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Structure type: polymer chemical repeating unit
Trivial name: K5 polysaccharide, K-antigen, N-acetyl heparosan, heparosan (N-acetylheparosan), heparosan, heparosan (glycosaminoglycan GAG), K5 CPS, heparosan (K5-antigen), N-acetylheparosan
Compound class: CPS, EPS, K-antigen, polysaccharide
Contained glycoepitopes: IEDB_115136,IEDB_140630,IEDB_141807,IEDB_151531,IEDB_153764,IEDB_423153

• Article ID: 250
  Hagner-McWhirter A, Hannesson HH, Campbell P, Westley J, Roden L, Lindahl U, Li JP "Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates" - Glycobiology 10(2) (2000) 159-171
  

  The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial (approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties

  biosynthesis, Escherichia coli, capsular polysaccharide

  NCBI PubMed ID: 10642607
  Journal NLM ID: 9104124
  Publisher: IRL Press at Oxford University Press
  Institutions: Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, S-751 23, Uppsala, Sweden
  
   
  
  Escherichia coli K5
  CSDB ID 6889 (all data & tools)
• Article ID: 261
  Hodson N, Griffiths G, Cook N, Pourhossein M, Gottfridson E, Lind T, Lidholt K, Roberts IS "Identification that KfiA, a protein essential for the biosynthesis of the Escherichia coli K5 capsular polysaccharide, is an a-UDP-GlcNAc glycosyltransferase - The formation of a membrane-associated K5 biosynthetic complex requires KfiA, KfiB, and KfiC" - Journal of Biological Chemistry 275(35) (2000) 27311-27315
  

  The Escherichia coli K5 capsular polysaccharide consists of the repeat structure -4)GlcA-β(1,4)-GlcNAc-α(1- and requires the KfiA, KfiB, KfiC, and KfiD proteins for its synthesis. Previously, the KfiC protein was shown to be a β-UDP-GlcA glycosyltransferase, and KfiD was shown to be a UDP-Glc dehydrogenase. Here, we demonstrate that KfiA is an α-UDP-GlcNAc glycosyltransferase and that biosynthesis of the K5 polysaccharide involves the concerted action of the KfiA and KfiC proteins. By site-directed mutagenesis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activity of KfiA. In addition, by Western blot analysis, we determined that association of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, whereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic membrane fractions when both KfiA and KfiC were present. These data suggest that the interaction between the KfiA, KfiB, and KfiC proteins is essential for the stable association of these proteins with the cytoplasmic membrane and the biosynthesis of the K5 polysaccharide.

  biosynthesis, Escherichia coli, capsular polysaccharide, glycosyltransferase, A-protein

  NCBI PubMed ID: 10859322
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: ISRobert@fs1.scg.man.ac.uk
  Institutions: School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom, Department of Medical Biochemistry and Microbiology, University of Uppsala, The BioMedical Center, Box 575, S-751 23 Uppsala, Sweden.
  
   
  
  Escherichia coli K5
  CSDB ID 7122 (all data & tools)
• Article ID: 320
  Manzoni M, Bergomi S, Rollini M "Influence of the culture conditions on extracellular lyase activity related to K5 polysaccharide" - Biotechnology Letters 22(1) (2000) 81-85
  

  The K5 capsular polysaccharide antigen of some Escherichia coli strains is the non-sulphated precursor in heparin biosynthesis. It is composed by two components, 16 000 and 1500 Da, whose ratio depends on the activity of the extracellular form of a lyase synthesized by the same K5 producer strain. The lyase activity on the K5 chain size was greatly influenced by the medium composition employed for the lyase production. The control of lyase activity results in defined ratios of the two components in the K5 polysaccharide that is suitable for the semisynthetic production of heparin-like molecules.

  polysaccharide, Escherichia coli, capsular polysaccharide, activity, extracellular, lyase, culture, heparin-like

  Journal NLM ID: 8008051
  Publisher: Kluwer Academic Publishers
  Correspondence: matilde.manzoni@unimi.it
  Institutions: Dipartimento di Scienze e Tecnologie Alimentari, Sezione di Microbiologia Industriale, Universita degli Studi, via Celoria 2, 20133 Milano, Italia
  
   
  
  Escherichia coli K5
  CSDB ID 7491 (all data & tools)
• Article ID: 348
  Petit C, Rigg GP, Pazzani C, Smith A, Sieberth V, Stevens M, Boulnois G, Jann K, Roberts IS "Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide" - Molecular Microbiology 17 (1995) 611-620
  

  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.

  biosynthesis, polysaccharide, Escherichia, Escherichia coli, capsular polysaccharide, cluster, gene cluster, region, capsule, proteins

  NCBI PubMed ID: 8801416
  Journal NLM ID: 8712028
  Publisher: Blackwell Publishing
  Correspondence: ISR@leics.ac.uk
  Institutions: Department of Microbiology and Immunology, Medical Sciences Building, University Road, University of Leicester, PO Box 138, Leicester LE1 9HN, UK, Max Planck-lnsUtut for Immunbiologie, D-7800 Freiburg-Zahringen, Germany
  Methods: genetic methods
  
   
  
  Escherichia coli K5
  CSDB ID 8005 (all data & tools)
• Article ID: 438
  Volpi N "Purification of the Escherichia coli K5 capsular polysaccharide and use of high-performance capillary electrophoresis to qualitative and quantitative monitor the process" - Electrophoresis 25(18-19) (2004) 3307-3312
  

  A rapid, highly sensitive, and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K5 bacteria Bi8337/41 010:K5:H4. This natural polysaccharide having the structure of a desulfo-heparin composed of -4)-αGlcUA-(1,4)-α-GlcNAc-(1- is separated (GlcUA = D-glucuronic acid; GlcNAc = D-glucosamine) and qualitatively and quantitatively determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient > approximately 0.99) was found for the polymer over a wide range of concentrations, from approx. 60 to 1500 ng, with a detection sensitivity of < approximately 60 ng. Furthermore, this qualitative and quantitative HPCE approach was applied to the K5 extraction and purification process from cultured bacteria in several stages. HPCE was also able to separate several molecular species mainly due to the presence of polysaccharides of distinct and increasing mean chain lengths. A linear relationship was found for migration time and log molecular mass of different K5 polysaccharide species, and this model was used to calculate the molecular mass of the main K5 species producing a result of approx. 17,000, also confirmed by high-performance size-exclusion chromatography analysis, yielding approx. 17 200.

  Escherichia coli, glycosaminoglycans, heparin, capillary electrophoresis, Heparan, K5 polysaccharide

  NCBI PubMed ID: 15472951
  Journal NLM ID: 8204476
  Publisher: Wiley-VCH
  Correspondence: volpi@unimo.it
  Institutions: Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy
  
   
  
  Escherichia coli K5
  CSDB ID 8902 (all data & tools)
• Article ID: 459
  Urbinati C, Bugatti A, Oreste P, Zoppetti G, Waltenberger J, Mitola S, Ribatti D, Presta M, Rusnati M "Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists" - FEBS Letters 568(1-3) (2004) 171-177
  

  The HIV-1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat-antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O-sulfated K5 with high sulfation degree (K5-OS(H)) and N,O-sulfated K5 with high (K5-N,OS(H)) or low (K5-N,OS(L)) sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV-LTR-transactivation. Also, K5-OS(H) and K5-N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor-2 on endothelial cell (EC) surface. Finally, K5-OS(H) inhibits [Formula: see text] integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5-OS(H) and K5-N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti-AIDS therapies.

  Escherichia coli, antagonist, extracellular, adhesion, heparan sulfate, Hiv-1, polysaccharide derivatives, proteoglycan, sulfation

  NCBI PubMed ID: 15196942
  Journal NLM ID: 0155157
  Publisher: Elsevier
  Correspondence: rusnati@med.unibs.it
  Institutions: Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy
  Methods: sulfation
  
   
  
  Escherichia coli K5
  CSDB ID 8931 (all data & tools)
• Article ID: 508
  Manzoni M, Rollini M, Piran E, Parini C "Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide" - Biotechnology Letters 26(4) (2004) 351-356
  

  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

  PCR, DNA, strain, structural, polysaccharide, analysis, chain, molecular, Escherichia, Escherichia coli, high, specific, mutant, activity, region, precursor, production, enzyme, component, time, rearrangement, lyase, action, membrane, heparin, fermentation, culture, characterisation, PDF, analogue, decrease, fraction, molecular weight, Ultrafiltration

  NCBI PubMed ID: 15055774
  Journal NLM ID: 8008051
  Publisher: Kluwer Academic Publishers
  Institutions: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Industriale, Uni- ersita degli Studi, Via G. Celoria 2, 20133 Milano, Italy
  
   
  
  Escherichia coli K5
  CSDB ID 9233 (all data & tools)
• Article ID: 1242
  Sieberth V, Rigg GP, Roberts IS, Jann K "Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes" - Journal of Bacteriology 177(15) (1995) 4562-4565
  

  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-β-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.

  expression, gene, characterization, polysaccharide, Escherichia coli, capsule, type-specific, dehydrogenase

  NCBI PubMed ID: 7635844
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Max-Planck-Institut fur Immunbiologie, D-79108 Freiburg, Germany, Department of Microbiology, University of Leicester, Leicester LE19HU, United Kingdom
  
   
  
  Escherichia coli K5
  CSDB ID 4902 (all data & tools)
• Article ID: 1378
  Borgenstrom M, Jalkanen M, Salmivirta M "Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling" - Journal of Biological Chemistry 278(50) (2003) 49882-49889
  

  Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling

  Bacterial, potential, capsular, polysaccharide, cell, molecule, degree, Escherichia, Escherichia coli, capsular polysaccharide, factor, high, regulation, response, Bacterial polysaccharide, activity, polysaccharides, modified, surface, interaction, cellular, modulation, preparation, cells, glycosaminoglycan, heparin, effect, species, heparin-like, heparan sulfate, proteoglycan, sulfation, analogue, receptors, receptor, sulfated, growth, sulfate, proliferation, fibroblast, fibroblasts, growth factor, inhibitor, inhibitory, physiological, stimulation

  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: markku.salmivirta@btk.utu.fi
  Institutions: Turku Centre for Biotechnology,Turku, Finland
  
   
  
  Escherichia coli K5
  CSDB ID 6270 (all data & tools)
• Article ID: 1414
  DeAngelis PL, Gunay NS, Toida T, Mao WJ, Linhardt RJ "Identification of the capsular polysaccharides of Type D and F Pasteurella multocida as unmodified heparin and chondroitin, respectively" - Carbohydrate Research 337(17) (2002) 1547-1552
  

  Pasteurella multocida is a pathogenic Gram-negative bacterial species that infects a wide variety of animals and humans. A notable morphological feature of many isolates is the extracellular capsule. The ability to remove the capsule by treatment with certain glycosidases has been utilized to discern various capsular types called A, D and F. Based on this preliminary evidence, these microbes have capsules made of glycosaminoglycans, linear polysaccharides composed of repeating disaccharide units containing an amino sugar. Glycosaminoglycans are also abundant components of the vertebrate extracellular matrix. It has been shown previously that the major Type A capsular material was hyaluronan (hyaluronic acid). We report that the Type D polymer is an unmodified heparin (N-acetylheparosan) with a →4)-β-D-Glcp-UA-(1→4)-α-D-Glcp-NAc-(1→ repeating unit and the Type F polymer is an unmodified chondroitin with a →4)-β-D-Glcp-UA-(1→3)-β-D-Galp-NAc-(1→ repeating unit. The monosaccharide compositions, disaccharide profiles, and 1H NMR analyses are consistent with these identifications. The molecular size of the Pasteurella polymers is approximately 100-300 kDa as determined by gel electrophoresis and multi-angle laser light scattering; this size is much greater than the 10-30 kDa size of the analogous polymers isolated from animal tissues. The glycosaminoglycan capsular polymers are relatively non-immunogenic virulence factors that enhance microbial pathogenicity.

  capsular polysaccharide, chondroitin, glycosaminoglycan, Pasteurella multocida, Heparosan

  NCBI PubMed ID: 12350324
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Correspondence: paul-deangelis@ouhsc.edu
  Institutions: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
  
   
  
  Pasteurella multocida
  CSDB ID 6569 (all data & tools)
• Article ID: 1456
  Hänfling P, Shashkov AS, Jann B, Jann K "Analysis of the enzymatic cleavage (b elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: a reexamination" - Journal of Bacteriology 178 (1996) 4747-4750
  

  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 (∆4,5GlcA) at their nonreducing end. Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase.

  structure, capsular, polysaccharide, analysis, Escherichia, Escherichia coli, capsular polysaccharide, enzymatic, cleavage, elimination, b-elimination

  NCBI PubMed ID: 8755913
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, Max-Planck-Institut für Immunbiologie, Stuübeweg 51, D-79108 Freiburg, Germany
  Methods: 13C NMR, 1H NMR, SDS-PAGE, b-elimination, enzymatic degradation, GPC
  
   
  
  Escherichia coli O18:K5
  CSDB ID 2404 (all data & tools)
• Article ID: 1630
  Lindahl U, Li JP, Kusche-Gullberg M, Salmivirta M, Alaranta S, Veromaa T, Emeis J, Roberts I, Taylor C, Oreste P, Zoppetti G, Naggi A, Torri G, Casu B "Generation of 'neoheparin' from E. coli K5 capsular polysaccharide" - Journal of Medicinal Chemistry 48(2) (2005) 349-352
  

  Heparin remains a major drug in prevention of thromboembolic disease. Concerns related to its animal source have prompted search for heparin analogues. The anticoagulant activity of heparin depends on a specific pentasaccharide sequence that binds antithrombin. We report the generation of a product with antithrombin-binding, anticoagulant, and antithrombotic properties similar to those of heparin, through combined chemical and enzymatic modification of a bacterial (E. coli K5) polysaccharide. The process is readily applicable to large-scale production.

  Escherichia coli, capsular polysaccharide, heparin, chemical synthesis, anticoagulant activity, enzymatic modification

  NCBI PubMed ID: 15658847
  Journal NLM ID: 9716531
  Publisher: Washington, DC: American Chemical Society
  Correspondence: ulf.lindahl@imbim.uu.se
  Institutions: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden
  Methods: biological assays, biosynthetic modifications, chemoenzymatic modifications
  
   
  
  Escherichia coli K5
  CSDB ID 10573 (all data & tools)
• Article ID: 1674
  Whitfield C, Valvano MA "Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria" - Advances in Microbial Physiology 35 (1993) 135-246
  

  This chapter provides an overview of the molecular mechanisms involved in synthesis and expression of cell-surface polysaccharides in Gram-negative bacteria. Biosynthesis of many cell-surface components, including polysaccharides, involves enzymes and enzyme complexes found in the cytoplasmic membrane. The peptidoglycan layer is located immediately external to the cytoplasmic membrane and this layer is required for cell shape and rigidity. Gram-negative bacteria possess a periplasm that contains a variety of proteins and enzymes, including some involved in import and export of macromolecules. Biosynthesis of bacterial cell-surface polysaccharides involves a series of sequential processes: (1) biosynthesis of activated precursors in the cytoplasm, (2) formation of repeating units, (3) polymerization of repeating units, and (d) export of polysaccharides to the cell surface. The assembly of polysaccharide repeating units and subsequent polymerization reactions occur at the cytoplasmic membrane, using precursors synthesized in the cytoplasm. Genes for biosynthesis of cell-surface polysaccharides are chromosomal and are arranged in clusters of one or more transcriptional units. The synthesis of lipopolysaccharide (LPS) may be subject to complex regulation, but on-off switching is not possible due to the essential structural requirement for the lipid A-core LPS molecule. Most bacteria use extracellular polysaccharides (EPSs) for protection, and many regulatory strategies are directed to modulating EPS synthesis in response to appropriate environmental cues. Application of genetic and biochemical approaches has facilitated detailed analysis of complex, multicomponent systems, such as those involved in synthesis of cell-surface polysaccharides.

  NCBI PubMed ID: 8310880
  Publication DOI: 10.1016/S0065-2911(08)60099-5
  Journal NLM ID: 0117147
  Institutions: Department of Microbiology, University of Guelph, Ontario, Canada, Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada, N6A 5C1
  
   
  
  Escherichia coli K5
  CSDB ID 101033 (all data & tools)
• Article ID: 2297
  Lidholt K, Fjelstad M, Jann K, Lindahl U "Biosynthesis of heparin, Part XXV. Substrate specificities of glycosyltransferases involved in formation of heparin precursor and E. coli K5 capsular polysaccharides" - Carbohydrate Research 255 (1994) 87-101
  
  Journal NLM ID: 0043535
  Publisher: Elsevier
  
   
  
  Escherichia coli K5
  CSDB ID 117746 (all data & tools)
• Article ID: 2314
  Casu B, Grazioli G, Razi N, Guerrini M, Naggi A, Torri G, Oreste P, Tursi F, Zoppetti G, Lindahl U "Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5" - Carbohydrate Research 263 (1994) 271-284
  

  O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure →4)-β-D-GlcA(1→4)-β-D-GlcNSO3(-)-(1→, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

  NCBI PubMed ID: 7805054
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Institutions: Istituto di Chimica e Biochimica G. Ronzoni, Milan, Italy
  
   
  
  Escherichia coli K5
  CSDB ID 118190 (all data & tools)
• Article ID: 2364
  Vann WF, Schmidt MA, Jann B, Jann K "The structure of the capsular polysaccharide (K5 antigen) of urinary tract-infective Escherichia coli O10:K5:H4" - European Journal of Biochemistry 116(2) (1981) 359-364
  

  The capsular polysaccharide was isolated from Escherichia coli 010:K5:H4; it could not be obtained from a uncapsulated (K5-) mutant. It contains N-acetylglucosamine and glucuronic acid in a molar ratio of 1:1. Acid hydrolysis of the acidic polysaccharide as well as Smith degradation and degradation by deamination of the carboxyl-reduced polysaccharide suggested that the polysaccharide is composed of a disaccharide repeating unit. The data obtained by methylation analysis and nuclear magnetic resonance spectroscopy indicated that the repeating sequence of the capsular polysaccharide is the 4-β-glucuronyl-1,4-α-N-acetylglucosaminyl unit. This structure is similar to that of desulfo-heparin.

  NCBI PubMed ID: 7018909
  Publication DOI: 10.1111/j.1432-1033.1981.tb05343.x
  Journal NLM ID: 0107600
  Publisher: Oxford, UK: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
  Institutions: Max-Planck-Institut fur Immunbiologie, Freiburg
  Methods: GC-MS
  
   
  
  Escherichia coli K5
  CSDB ID 122543 (all data & tools)
• Article ID: 2498
  Hahnenberger R, Jakobson AM, Ansari A, Wehler T, Svahn CM, Lindahl U "Low-sulfated oligosaccharides derived from heparan sulfate inhibit normal angiogenesis" - Glycobiology 3 (1993) 567-573
  
  Journal NLM ID: 9104124
  Publisher: IRL Press at Oxford University Press
  
   
  
  Escherichia coli K5
  CSDB ID 126836 (all data & tools)
• Article ID: 3259
  Whitfield C "Biosynthesis and Assembly of Capsular Polysaccharides in Escherichia coli" - Annual Review of Biochemistry 75(1) (2006) 39-68
  

  Capsules are protective structures on the surfaces of many bacteria. The remarkable structural diversity in capsular polysaccharides is illustrated by almost 80 capsular serotypes in Escherichia coli. Despite this variation, the range of strategies used for capsule biosynthesis and assembly is limited, and E. coli isolates provide critical prototypes for other bacterial species. Related pathways are also used for synthesis and export of other bacterial glycoconjugates and some enzymes/processes have counterparts in eukaryotes. In gram-negative bacteria, it is proposed that biosynthesis and translocation of capsular polysaccharides to the cell surface are temporally and spatially coupled by multiprotein complexes that span the cell envelope. These systems have impact on both a general understanding of membrane trafficking in bacteria and on bacterial pathogenesis. Expected online publication date for the Annual Review of Biochemistry Volume 75 is June 2, 2006. Please see http://www.annualreviews.org/catalog/pub_dates.asp for revised estimates.

  glycosyltransferases, cell-surface biogenesis, trans-envelope assembly complex, polysaccharide export

  NCBI PubMed ID: 16756484
  Publication DOI: 10.1146/annurev.biochem.75.103004.142545
  Journal NLM ID: 2985150R
  Correspondence: cwhitfie@uoguelph.ca
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 Canada
  
   
  
  Escherichia coli K5
  CSDB ID 395 (all data & tools)
• Article ID: 3617
  Bronner D, Sieberth V, Pazzani C, Roberts IS, Boulnois GJ, Jann B, Jann K "Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster" - Journal of Bacteriology 175 (1993) 5984-5992
  

  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.

  antigen, expression, gene, capsular, polysaccharide, analysis, chain, group, Escherichia, Escherichia coli, acid, capsular polysaccharide, antigens, cluster, gene cluster, mutant, mutants, activity, polysaccharides, region, biochemical, reduced, surface, mutation, synthetase, extracellular, transport, capacity, chain length, Microscopy, polymerization, electron microscopy, D

  NCBI PubMed ID: 8397188
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Max-Planck-Institut fur Immunbiologie, Freiburg, Germany.
  Methods: genetic methods, biochemical methods, microscopy
  
   
  
  Escherichia coli K5
  CSDB ID 24809 (all data & tools)
• Article ID: 3618
  Bronner D, Sieberth V, Pazzani C, Smith A, Boulnois G, Roberts I, Jann B, Jann K "Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli" - FEMS Microbiology Letters 113(3) (1993) 279-284
  

  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 genes were studied using immunoelectron microscopy. Membranes from these bacteria were incubated with UDP[14C]GlcA and UDPGlcNAc 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 kpsC and kpsS) and from region 3 (notably kpsT) in the K5 polysaccharide synthesis was apparent and is discussed.

  biosynthesis, synthesis, common, expression, gene, strain, capsular, polysaccharide, group, polymer, Escherichia, Escherichia coli, capsular polysaccharide, type, cluster, gene cluster, Genes, specific, bacteria, region, surface, capsule, membrane, membranes, recombinant, transport, type-specific, Microscopy, Gene region, immunoelectron microscopy, K5

  NCBI PubMed ID: 8270192
  Journal NLM ID: 7705721
  Publisher: Blackwell Publishing
  Institutions: Max-Planck-Institut fur Immunobiologie, Freiburg, FRG.
  Methods: genetic methods, biochemical methods, microscopy
  
   
  
  Escherichia coli K5
  CSDB ID 24810 (all data & tools)
• Article ID: 3656
  Sismey-Ragatz AE, Green DE, Otto NJ, Rejzek M, Field RA, DeAngelis PL "Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases: monodisperse polymers and unnatural structures" - Journal of Biological Chemistry 282(39) (2007) 28321-28327
  

  Heparosan (-GlcUA-β1,4-GlcNAc-α1,4-)(n) is a member of the glycosaminoglycan polysaccharide family found in the capsule of certain pathogenic bacteria as well as the precursor for the vertebrate polymers, heparin and heparan sulfate. The two heparosan synthases from the Gram-negative bacteria Pasteurella multocida, PmHS1 and PmHS2, were efficiently expressed and purified using maltose-binding protein fusion constructs. These relatively homologous synthases displayed distinct catalytic characteristics. PmHS1, but not PmHS2, was able to produce large molecular mass (100-800 kDa) monodisperse polymers in synchronized, stoichiometrically controlled reactions in vitro. PmHS2, but not PmHS1, was able to utilize many unnatural UDP-sugar analogs (including substrates with acetamido-containing uronic acids or longer acyl chain hexosamine derivatives) in vitro. Overall these findings reveal potential differences in the active sites of these two Pasteurella enzymes. In the future, these catalysts should allow the creation of a variety of heparosan and heparinoids with utility for medical applications.

  synthesis, glycosyltransferases, glycosaminoglycan, heparin, heparan sulfate, Pasteurella multocida, chemical synthesis, synthase, Uridine Diphosphate Sugars

  NCBI PubMed ID: 17627940
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: paul-deangelis@ouhsc.edu
  Institutions: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
  Methods: MALDI-TOF MS, genetic methods, biochemical methods
  
   
  
  Pasteurella multocida
  CSDB ID 23633 (all data & tools)
• Article ID: 4072
  Masuko S, Higashi K, Wang Z, Bhaskar U, Hickey AM, Zhang F, Toida T, Dordick JS, Linhardt RJ "Ozonolysis of the double bond of the unsaturated uronate residue in low-molecular-weight heparin and K5 heparosan" - Carbohydrate Research 346(13) (2011) 1962-1966
  

  Ozone is known to add across and cleave carbon-carbon double bonds. Ozonolysis is widely used for the preparation of pharmaceuticals, for bleaching substances and for killing microorganisms in air and water sources. Some polysaccharides and oligosaccharides, such as those prepared using chemical or enzymatic beta-elimination, contain a site of unsaturation. We examined ozonolysis of low-molecular-weight heparins (LMWHs), enoxaparin and logiparin, and heparosan oligo- and polysaccharides for the removal of the nonreducing terminal unsaturated uronate residue. 1D (1)H NMR showed that these ozone-treated polysaccharides retained the same structure as the starting polysaccharide, except that the C4-C5 double bond in the nonreducing end unsaturated uronate had been removed. The anticoagulant activity of the resulting product from enoxaparin and logiparin was comparable to that of the starting material. These results demonstrate that ozonolysis is an important tool for the removal of unsaturated uronate residues from LMWHs and heparosan without modification of the core polysaccharide structure or diminution of anticoagulant activity. This reaction also has potential applications in the chemoenzymatic synthesis of bioengineered heparin from Escherichia coli-derived K5 heparosan.

  heparin, Heparosan, anticoagulant activity, ozone, unsaturated uronic acid, low-molecular-weight heparin, b Elimimnation

  NCBI PubMed ID: 21742314
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Correspondence: linhar@rpi.edu (R.J. Linhardt)
  Institutions: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
  Methods: 1H NMR, ESI-MS, NMR-1D, ozonolysis, enzymatic digestion
  
   
  
  Escherichia coli K5 (ATCC 23506)
  CSDB ID 26283 (all data & tools)
• Article ID: 4079
  Otto NJ, Solakyildirim K, Linhardt RJ, DeAngelis PL "Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog" - Glycobiology 21(10) (2011) 1331-1340
  

  Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-D-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-D-GlcUA-α1,4-D-GlcNAc-α1-](n) was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications

  polysaccharide, glycosyltransferase, glycosaminoglycan, Pasteurella multocida, Heparosan, Comamonas

  NCBI PubMed ID: 21610195
  Publication DOI: 10.1093/glycob/cwr072
  Journal NLM ID: 9104124
  Publisher: IRL Press at Oxford University Press
  Correspondence: paul-deangelis@ouhse.edu
  Institutions: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma Center for Medical Glycobiology, Oklahoma City, OK 73126, USA, Department of Chemistry and Chemical Biology Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
  Methods: 13C NMR, 1H NMR, NMR-2D, PCR, TLC, acid hydrolysis, MALDI-TOF MS, NMR-1D, genetic methods, enzymatic digestion, cloning
  
   
  
  Comamonas testosteroni KF-1
  CSDB ID 26900 (all data & tools)
• Article ID: 4132
  Wang Z, Dordick JS, Linhardt RJ "Escherichia coli K5 heparosan fermentation and improvement by genetic engineering" - Bioengineered Bugs 2(1) (2011) 63-67
  

  N-acetyl heparosan is the precursor for the biosynthesis of the important anticoagulant drug heparin. The E. coli K5 capsular heparosan polysaccharide provides a promising precursor for in vitro chemoenzymatic production of bioengineered heparin. This article explores the improvements of heparosan production for bioengineered heparin by fermentation process engineering and genetic engineering.

  heparin, fermentation, Heparosan, K5 capsule polysaccharide

  NCBI PubMed ID: 21636991
  Journal NLM ID: 101525147
  Publisher: Austin, TX: Landes Bioscience
  Correspondence: dordick@rpi.edu
  Institutions: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA
  Methods: fermentation
  
   
  
  Escherichia coli K5
  CSDB ID 26386 (all data & tools)
• Article ID: 4133
  Wang Z, Yang B, Zhang Z, Ly M, Takieddin M, Mousa S, Liu J, Dordick JS, Linhardt RJ "Control of the heparosan N-deacetylation leads to an improved bioengineered heparin" - Applied Microbiology and Biotechnology 91(1) (2011) 91-99
  

  The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.

  heparin, deacetylation, Heparosan, porcine intestine

  NCBI PubMed ID: 21484210
  Journal NLM ID: 8406612
  Publisher: Springer
  Correspondence: linhar@rpi.edu
  Institutions: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA, Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA, The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY 12208, USA, Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
  Methods: 1H NMR, de-N-acetylation, enzymatic modification, PAGE, LC-MS, SEC, fermentation, anticoagulation activity, N-sulfonation
  
   
  
  Escherichia coli K5
  CSDB ID 26387 (all data & tools)
• Article ID: 4431
  Ovodov YS "Bacterial capsular antigens. Structural patterns of capsular antigens" - Biochemistry (Moscow) 71(9) (2006) 937-954
  

  Structural patterns of bacterial capsular antigens including capsular polysaccharides and exoglycans are given in this review. In addition, the immunological activity of capsular antigens and their role in type specificity of bacteria are discussed.

  structure, capsular polysaccharides, bacterial capsular antigens, bacterial exoglycans, immunological activity, type specificity

  NCBI PubMed ID: 17009947
  Publication DOI: 10.1134/S000629790609001X
  Journal NLM ID: 0376536
  Publisher: Nauka/Interperiodica
  Correspondence: ovoys@physiol.komisc.ru
  Institutions: Institute of Physiology, Komi Science Center, Urals Branch of the Russian Academy of Sciences, Syktyvkar 167982, Russia
  
   
  
  Escherichia coli K5
  CSDB ID 28740 (all data & tools)
• Article ID: 4626
  Thompson JE, Pourhossein M, Waterhouse A, Hudson T, Goldrick M, Derrick JP, Roberts IS "The K5 lyase KflA combines a viral tail spike structure with a bacterial polysaccharide lyase mechanism" - Journal of Biological Chemistry 285(31) (2010) 23963-23969
  

  K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-βGlcA-(1,4)-αGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

  Escherichia coli, capsular polysaccharide, crystal structure, protein structure, K5 lyase A (KflA)

  NCBI PubMed ID: 20519506
  Publication DOI: 10.1074/jbc.M110.127571
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: i.s.roberts@manchester.ac.uk
  Institutions: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
  Methods: X-ray, SDS-PAGE, genetic methods, crystallization
  
   
  
  Escherichia coli K5
  CSDB ID 29362 (all data & tools)
• Article ID: 4640
  Willis LM, Whitfield C "Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways" - Carbohydrate Research 378 (2013) 35-44
  

  Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.

  biosynthesis, capsular polysaccharides, glycosyltransferases, gram negative bacteria, export, ABC transporters

  NCBI PubMed ID: 23746650
  Publication DOI: 10.1016/j.carres.2013.05.007
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Correspondence: C. Whitfield
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
  
   
  
  Escherichia coli K5, Pasteurella multocida type D
  CSDB ID 29818 (all data & tools)
• Article ID: 4804
  Teng L, Fu H, Wang M, Deng C, Song Z, Chen J "Immunomodulatory activity of heparan sulfate mimetics from Escherichia coli K5 capsular polysaccharide in vitro" - Carbohydrate Polymers 115 (2015) 643-650
  

  Escherichia coli K5 capsular polysaccharide (K5PS) has been used as starting material to synthesize heparan sulfate (HS) mimetics with various biological properties. This study determined the immunomodulatory activities of K5PS through the sulfation process. The immunomodulatory effects of sulfated K5 polysaccharide derivatives were evaluated in vitro on murine macrophages and lymphocytes. Results indicated that HS mimetics with high sulfation in the O-position stimulated cell proliferation of macrophages and lymphocytes, significantly enhanced cytokine secretion and upregulated the cytotoxicity of NK cells. This study suggests that high sulfation in O-position of HS is required for the immunomodulatory activities.

  Escherichia coli, sulfation, sulfate, immunomodulatory activity, K5 capsular polysaccharide, Structure-activity correlation

  NCBI PubMed ID: 25439943
  Publication DOI: 10.1016/j.carbpol.2014.08.119
  Journal NLM ID: 8307156
  Publisher: Elsevier
  Correspondence: jhchenwhut@126.com
  Institutions: Wuxi Medical School, Jiangnan University, Wuxi 214122, PR China, Changshan Biochem Pharm (Jiangsu) Co., Ltd., Changzhou 213149, PR China, The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, PR China, School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, PR China
  Methods: 13C NMR, ELISA, chemical synthesis, biological assays, statistical analysis, immunological assays
  
   
  
  Escherichia coli O10:K5:H4
  CSDB ID 30682 (all data & tools)
• Article ID: 4882
  Senchenkova SN, Zhang Y, Perepelov AV, Guo X, Shashkov AS, Liu B, Knirel YA "Structure and biosynthesis gene cluster of the O-antigen of Escherichia coli O12" - Biochemistry (Moscow) 81(4) (2016) 401-406
  

  Two polysaccharides were isolated from Escherichia coli O12, the major being identified as the O12-antigen and the minor as the K5-antigen. The polysaccharides were studied by sugar analysis, Smith degradation, and one- and two-dimensional (1)H and (13)C NMR spectroscopy. As a result, the following structure of the O12-polysaccharide was elucidated, which, to our knowledge, has not been hitherto found in bacterial carbohydrates: →2)-β-d-Glcp-(1→6)-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→. The →4)-β-d-GlcpA-(1→4)-α-d-GlcpNAc-(1→ structure established for the K5-polysaccharide (heparosan) is previously known. Functions of genes in the O-antigen biosynthesis gene cluster of E. coli O12 were assigned by comparison with sequences in the available databases and found to be consistent with the O12-polysaccharide structure.

  Escherichia coli, glycosyltransferase, O-antigen gene cluster, O-Polysaccharide structure

  NCBI PubMed ID: 27293097
  Publication DOI: 10.1134/S0006297916040106
  Journal NLM ID: 0376536
  Publisher: Nauka/Interperiodica
  Correspondence: yknirel@gmail.com
  Institutions: Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, Nankai University, TEDA Institute of Biological Sciences and Biotechnology,300457 Tianjin, P. R. China
  Methods: 13C NMR, 1H NMR, NMR-2D, sugar analysis, acid hydrolysis, GLC, Smith degradation, NMR-1D, GPC, mild acid degradation, function analysis of gene clusters
  
   
  
  Escherichia coli O12 (G1280)
  CSDB ID 11247 (all data & tools)
• Article ID: 5443
  Guan L, Xue Y, Ding W, Zhao Z "Biosynthesis and regulation mechanisms of the Pasteurella multocida capsule" - Research in Veterinary Science 127 (2019) 82-90
  

  Pasteurella multocida possesses a polysaccharide capsule composed of a viscous surface layer that acts as a critical structural component and virulence factor. Capsular polysaccharides are structurally similar to vertebrate glycosaminoglycans, providing an immunological mechanism for bacterial molecular mimicry, resistance to phagocytosis, and immune evasion during the infection process. In recent years, a series of important research advances have been made in understanding the biosynthesis and regulatory aspects of the P.multocida capsule. This review systematically examines the serogroups, polysaccharide composition and structures, biosynthetic loci and functions, biosynthesis pathways, and expression regulation mechanisms of the P.multocida capsule, supplying a theoretical basis for the molecular pathogenesis of the P.multocida capsule and the future development of capsular polysaccharide vaccines.

  biosynthesis, capsule, glycosaminoglycan, Pasteurella multocida, Expression regulation

  NCBI PubMed ID: 31678457
  Publication DOI: 10.1016/j.rvsc.2019.10.011
  Journal NLM ID: 0401300
  Publisher: British Veterinary Association
  Correspondence: Z. Zhao
  Institutions: Laboratory of Veterinary Biologics Engineering, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China, Key-Disciplines Lab of Safety of Environment and Animal Product, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
  
   
  
  Pasteurella multocida D
  CSDB ID 762 (all data & tools)
• Article ID: 5791
  Knirel YA, Van Calsteren M "Bacterial exopolysaccharides" - Book: Comprehensive Glycoscience: From Chemistry to Systems Biology. Reference Module in Chemistry, Molecular Sciences and Chemical Engineering (2021) 1-75
  

  Bacterial extracellular polysaccharides are known as a cell-bound capsule, a sheath, or a slime, which is excreted into the environment. They play an important role in virulence of medical bacteria and plant-to-symbiont interaction and are used for serotyping of bacteria and production of vaccines. Some exopolysaccharides have commercial applications in industry, and claims of health benefits have been documented for an increasing number of them. Exopolysaccharides have diverse composition and structure, and some contain sugar and non-sugar components that are found in bacterial carbohydrates only. The present article provides an updated collection of the data on exopolysaccharides of various classes of gram-negative and gram-positive bacteria reported until the end of 2019. When known, biosynthesis pathways of exopolysaccharides are treated in a summary manner. References are made to structure and biosynthesis relatedness between exopolysaccharides of different bacterial taxa as well as between bacterial polysaccharides and mammalian glycosaminoglycans.

  polysaccharide structure, Gram-negative bacteria, capsule, Biofilm, polysaccharide biosynthesis, gram-positive bacteria, Monosaccharide composition, Bacterial exopolysaccharide, non-sugar component

  Publication DOI: 10.1016/B978-0-12-819475-1.00005-5
  Publisher: Elsevier
  Correspondence: marie-rose.vancalsteren@canada.ca; yknirel@gmail.com
  Editors: Barchi J, Kamerling H
  Institutions: N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, QC, Canada
  
   
  
  Pasteurella multocida D
  CSDB ID 6676 (all data & tools)
• Article ID: 6088
  Knirel YA, Kenyon JJ "Biosynthesis of bacterial polysaccharides" - Book: Polysaccharides of Microbial Origin. Biomedical Applications (2021) 453-479
  

  The present chapter is devoted to biosynthesis pathways of various bacterial surface polysaccharides, including slime exopolysaccharides and capsular polysaccharides, as well as the O-antigen polysaccharide chains of lipopolysaccharides. The polysaccharides are extraordinarily diverse in structure and define the specificity of interaction between bacteria and their environments, including interactions with host organisms, bacteriophages, and natural predators. As such, both homopolysaccharides and heteropolysaccharides produced by bacteria have an important function in bacterial lifestyle, particularly in protection of human pathogens against the immune system response and some antimicrobials. In both gram-negative and gram-positive bacteria, biosynthesis of most cell-surface heteropolysaccharides is accomplished by the Wzy-dependent pathway, whereas some exopolysaccharides, such as a β-(1-6)-linked polymer of D-GlcpNAc called PNAG, are synthesized by the synthase-dependent pathway. For synthesis of homopolysaccharides and some heteropolysaccharides with a small (di- or tri-saccharide) repeating unit, gram-negative bacteria use the ATP-binding cassette (ABC) transporter-dependent pathway. A fourth pathway is utilized for the extracellular synthesis of several glucans and fructus by a single sucrase protein. The specific mechanisms of these pathways, including a brief view on the genetics of the polysaccharide biosynthesis, is provided.

  lipopolysaccharides, capsular polysaccharides, bacteria, exopolysaccharides, gram negative bacteria, biosynthesis pathway

  Publication DOI: 10.1007/978-3-030-42215-8_28
  Publisher: Springer
  Correspondence: Y.A. Knirel ; johanna.kenyon@qut.edu.au
  Editors: J.Oliveira HRRLR
  Institutions: N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia, Centre for Immunolgy and Infection Control University of Minho, (CIIC), School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia URWPalladioL-Rom
  
   
  
  Pasteurella multocida type D, Escherichia coli K5
  CSDB ID 5890 (all data & tools)
• Article ID: 6167
  Yakovleva L, Fulleborn JA, Walvoort MTC "Opportunities and Challenges of Bacterial Glycosylation for the Development of Novel Antibacterial Strategies" - Frontiers in Microbiology 12 (2021) 745702
  

  Glycosylation is a ubiquitous process that is universally conserved in nature. The various products of glycosylation, such as polysaccharides, glycoproteins, and glycolipids, perform a myriad of intra- and extracellular functions. The multitude of roles performed by these molecules is reflected in the significant diversity of glycan structures and linkages found in eukaryotes and prokaryotes. Importantly, glycosylation is highly relevant for the virulence of many bacterial pathogens. Various surface-associated glycoconjugates have been identified in bacteria that promote infectious behavior and survival in the host through motility, adhesion, molecular mimicry, and immune system manipulation. Interestingly, bacterial glycosylation systems that produce these virulence factors frequently feature rare monosaccharides and unusual glycosylation mechanisms. Owing to their marked difference from human glycosylation, bacterial glycosylation systems constitute promising antibacterial targets. With the rise of antibiotic resistance and depletion of the antibiotic pipeline, novel drug targets are urgently needed. Bacteria-specific glycosylation systems are especially promising for antivirulence therapies that do not eliminate a bacterial population, but rather alleviate its pathogenesis. In this review, we describe a selection of unique glycosylation systems in bacterial pathogens and their role in bacterial homeostasis and infection, with a focus on virulence factors. In addition, recent advances to inhibit the enzymes involved in these glycosylation systems and target the bacterial glycan structures directly will be highlighted. Together, this review provides an overview of the current status and promise for the future of using bacterial glycosylation to develop novel antibacterial strategies.

  glycosylation, pathogenic bacteria, metabolic oligosaccharide engineering, antibacterial strategies, antivirulence

  NCBI PubMed ID: 34630370
  Publication DOI: 10.3389/fmicb.2021.745702
  Journal NLM ID: 101548977
  Publisher: Lausanne: Frontiers Research Foundation
  Correspondence: Marthe T.C. Walvoort
  Institutions: Faculty of Science and Engineering, Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands
  
   
  
  Escherichia coli K5
  CSDB ID 10417 (all data & tools)
• Article ID: 6185
  Clarke BR, Esumeh F, Roberts IS "Cloning, expression, and purification of the K5 capsular polysaccharide lyase (KflA) from coliphage K5A: evidence for two distinct K5 lyase enzymes" - Journal of Bacteriology 182(13) (2000) 3761-3766
  

  The Escherichia coli K5 capsular polysaccharide [-4)-βGlcA-(1,4)-αGlcNAc-(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.

  capsular polysaccharide, Escherichia coli K5, bacteriophage K5A

  NCBI PubMed ID: 10850992
  Publication DOI: 10.1128/JB.182.13.3761-3766.2000
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: ISRobert@fs1.scg.man.ac.uk
  Institutions: School of Biological Sciences, University of Manchester, Manchester, UK
  Methods: PCR, DNA sequencing, SDS-PAGE, DNA techniques, Western blotting, electrophoresis, extraction, cell growth, enzymatic assay, precipitation, spectrophotometry, centrifugation, optical density measurement, protein determination, protein expression, BLAST
  
   
  
  Escherichia coli K5
  CSDB ID 65169 (all data & tools)