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Biosynthesis of Ganglioside Mimics in Campylobacter jejuni OH4384

2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês

10.1074/jbc.275.6.3896

1083-351X

Michel Gilbert, Jean‐Robert Brisson, Marie-France Karwaski, J.J. Michniewicz, Anna‐Maria Cunningham, Yuyang Wu, N. Martin Young, Warren W. Wakarchuk,

Bacteriophages and microbial interactions

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS. We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS. lipooligosaccharide cytidine monophosphate-N-acetylneuraminic acid correlated spectroscopy 6-(5-fluorescein-carboxamido)-hexanoic acid succimidyl ester heteronuclear multiple bond coherence heteronuclear single quantum coherence lipopolysaccharide nuclear Overhauser effect NOE spectroscopy total correlation spectroscopy amino acid(s) kilobase pair(s) open reading frame polymerase chain reaction 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid isopropyl-1-thio-β-d-galactopyranoside matrix-assisted laser desorption ionization/time of flight Since the late 1970s, Campylobacter jejuni has been recognized as an important cause of acute gastroenteritis in humans (1.Skirrow M.B. Br. Med. J. 1977; 2: 9-11Crossref PubMed Scopus (902) Google Scholar). Epidemiological studies have shown that Campylobacterinfections are more common in developed countries thanSalmonella infections, and they are also an important cause of diarrheal diseases in developing countries (2.Ketley J.M. Microbiology. 1997; 143: 5-21Crossref PubMed Scopus (309) Google Scholar). In addition to causing acute gastroenteritis, C. jejuni infection has been implicated as a frequent antecedent to the development of Guillain-Barré syndrome, a form of neuropathy that is the most common cause of generalized paralysis (3.Ropper A.H. N. Engl. J. Med. 1992; 326: 1130-1136Crossref PubMed Scopus (638) Google Scholar). One of the most commonC. jejuni serotypes associated with Guillain-Barrésyndrome is O:19 (4.Kuroki S. Saida T. Nukina M. Haruta T. Yoshioka M. Kobayashi Y. Nakanishi H. Ann. Neurol. 1993; 33: 243-247Crossref PubMed Scopus (223) Google Scholar), and this prompted detailed study of the LOS structure of strains belonging to this serotype, including strains OH4382 and OH4384, which were isolated from two siblings who developed the Guillain-Barré syndrome (5.Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Mills S.D. Kurjanczyk L.A. Penner J.L. J. Bacteriol. 1992; 174: 1324-1332Crossref PubMed Scopus (77) Google Scholar, 6.Aspinall G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Biochemistry. 1994; 33: 241-249Crossref PubMed Scopus (174) Google Scholar, 7.Aspinall G.O. McDonald A.G. Pang H. Biochemistry. 1994; 33: 250-255Crossref PubMed Scopus (62) Google Scholar, 8.Aspinall G.O. Fujimoto S. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Infect. Immun. 1994; 62: 2122-2125Crossref PubMed Google Scholar). The core oligosaccharides of low molecular weight LOS1of O:19 strains were shown to exhibit molecular mimicry of gangliosides (Fig. 1). Terminal oligosaccharide moieties identical to those of GM1, GD1a, GD3, and GT1a 2The abbreviated designations of glycolipids are according to IUPAC-IUC nomenclature (see Ref. 31.Chester M.A. Eur. J. Biochem. 1998; 257: 293-298Crossref PubMed Scopus (180) Google Scholar). gangliosides have been found in various O:19 strains. The most extensive structure, a trisialylated ganglioside mimic of GT1a, has been observed in the strain OH4384. Molecular mimicry of host structures by the saccharide portion of LOS is considered to be a virulence factor of various mucosal pathogens, which could use this strategy to evade the immune response (9.Moran A.P. Prendergast M.M. Appelmelk B.J. FEMS Immunol. Med. Microbiol. 1996; 16: 105-115Crossref PubMed Google Scholar, 10.Moran A.P. Appelmelk B.J. Aspinall G.O. J. Endotoxin Res. 1996; 3: 521-531Crossref Scopus (47) Google Scholar). Consequently, the identification of the genes involved in LOS synthesis and the study of their regulation is of considerable interest for a better understanding of the pathogenesis mechanisms used by these bacteria. The cloning and characterization of a gene (heptosyltransferase I) involved in the synthesis of the LOS inner core has been reported (11.Klena J.D. Gray S.A. Konkel M.E. Gene (Amst.). 1998; 222: 177-185Crossref PubMed Scopus (27) Google Scholar), while two other groups (12.Fry B.N. Korolik V. ten Brinke J.A. Pennings M.T.T. Zalm R. Teunis B.J.J. Coloe P.J. van der Zeijst B.A.M. Microbiology. 1998; 144: 2049-2061Crossref PubMed Scopus (71) Google Scholar, 13.Wood A.C. Oldfield N.J. O'Dwyer C.A. Ketley J.M. Microbiology. 1999; 145: 379-388Crossref PubMed Scopus (28) Google Scholar) have reported the cloning of LPS biosynthesis genes. Some of these genes are homologous to bacterial glycosyltransferases, but none have been linked unequivocally to the synthesis of the LOS outer core. The genes reported by Fry et al. (12.Fry B.N. Korolik V. ten Brinke J.A. Pennings M.T.T. Zalm R. Teunis B.J.J. Coloe P.J. van der Zeijst B.A.M. Microbiology. 1998; 144: 2049-2061Crossref PubMed Scopus (71) Google Scholar) and Wood et al. (13.Wood A.C. Oldfield N.J. O'Dwyer C.A. Ketley J.M. Microbiology. 1999; 145: 379-388Crossref PubMed Scopus (28) Google Scholar) could be involved in the synthesis of the O-chain or in the synthesis of another cell-associated carbohydrate. Recently, the genome sequence of the C. jejunistrain NCTC 11168 has been completed by the Sanger Centre. The serotype of this strain is O:2, but its core oligosaccharide structure is not known. This genome sequence therefore represents a source of information for the identification of the genes involved in the synthesis of the outer core of the LOS. In addition to their importance for pathogenesis studies, bacterial glycosyltransferases have been shown to be tools for the chemo-enzymatic syntheses of oligosaccharides with biological activity (14.Gilbert M. Cunningham A.-M. Watson D.C. Martin A. Richards J.C. Wakarchuk W.W. Eur. J. Biochem. 1997; 249: 187-194Crossref PubMed Scopus (68) Google Scholar, 15.Gilbert M. Bayer R. Cunningham A-M. Defrees S. Gao Y. Watson D.C. Young N.M. Wakarchuk W.W. Nat. Biotechnol. 1998; 16: 769-772Crossref PubMed Scopus (120) Google Scholar). Since many bacterial glycosyltransferases catalyze the formation of oligosaccharides identical to mammalian structures and they are easier to produce in quantity (15.Gilbert M. Bayer R. Cunningham A-M. Defrees S. Gao Y. Watson D.C. Young N.M. Wakarchuk W.W. Nat. Biotechnol. 1998; 16: 769-772Crossref PubMed Scopus (120) Google Scholar, 16.Wakarchuk W.W. Cunningham A.-M. Watson D.C. Young N.M. Protein Eng. 1998; 11: 295-302Crossref PubMed Scopus (80) Google Scholar), they are attractive alternatives to the equivalent mammalian glycosyltransferases. The ganglioside mimics synthesized by many C. jejuni O:19 strains contain α-2,3- and α-2,8-linked sialic acids; this organism is then a source of both α-2,3- and α-2,8- sialyltransferases. In this work, we report the sequencing of a locus involved in the biosynthesis of the LOS outer core and the cloning and expression of four glycosyltransferases, which encode enzyme activities required for the biosynthesis of ganglioside mimics by C. jejuni OH4384, which has been implicated in Guillain-Barré syndrome. One of these enzymes is a novel bifunctional sialyltransferase, which makes both α-2,3 and α-2,8-sialic acid linkages. The 1H and13C NMR analysis was done on nanomole amounts of enzymatic product using homonuclear and heteronuclear methods. For one compound, the α-2,3 and α-2,8-sialic acid linkages were confirmed by using a gradient inverse broadband nano-NMR probe to detect the3J(C,H) correlation across the glycosidic bonds. The following C. jejunistrains were used in this study: serostrain O:19 (ATCC 43446); serotype O:19, strains OH4382 and OH4384 were obtained from the Laboratory Center for Disease Control (Health Canada, Winnipeg, Manitoba, Canada); and serotype O:2 (NCTC 11168). Escherichia coli DH5α was used for the HindIII library, while E. coli AD202 (CGSG 7297) was used to express the different cloned glycosyltransferases. Genomic DNA isolation from theC. jejuni strains was performed using Qiagen Genomic-tip 500/G (Qiagen Inc., Valencia, CA). Plasmid DNA isolation, restriction enzyme digestions, purification of DNA fragments for cloning, ligations, and transformations were performed as recommended by the enzyme supplier, or the manufacturer of the kit used for the particular procedure. Long PCR reactions (>3 kb) were performed using the ExpandTM long template PCR system as described by the manufacturer (Roche Molecular Biochemicals). PCR reactions to amplify specific open reading frames (ORFs) were performed using thePwo DNA polymerase as described by the manufacturer (Roche Molecular Biochemicals). Restriction and DNA modification enzymes were purchased from New England Biolabs Ltd. (Mississauga, Ontario, Canada). DNA sequencing was performed using an Applied Biosystems (Montreal, Quebec, Canada) model 370A automated DNA sequencer and the manufacturer's cycle sequencing kit. The genomic library was prepared using a partial HindIII digest of the chromosomal DNA of C. jejuni OH4384. The partial digest was purified on a QIAquick column (QIAGEN Inc.) and ligated withHindIII-digested pBluescript SK−. E. coli DH5α was electroporated with the ligation mixture and the cells were plated on LB medium with 150 μg/ml ampicillin, 0.05 mm IPTG, and 100 μg/ml 5-bromo-4-chloro-indolyl-β-d-galactopyranoside. White colonies were picked in pools of 100 and were resuspended in 1 ml of medium with 15% glycerol. Twenty μl of each pool were used to inoculate 1.5 ml of LB medium supplemented with 150 μg/ml ampicillin. After 2 h of growth at 37 °C, IPTG was added to 1 mm and the cultures were grown for another 4.5 h. The cells were recovered by centrifugation, resuspended in 0.5 ml of 50 mm Mops (pH 7, 10 mm MgCl2) and sonicated for 1 min. The extracts were assayed for sialyltransferase activity as described below, except that the incubation time and temperature were 18 h and 32 °C, respectively. The positive pools were plated for single colonies, and 200 colonies were picked and tested for activity in pools of 10. Finally the colonies of the positive pools were tested individually, which led to the isolation of a two positive clones, pCJH9 (5.3-kb insert) and pCJH101 (3.9-kb insert). Using several subcloned fragments and custom-made primers, the inserts of the two clones were completely sequenced on both strands. The clones with individual HindIII fragments were also tested for sialyltransferase activity, and the insert of the only positive one (a 1.1-kb HindIII fragment cloned in pBluescript SK−) was transferred to pUC118 using KpnI andPstI sites in order to obtain the insert in the opposite orientation with respect to the plac promoter. The primers used to amplify the LOS biosynthesis locus of C. jejuni OH4384 were based on preliminary sequences from the complete genome of the strain NCTC 11168 (available via the World Wide Web from the Sanger Center). The primers CJ-42 (5′-GCCATTACCGTATCGCCTAACCAGG-3′; 25-mer) and CJ-43 (5′-AAAGAATACGAATTTGCTAAAGAGG-3′; 25-mer) were used to amplify an 11.47-kb locus using the ExpandTM long template PCR system. The PCR product was purified on a S-300 spin column (Amersham Pharmacia Biotech) and completely sequenced on both strands using a combination of primer walking and subcloning of HindIII fragments. Specific ORFs were amplified using the Pwo DNA polymerase. The PCR products were digested using the appropriate restriction enzymes and were cloned in the expression vector pCWori+ (17.Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (286) Google Scholar). Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce). For all of the enzymatic assays, 1 unit of activity was defined as the amount of enzyme that generated 1 μmol of product/min. FCHASE-labeled oligosaccharides were prepared as described previously (18.Wakarchuk W.W. Martin A. Jennings M.P. Moxon E.R. Richards J.C. J. Biol. Chem. 1996; 271: 19166-19173Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The screening assay for α-2,3-sialyltransferase activity in pools of clones contained 1 mm Lac-FCHASE, 0.2 mm CMP-Neu5Ac, 50 mm Mops, pH 7, 10 mm MnCl2, and 10 mm MgCl2 in a final volume of 10 μl. The various subcloned ORFs were tested for the expression of glycosyltransferase activities following a 4-h induction of the cultures with 1 mm IPTG. Extracts were made by sonication and the enzymatic reactions were performed overnight at 32 °C. The β-1,3-galactosyltransferase was assayed using 0.2 mmGM2-FCHASE (a generous gift of Dr. Eric Sjoberg, Cytel Corp.), 1 mm UDP-Gal, 50 mm Mes, pH 6, 10 mmMnCl2, and 1 mm dithiothreitol. The β-1,4-GalNAc transferase was assayed using 0.5 mmGM3-FCHASE, 1 mm UDP-GalNAc, 50 mm Hepes, pH 7, and 10 mm MnCl2. The α-2,3-sialyltransferase was assayed using 0.5 mm Lac-FCHASE, 0.2 mmCMP-Neu5Ac, 50 mm Hepes, pH 7, and 10 mmMgCl2. The α-2,8-sialyltransferase was assayed using 0.5 mm GM3-FCHASE, 0.2 mm CMP-Neu5Ac, 50 mm Hepes, pH 7, and 10 mm MnCl2. The reaction mixes were diluted appropriately with 10 mmNaOH and analyzed by capillary electrophoresis performed using the separation and detection conditions as described previously (19.Gilbert M. Watson D.C. Cunningham A.-M. Jennings M.P. Young N.M. Wakarchuk W.W. J. Biol. Chem. 1996; 271: 28271-28276Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The peaks from the electropherograms were analyzed using manual peak integration with the P/ACE Station software. For rapid detection of enzyme activity, samples from the transferase reaction mixtures were examined by thin layer chromatography on Silica-60 TLC plates (Merck) as described previously (19.Gilbert M. Watson D.C. Cunningham A.-M. Jennings M.P. Young N.M. Wakarchuk W.W. J. Biol. Chem. 1996; 271: 28271-28276Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). NMR experiments were performed on a Varian INOVA 600 NMR spectrometer. Most experiments were done using a 5-mm Z gradient triple resonance probe. NMR samples were prepared from 0.3 to 0.5 mg (200–500 nmol) of FCHASE-glycoside. The compounds were dissolved in H2O, and the pH was adjusted to 7.0 with dilute NaOH. After freeze-drying, the samples were dissolved in 600 μl of D2O. All NMR experiments were performed as described previously (20.Pavliak V. Brisson J.R. Michon F. Uhrin D. Jennings H.J. J. Biol. Chem. 1993; 268: 14146-14152Abstract Full Text PDF PubMed Google Scholar, 21.Brisson J.R. Uhrinova S. Woods R.J. van der Zwan M. Jarrell H.C. Paoletti L.C. Kasper D.L. Jennings H.J. Biochemistry. 1997; 36: 3278-3292Crossref PubMed Scopus (98) Google Scholar) using standard techniques such as COSY, TOCSY, NOESY, one-dimensional NOESY, one-dimensional TOCSY, and HSQC. For the proton chemical shift reference, the methyl resonance of internal acetone was set at 2.225 ppm (1H). For the13C chemical shift reference, the methyl resonance of internal acetone was set at 31.07 ppm relative to external dioxane at 67.40 ppm. Homonuclear experiments were on the order of 5–8 h each. The one-dimensional NOESY experiments for GD3-FCHASE (0.3 mm), with 8000 scans and a mixing time of 800 ms was done for a duration of 8.5 h each and processed with a line broadening factor of 2–5 Hz. For the one-dimensional NOESY of the resonances at 4.16 ppm, 3000 scans were used. The following parameters were used to acquire the HSQC spectrum: relaxation delay of 1.0 s, spectral widths in F2 and F1 of 6000 and 24147 Hz, respectively, acquisition times in t 2 of 171 ms. For the t 1 dimension, 128 complex points were acquired using 256 scans/increment. The sign discrimination in F1 was achieved by the States method. The total acquisition time was 20 h. For GM2-FCHASE, due to broad lines, the number of scans per increment was increased so that the HSQC was performed for 64 h. The phase-sensitive spectrum was obtained after zero filling to 2048 × 2048 points. Unshifted gaussian window functions were applied in both dimensions. The HSQC spectra were plotted at a resolution of 23 Hz/point in the 13C dimension and 8 Hz/point in the proton dimension. For the observation of the multiplet splittings, the 1H dimension was reprocessed at a resolution of 2 Hz/point using forward linear prediction and a π/4-shifted squared sinebell function. All the NMR data were acquired using Varian's standard sequences provided with the VNMR 5.1 or VNMR 6.1 software. The same program was used for processing. A gradient inverse broadband nano-NMR probe (Varian) was used to perform the gradient HMBC (22.Bax A. Summers M.F. J. Am. Chem. Soc. 1986; 108: 2093-2094Crossref Scopus (3272) Google Scholar, 23.Parella T. Sanchez-Ferrando F. Virgili A. J. Mag. Reson. A. 1995; 112: 241-245Crossref Scopus (29) Google Scholar) experiment for the GD3-FCHASE sample. The nano-NMR probe, which is a high resolution, magic angle spinning probe, produces high resolution spectra of liquid samples dissolved in only 40 μl (24.Manzi A. Salimath P.V. Spiro R.C. Keifer P.A. Freeze H.H. J. Biol. Chem. 1995; 270: 9154-9163Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The GD3-FCHASE sample (mass = 1486.33 Da) was prepared by lyophilizing the original 0.6-ml sample (200 nmol) and dissolving it in 40 μl of D2O for a final concentration of 5 mm. The final pH of the sample could not be measured. The gradient HMBC experiment was done at a spin rate of 2990 Hz, 400 increments of 1024 complex points, 128 scans per increment, acquisition time of 0.21 s, 1J(C,H) = 140 Hz, and n J(C,H) = 8 Hz, for a duration of 18.5 h. All mass measurements were obtained using a Perkin-Elmer Biosystems (Fragmingham, MA) Elite-STR MALDI-TOF instrument. Approximately 2 μg of each oligosaccharide was mixed with a matrix containing a saturated solution of dihydroxybenzoic acid. Positive and negative mass spectra were acquired using the reflector mode. Before the cloning of the glycosyltransferases, we examined C. jejuni OH4384 and NCTC 11168 cells for various enzymatic activities. When an enzyme activity was detected, we then optimized the assay conditions (described under "Experimental Procedures") to ensure maximal activity. The capillary electrophoresis assay we employed was extremely sensitive and allowed detection of enzyme activity in the microunits/ml range (19.Gilbert M. Watson D.C. Cunningham A.-M. Jennings M.P. Young N.M. Wakarchuk W.W. J. Biol. Chem. 1996; 271: 28271-28276Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). We examined both the sequenced strain NCTC 11168 and the Guillain-Barré syndrome åssociated strain OH4384 for the enzymes required for the GT1a ganglioside mimic synthesis. As predicted, strain OH4384 possessed the enzyme activities required for the synthesis of this structure: β-1,4-N-acetylgalactosaminyltransferase, β-1,3-galactosyltransferase, α-2,3-sialyltransferase, and α-2,8-sialyltransferase. The genome strain, NCTC 11168, lacked the β-1,3-galactosyltransferase and the α-2,8-sialyltransferase activities. Since the LOS structure for NCTC 11168 has not yet been reported, we do not yet know if the presence or absence of a particular enzyme activity in this strain correlates with the structure of its LOS outer core oligosaccharide. A plasmid library made from an unfractionated partial HindIII digestion of chromosomal DNA from C. jejuni OH4384 yielded 2,600 white colonies which were picked to form pools of 100. We used a "divide and conquer" screening protocol from which two positive clones were obtained and designated pCJH9 (5.3-kb insert, 3 HindIII sites) and pCJH101 (3.9-kb insert, 4 HindIII sites). ORF analysis and PCR reactions with C. jejuni OH4384 chromosomal DNA (data not shown) indicated that pCJH9 contained inserts that were not contiguous in the chromosomal DNA. The sequence downstream of nucleotide 1440 in pCJH9 was not further studied, while the first 1439 nucleotides were found to be completely contained within the sequence of pCJH101. The ORF analysis and PCR reactions with chromosomal DNA indicated that all of the pCJH101 HindIII fragments were contiguous in C. jejuni OH4384 chromosomal DNA. Four ORFs, two partial and two complete, were found in the sequence of pCJH101 (Fig. 2). The first 812 nucleotides encode a polypeptide that is 69% identical with the last 265 amino acid (aa) residues of the peptide chain release factor RF-2 (prfB gene, GenBank AE000537) from Helicobacter pylori. The last base of the TAA stop codon of the chain release factor is also the first base of the ATG start codon of an open reading frame that spans nucleotides 812–2104 in pCJH101. This ORF was designated cst-I( C ampylobacter sialyltransferase I) and encodes a 430-aa polypeptide that is homologous with a putative ORF fromHaemophilus influenzae (GenBank accession no. U32720). The putative H. influenzae ORF encodes a 231-aa polypeptide that is 39% identical to the middle region of the Cst-I polypeptide (aa residues 80–330). The sequence downstream of cst-I includes an ORF and a partial ORF that encode polypeptides that are homologous (>60% identical) with the two subunits, CysD and CysN, of theE. coli sulfate adenylyltransferase (GenBank accession no.AE000358). In order to confirm that the cst-I ORF encodes sialyltransferase activity, we subcloned it and overexpressed it inE. coli. The expressed enzyme was used to add sialic acid to Gal-β-1,4-Glc-β-FCHASE (Lac-FCHASE). This product (GM3-FCHASE) was analyzed by NMR to confirm the Neu5Ac-α-2,3-Gal linkage specificity of Cst-I (see text below, Table I, and Figs. 3 and 5).Table IProton NMR chemical shifts for the fluorescent derivatives of the ganglioside mimics synthesized using the cloned glycosyltransferasesResidueChemical shift (ppm)aProton NMR chemical shifts are given in ppm from HSQC spectrum obtained at 600 MHz, D2O, pH 7, 28 °C for Lac-, 25 °C for GM3-, 16 °C for GM2-, 24 °C for GM1a-, and 24 °C GD3-FCHASE. The methyl resonance of internal acetone is at 2.225 ppm (1H). The error is ±0.02 ppm for 1H chemical shifts and ±5 °C for the sample temperature. The error is ±0.1 ppm for the H-6 resonances of residues a, b, d, and e due to overlap.HLac-GM3-GM2-GM1a-GD3-βGlc (a)14.574.704.734.764.7623.233.323.273.303.3833.473.543.563.583.5743.373.483.393.433.5653.303.443.443.463.5063.733.813.803.813.856′3.223.383.263.353.50βGal(1–4) (b)14.324.434.424.444.4623.593.603.393.393.6033.694.134.184.184.1043.973.994.174.174.0053.813.773.843.833.7863.863.813.793.783.786′3.813.783.793.783.78αNeu5Ac(2–3) (c)3ax1.811.971.961.783eq2.762.672.682.6743.693.783.793.6053.863.843.833.8263.653.493.513.6873.593.613.603.8783.913.773.774.1593.883.903.894.189′3.653.633.643.74NAc2.032.042.032.07βGalNAc(1–4) (d)14.774.8123.944.0733.703.8243.934.1853.743.7563.863.846′3.863.84NAc2.042.04βGal(1–3) (e)14.5523.5333.6443.9253.6963.786′3.74αNeu5Ac(2–8) (f)3ax1.753eq2.7643.6653.8263.6173.5883.9193.889′3.64NAc2.02a Proton NMR chemical shifts are given in ppm from HSQC spectrum obtained at 600 MHz, D2O, pH 7, 28 °C for Lac-, 25 °C for GM3-, 16 °C for GM2-, 24 °C for GM1a-, and 24 °C GD3-FCHASE. The methyl resonance of internal acetone is at 2.225 ppm (1H). The error is ±0.02 ppm for 1H chemical shifts and ±5 °C for the sample temperature. The error is ±0.1 ppm for the H-6 resonances of residues a, b, d, and e due to overlap. Open table in a new tab Figure 5Identification of the glycosidation site from a comparison of the HSQC spectra of FCHASE-glycosides. The two spectra of the precursor and enzymatic product are overlaid, except for GD3, which is plotted with GM3. Only one contour is drawn for each spectrum. The two cross-peaks for the same common atom in the two compounds are indicated by boxes around the two cross-peaks or arrows. The linkage site is identified from the large13C downfield glycosidation shift, indicated byfilled cross-peaks. The proton chemical shift axis is F2, and the 13C chemical shift axis is F1. Cross-peaks are labeled using a for Glc, b for Gal(1–4), c for Neu5Ac(2–3), d for GalNAc(1–4), e for Gal(1–3), and f for Neu5Ac(2–8), and by the atom number as in Tables I and III.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of the sequence data available at the website of the C. jejuni NCTC 11168 sequencing group (Sanger Centre) revealed that the two heptosyltransferases involved in the synthesis of the inner core of the LPS were readily identifiable by sequence homology with other bacterial heptosyltransferases. The region between the two heptosyltransferases spans 13.49 kb in NCTC 11168 and includes at least seven potential glycosyltransferases based on BLAST searches in GenBank. Since no structure is available for the LOS outer core of NCTC 11168, it was impossible to suggest functions for the putative glycosyltransferase genes in that strain. Based on conserved regions in the heptosyltransferases sequences, we designed primers (CJ-42 and CJ-43) to amplify the region between them. We obtained a PCR product of 13.49 kb using chromosomal DNA fromC. jejuni NCTC 11168 and a PCR product of 11.47 kb using chromosomal DNA from C. jejuni OH4384. The size of the PCR product from strain NCTC 11168 was consistent with the Sanger Center data. The smaller size of the PCR product from strain OH4384 indicated heterogeneity between the strains in the region between the two heptosyltransferase genes and suggested that the genes for some of the glycosyltransferases specific to strain OH4384 could be present in that location. We sequenced the 11.47-kb PCR product using a combination of primer walking and subcloning of HindIII fragments (GenBank accession no. AF130984). The G/C content of the DNA was 27%, typical of DNA from Campylobacter. Analysis of the sequence suggests the presence of 11 complete ORFs in addition to the two partial ORFs encoding the two heptosyltransferases (Fig. 2, TableII). When comparing the deduced amino acid sequences, we found that the two strains share six genes that are above 80% identical and four genes that are between 52 and 68% identical (Table II). Four genes are unique to C. jejuniNCTC 11168, while one gene is unique to C. jejuni OH4384 (Fig. 2). Two genes that are