2252-11U-cells, the Ter-15 mutant, the Ter-15 (F'-lac) and the Ter-15 (F+) cells, lipopolysaccharides were isolated and the primary structure of its core oligosaccharides was elucidated. When the F'-lac episome is transferred to the Ter-15 mutant by conjugation, the structure of the glucose III(1 leads to 3)glucose II(1 leads to 3)glucose I residue and the galactose I(1 leads to 2)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 mutant changes into the structure of the glucose IV(1 leads to 6)glucose III(1 leads to 2)glucose II(1 leads to 3)glucose I residue and the galactose I (1 leads to 6)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 (F'-lac) cells, but the core oligosaccharide in the Ter-15 (F+) cells is the same structure with that of the core oligosaccharide from the Ter-15 mutant when F+ episome is transferred to the Ter-15 mutant. Also, the core oligosaccharide from the Ter-15 (F'-lac) cells shows the same structure with that of the core oligosaccharide from E. coli K12 W2252-11U- cells (the parent cells). As the result, the ability to produce the structure of the core oligosaccharide in E. coli K12 W2252-11U- cells is recovered in the Ter-15 (F'-lac) cells by the dominant expression of lac gene or its containing DNA segment in F'-lac episome.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

Structural investigation of the capsular polysaccharide from Escherichia coli O9:K37 (A 84a)

The structure of the capsular polysaccharide from E. coli O9:K37 (A 84a) has been studied, using methylation analysis, Smith degradation, and graded acid hydrolysis. The configurations at the anomeric centres were assigned by 1H-n.m.r. spectroscopy of the polysaccharide and its derivatives and oligosaccharide fragments. The polysaccharide has the following trisaccharide repeating-unit which is unique in the E. coli series of capsular polysaccharides in possessing a 1-carboxyethylidene group as the sole acidic function. (Formula: see text) E. coli capsular polysaccharides have been classified into seventy-four serotypes. The structures of about twenty of these polysaccharides have been elucidated, one of which, K29, has been reported to contain a 1-carboxyethylidene group. In continuation of a programme aimed at establishing the structural basis for the serology and immunochemistry of the E. coli capsular antigens, we now report on the structure of the capsular polysaccharide from E. coli O9:K37.     

Bacteriophage-associated glycan hydrolases specific for Escherichia coli capsular serotype K12

Four bacteriophages were identified, which carry glycan hydrolases specific for the Escherichia coli K12 capsular polysaccharide. All these glycanases catalyze the hydrolysis of the alpha-L-rhamnosyl-1,5-beta-3-deoxy-D-manno-2-octulosonic acid linkage as demonstrated with a special thiobarbituric acid assay procedure, which discriminates between the C5 substituted and unsubstituted 3-deoxy-D-manno-2-octulosonic acid (dOclA). This assay, together with gel filtration, 1H-NMR and 13C-NMR spectroscopy showed that depolymerization led to the dimer of the K12 repeating unit, (,5-beta-dOcl1Ap-2,3-alpha-LRhap-1,2-alpha LRhap-1,)2, as the primary degradation product. The phages (phi 12-W, phi 12-S, phi 82-W1, phi 82-W2) were tested for their ability to infect Escherichia coli strains Su65-42 (O4:K12:H-) and CDC63-57 [O139:K82(12):H1]. phi 12-W and phi 12-S, respectively, infected strain Su65-42 only, phi 82-W2 CDC63-57 only, and phi 82-W1 both bacterial strains. These distinct host specificities cannot be explained by differences in the action of the glycanases, which depolymerize the capsules of both strains.     

Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone

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.     

Two different Escherichia coli capsular polysaccharide depolymerases each associated with one of the coliphage phi K5 and phi K20

The Escherichia coli capsular polysaccharides (K antigens) K5 and K20 are known as primary receptors for the coliphage phi K5 and phi K20, respectively. A host range study of the phage revealed that E. coli K5 strains were not only lysed by phi K5 but also by phi K20, and furthermore that the E. coli K95 test strain was attacked by phi K5 in addition to K5 strains. In order to find out whether the phage can degrade the K antigens, the interaction of the phage with isolated polysaccharides was studied. It could be demonstrated that phi K5 was able to depolymerize the K5 and K95 polysaccharides and that phi K20 showed degrading activity towards the antigens K20 and K5. Obviously, each of the phages was associated with two different enzyme systems which enabled them to recognize and depolymerize chemically unrelated polysaccharides.     

Structural studies on the hexose region of the Enterobacteriaceae type R3 core polysaccharide.

The structure of the hexose region of the Enterobacteriaceae type R3 core polysaccharide, which is the common core in Shigella flexneri and some Escherichia coli lipopolysaccharides (LPS), has been investigated. The principal method used in these studies involved methylation analysis of the original and modified materials. The polysaccharide was modified by N-deacetylation-deamination and by Smith degradation. As a result of these studies, the following structure is proposed: alpha-D-Glcp-(1 leads to 2)-alpha-D-Glcp-(1 leads to 2)-[alpha-D-GlcNAcp-(1 leads to 3)]-alpha-D-Galp-(1 leads to 3)-alpha-D-Glcp-(1 leads to.     

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.     

Escherichia coli serotype K44: an acidic capsular polysaccharide containing two 2-acetamido-2-deoxyhexoses

The structure of the capsular polysaccharide from Escherichia coli O8:K44 (A):H- (K44 antigen) has been established using the techniques of methylation, beta-elimination, deamination, and Smith degradation. N.m.r. spectroscopy (13C and 1H) was used extensively to establish the nature of the anomeric linkages of the polysaccharide and of oligosaccharides derived through degradative procedures. The K antigen is comprised of repeating units of the linear tetrasaccharide shown. This acidic polysaccharide represents the first instance of an E. coli K antigen in this series (group A) that has been found to contain two different 2-acetamido-2-deoxyhexoses.