A simple method for purification of Shiga or Shiga-like toxin from Shigella dysenteriae and Escherichia coli O157: H7 by immunoaffinity column chromatography

Abstract A simple method was developed for purifying Shiga toxin and Shiga-like toxin from the culture supernatants and cell lysates of Shigella dysenteriae type 1 and Escherichia coli O157 : H7 grown in modified syncase media. Two steps, DEAE cellulose column chromatography and immunoaffinity column chromatography, were sufficient for obtaining purified toxin. By this procedure, about 0.32–0.75 mg of purified toxin was obtained from 5 1 of culture with high recovery rate (53–62%). The toxins purified by this method from the culture supernatants and cell lysates of S. dysenteriae and E. coli O15 : H7 were immunologically, biologically and structurally indistinguishable.     

Structure of the O-polysaccharide of Vibriocholerae O43 containing a new monosaccharide derivative, 4-(N-acetyl-l-allothreonyl)amino-4,6-dideoxy-d-glucose

The O-polysaccharide of Vibriocholerae O43 was studied using chemical analyses, triflic acid solvolysis and 2D NMR spectroscopy, including ¹H/¹H COSY, TOCSY, NOESY and ¹H/¹³C gradient-selected HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:→3)-β-d-Quip4NAcyl-(1→3)-α-d-GalpNAcA-(1→4)-α-d-GalpNAc-(1→3)-α-d-QuipNAc-(1→where d-QuiNAc stands for 2-acetamido-2,6-dideoxy-d-glucose, d-Qui4NAcyl for 4-(N-acetyl-l-allothreonyl)amino-4,6-dideoxy-d-glucose and d-GalNAcA for 2-acetamido-2-deoxy-d-galacturonic acid.     

Production of Shiga toxin and a cytolethal distending toxin (CLDT) by serogroups of Shigella spp.

Abstract 56 strains of Shigella including 12 Shigella dysenteriae (serotypes 1, 2, 9, 11 and 12), 23 Shigella flexneri (serotypes 1, 2, 3, 4, 6, var. X and var. Y), 19 Shigella boydii (serotypes 1, 2, 4, 5, 7, 11, 13, 14, 15 and 18), and 2 Shigella sonnei were screened for their ability to produce both classic Shiga toxin and a new heat-labile cytolethal distending toxin (CLDT). Whereas extracellular Shiga toxin was only detectable in filtrates of five S. dysenteriae type 1 strains, CLDT was produced by four strains of S. dysenteriae type 2 and an isolate of S. boydii type 7. No cytotonic enterotoxins similar to Escherichia coli LT were observed in this study. None of the S. flexneri or S. sonnei isolates tested were found to produce extracellular cytotoxic factors. The Shiga toxin produced by the S. dysenteriae type 1 was neutralizable by anti-toxin to verotoxin 1 of E. coli O157 : H7. The Shigella CLDT was neutralizable by antisera prepared to a CLDT-producing E. coli O55 : H4.     

Structure of the O-specific polysaccharide chain of Shigella dysenteriae type 7 lipopolysaccharide

The structure of the O-specific polysaccharide chain of the Shigella dysenteriae type 7 lipopolysaccharide has been established mainly by 13C NMR analysis of the intact and modified (acetylated and de-O-acetylated) polymers, as well as of products of its solvolysis with anhydrous hydrogen fluoride. The polysaccharide contains two unusual sugar derivatives. N-acetyl-D-galactosaminuronamide and 4-(N-acetylglycyl)amido-4,6-dideoxy-D-glucose (GalNAcAN and Qui4N----GlyAc, respectively) and is built up of tetrasaccharide repeating units of the following structure: (Formula: see text). Serological cross-reaction of S. dysenteriae type 7 and Pseudomonas aeruginosa O4 (Lányl) is accounted for by the similarity of their O-specific polysaccharides.     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of Shigella dysenteriae types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of nonstoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, the position of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by 13C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner. [Formula: see text].     

Structural and genetic characterization of the O-antigen of Salmonella enterica O56 containing a novel derivative of 4-amino-4,6-dideoxy-d-glucose

Based on the O-antigens (O-polysaccharides), one of the most variable cell constituents, 46 O-serogroups have been recognized in the Kauffmann-White serotyping scheme for Salmonella enterica. In this work, the structure of the O-polysaccharide and the genetic organization of the O-antigen gene cluster of S. enterica O56 were investigated. As judged by sugar and methylation analyses, along with NMR spectroscopic data, the O-polysaccharide has a linear tetrasaccharide O-unit, which consists of one residue each of d-ribofuranose, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, and a novel sugar derivative, 4-(N-acetyl-l-seryl)amino-4,6-dideoxy-d-glucose (d-Qui4NSerAc). The following structure of the O-polysaccharide was established:→3)-β-d-Quip4NSerAc-(1→3)-β-d-Ribf-(1→4)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen gene cluster of S. enterica O56 having 12 open reading frames was found between the housekeeping genes galF and gnd. A comparison with databases and using the O-antigen structure data enabled us to ascribe functions to genes for (i) synthesis of d-GalNAc and d-Qui4NSerAc, (ii) sugar transfer, and (iii) O-antigen processing, including genes for O-unit flippase (Wzx) and O-antigen polymerase (Wzy).     

Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose

Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.     

Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains.

Introduction of the rol genes of Shigella dysenteriae 1 and Escherichia coli K-12 into Shigella flexneri carrier strains expressing the heterologous S. dysenteriae type 1 lipopolysaccharide resulted in the formation of longer chains of S. dysenteriae 1 O antigen. In bacteria producing both homologous and heterologous O antigen, this resulted in a reduction of the masking of heterologous O antigen by homologous lipopolysaccharide and an increased immune response induced by intraperitoneal immunization of mice by recombinant bacteria. The rol genes of S. dysenteriae 1 and E. coli K-12 were sequenced, and their gene products were compared with the S. flexneri Rol protein. The primary sequence of S. flexneri Rol differs from both E. coli K-12 and S. dysenteriae 1 Rol proteins only at positions 267 and 270, which suggests that this region may be responsible for the difference in biological activities.     

Structure of the O-polysaccharide and serological cross-reactivity of the Providencia stuartii O33 lipopolysaccharide containing 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp)-(1-->, where d-Qui4N(Ac-D-Asp) is 4-(N-acetyl-D-aspart-4-yl)amino-4,6-dideoxy-D-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O-D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4N(Ac-D-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral beta-D-Qui4N(Ac-L-Asp) residue.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

A rapid subtractive immunization method to prepare discriminatory monoclonal antibodies for food E. coli O157:H7 contamination

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1:10(6) with E. coli O157:H7 and affinity constants ranged from 1.57 × 10(10) to 2.79 × 10(10) L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.     

Structure and gene cluster of the O-antigen of Escherichia coli O109; chemical and genetic evidences of the presence of L-RhaN3N derivatives in the O-antigens of E. coli O109 and O119

O-antigen representing the O-polysaccharide chain of the lipopolysaccharide is the most variable constituent on the cell surface of Gram-negative bacteria and a player in their pathogenicity. The O-polysaccharide of Escherichia coli O109 was studied by sugar analysis and nuclear magnetic resonance spectroscopy and found to contain a rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy-l-mannose (l-RhaNAc3NAc). The following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established, which is closely related to that of Proteus penneri O66: Ac--4-β-L-RhapNAc3NAc -->4)-α-D-Glcp-(1-->3)-α-L-6dTalp-(1-->3)-β-D-GlcpNAc-(1-->. The O-antigen gene cluster of E. coli O109 was sequenced and all 14 genes found were assigned functions based on their similarity to genes from the available databases. Putative genes for synthesis of l-RhaN3N were found in E. coli O109 and their homologues in E. coli O119, whose O-antigen has been reported earlier to contain 2-acetamido-2,3,6-trideoxy-3-formamido-d-mannose (d-RhaNAc3NFo). Analysis by GLC of the (S)-2-octyl glycosides confirmed that the absolute configuration of RhaN3N in E. coli O119 should be revised from D TO L.     

Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.     

Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.

Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars

A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10(10) and 10(4) cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10(6) to 10(9) CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as L-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of "nonfermentable" sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.     

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.     

Antibacterial activity of fresh and processed red muscadine juice and the role of their polar compounds on Escherichia coli O157:H7

Aims:  The objectives of this research were to show the anti- Escherichia coli O157:H7 effect of fresh (FRMJ) and processed red muscadine (V itis rotundifolia ) juice (PRMJ) and to discern the active compounds responsible for anti- E . coli O157:H7.
Methods and Results:  Polar and phenolic compounds of FRMJ and PRMJ were analysed by high-performance liquid chromatography. Antibacterial activity of FRMJ, PRMJ, their polar and polyphenol fractions, individual synthetic acids and their mixture with or without sugars were investigated on E . coli O157:H7. FRMJ and PRMJ inactivated ( P  ≤ 0·05) 5-log cocktail cells of E. coli O157:H7 within 4 h at 37°C. Polar fractions that contained malic, tartaric and tannic acids showed strong antimicrobial activity ( P  ≤ 0·05) against E . coli O157:H7. Tannic acid among the synthetic acids showed the highest antimicrobial activity against E. coli O157:H7.
Conclusions:  FRMJ, PRMJ and their polar compounds showed strong anti- E . coli O157:H7 activity.
Significance and Impact of the Study:  Earlier findings have failed to show any anti -E . coli O157:H7 effect of grape juice without adding preservatives. Our findings show that red muscadine juice has natural antibacterial substances and suggest that these can be used as active antimicrobial ingredients against E . coli O157:H7 in nonalcoholic beverages.     

Identification of the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in synthesis of enterobacterial common antigen in Escherichia coli K-12.

The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the wec gene cluster located at ca. 85.4 min on the Escherichia coli chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the wec gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the wec gene cluster in the region between o416 and wecG revealed that it contained three open reading frames: o74, o204, and o450. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames, o359 and o450. Mutants of E. coli possessing null mutations in o359 were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [(3)H]FucNAc from TDP-[(3)H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type o359 allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified o359 gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [(3)H]Fuc4NAc from TDP-[(3)H]Fuc4NAc to lipid II. Our data support the conclusion that o359 of the wec gene cluster of E. coli is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.