Title: The Genetic Bases for the Variation in the Lipo-oligosaccharide of the Mucosal Pathogen, Campylobacter jejuni
Abstract: We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejunistrains expressing a total of 8 different ganglioside mimics in their LOS outer cores. Based on the organization of the genes, the 11 corresponding loci could be classified into three classes, with one of them being clearly an intermediate evolutionary step between the other two. Comparative genomics and expression of specific glycosyltransferases combined with in vitro activity assays allowed us to identify at least five distinct mechanisms that allowC. jejuni to vary the structure of the LOS outer core as follows: 1) different gene complements; 2) phase variation because of homopolymeric tracts; 3) gene inactivation by the deletion or insertion of a single base (without phase variation); 4) single mutation leading to the inactivation of a glycosyltransferase; and 5) single or multiple mutations leading to "allelic" glycosyltransferases with different acceptor specificities. The differences in the LOS outer core structures expressed by the 11 C. jejuni strains examined can be explained by one or more of the five mechanisms described in this work. We have compared the lipo-oligosaccharide (LOS) biosynthesis loci from 11 Campylobacter jejunistrains expressing a total of 8 different ganglioside mimics in their LOS outer cores. Based on the organization of the genes, the 11 corresponding loci could be classified into three classes, with one of them being clearly an intermediate evolutionary step between the other two. Comparative genomics and expression of specific glycosyltransferases combined with in vitro activity assays allowed us to identify at least five distinct mechanisms that allowC. jejuni to vary the structure of the LOS outer core as follows: 1) different gene complements; 2) phase variation because of homopolymeric tracts; 3) gene inactivation by the deletion or insertion of a single base (without phase variation); 4) single mutation leading to the inactivation of a glycosyltransferase; and 5) single or multiple mutations leading to "allelic" glycosyltransferases with different acceptor specificities. The differences in the LOS outer core structures expressed by the 11 C. jejuni strains examined can be explained by one or more of the five mechanisms described in this work. lipo-oligosaccharides cytidine monophosphate-N-acetylneuraminic acid 6-(5-fluorescein-carboxamido)-hexanoic acid succimidyl ester open reading frame heptose nucleotide. The abbreviated designations of glycolipids are according to IUPAC-IUC nomenclature Many pathogenic bacteria have variable cell-surface glycoconjugates such as capsules in Streptococcus spp. andNeisseria meningitidis (1Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (515) Google Scholar), lipopolysaccharides in Gram-negative bacteria (2Preston A. Mandrell R.E. Gibson B.W. Apicella M.A. Crit. Rev. Microbiol. 1996; 22: 139-180Crossref PubMed Scopus (235) Google Scholar), and glycosylated surface-layer proteins (3Castric P. Cassels F.J. Carlson R.W. J. Biol. Chem. 2001; 276: 26479-26485Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). In mucosal pathogens, the variability of cell-surface polysaccharides has been shown to play a major role in virulence (4Moxon E.R. Kroll J.S. Curr. Top. Microbiol. Immunol. 1990; 150: 65-85PubMed Google Scholar). This variation is caused by the diversity of monosaccharide components and the linkages between them, derivatization with noncarbohydrate moieties, and in some cases, by the length and sequence of the repeating units. The variation of these glycan structures can sometimes be correlated with a specific gene complement, but it is probable that other genetic mechanisms are also employed to create variable cell-surface glycoconjugates. The DNA sequencing of the relevant genetic loci from multiple strains of a pathogen can provide insights into the genetic origins of important strain variable traits such as cell-surface glycoconjugates. The mucosal pathogen Campylobacter jejuni has been recognized as an important cause of acute gastroenteritis in humans (5Allos B.M. Clin. Infect. Dis. 2001; 32: 1201-1206Crossref PubMed Scopus (863) Google Scholar) and has been shown to have variable cell-surface carbohydrates that are associated with virulence (6Bacon D.J. Szymanski C.M. Burr D.H. Silver R.P. Alm R.A. Guerry P. Mol. Microbiol. 2001; 40: 769-777Crossref PubMed Scopus (226) Google Scholar, 7Guerry P. Szymanski C.M. Prendergast M.M. Hickey T.E. Ewing C.P. Pattarini D.L. Moran A.P. Infect. Immun. 2002; (in press)Google Scholar). Epidemiological studies have shown that Campylobacter infections are more common thanSalmonella infections in developed countries, and they are also an important cause of diarrheal diseases in developing countries.C. jejuni is also considered the most frequent antecedent infection to the development of Guillain-Barré syndrome, a form of neuropathy that is the most common cause of generalized paralysis since the eradication of poliomyelitis in developed countries (8Jacobs B.C. Rothbarth P.H. van der Meché F.G.A. Herbrink P. Schmitz P.I.M. de Klerk M.A. van Doorn P.A. Neurology. 1998; 51: 1110-1115Crossref PubMed Scopus (618) Google Scholar). The core oligosaccharides of low molecular weight lipo-oligosaccharides (LOS)1 of many C. jejuni strains have been shown to exhibit molecular mimicry of the carbohydrate moieties of gangliosides (Fig. 1). Terminal oligosaccharides identical to those of GM1a, GM2, GM3, GD1a, GD1c, GD3, and GT1a gangliosides have all been found in various C. jejuni strains (see Table I for references). Molecular mimicry of host structures by the saccharide portion of LOS is considered to be a virulence factor of various mucosal pathogens, which may use this strategy to evade the immune response (9Moran A.P. Prendergast M.M. Appelmelk B.J. FEMS Immunol. Med. Microbiol. 1996; 16: 105-115Crossref PubMed Google Scholar). The molecular mimicry between C. jejuni LOS outer core structures and gangliosides has also been suggested to act as a trigger for autoimmune mechanisms in the development of Guillain-Barré syndrome (10Yuki N. Handa S. Taki T. Kasama T. Takahashi M. Saito K. Miyatake T. Biomed. Res. 1992; 13: 451-453Crossref Scopus (87) Google Scholar).Table IDescription of the C. jejuni strains used in this workStrain no.Penner serotypeLOS outercore type1-aSee Fig. 1 for the description of the structures.(ganglioside mimic)Reference for the LOS outer core structureLOS biosynthesis locus class (GenBank™ accession no.)ATCC 43438O:10 (type strain)I (GD1c) 15Nam Shin J.E. Ackloo S. Mainkar A.S. Monteiro M.A. Pang H. Penner J.L. Aspinall G.O. Carbohydr. Res. 1998; 305: 223-232Crossref Scopus (43) Google Scholar A (AF400048)ATCC 43432O:4 (type strain)II (GD1a) 11Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Moran A.P. Penner J.L. Eur. J. Biochem. 1993; 213: 1017-1027Crossref PubMed Scopus (118) Google Scholar A (AF215659)ATCC 43446O:19 (type strain)II (GD1a) 13Aspinall G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Biochemistry. 1994; 33: 241-249Crossref PubMed Scopus (171) Google Scholar A (AF167344)OH4384 (GBS strain)O:19III (GT1a) 13Aspinall G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Biochemistry. 1994; 33: 241-249Crossref PubMed Scopus (171) Google Scholar A (AF130984)OH4382 (GBS strain)O:19IV (GD3) 13Aspinall G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Biochemistry. 1994; 33: 241-249Crossref PubMed Scopus (171) Google Scholar A (AF167345)ATCC 43460O:41 (type strain)?None A (AY044868)ATCC 43449O:23 (type strain)V (GM2) 11Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Moran A.P. Penner J.L. Eur. J. Biochem. 1993; 213: 1017-1027Crossref PubMed Scopus (118) Google Scholar B (AF401529)ATCC 43456O:36 (type strain)V (GM2) 11Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Moran A.P. Penner J.L. Eur. J. Biochem. 1993; 213: 1017-1027Crossref PubMed Scopus (118) Google Scholar B (AF401528)NCTC 11168 ("Genome" strain)O:2VI (GM1a) 22Linton D. Gilbert M. Hitchen P.G. Dell A. Morris H.R. Wakarchuk W.W. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 37: 501-514Crossref PubMed Scopus (169) Google Scholar1-bLinton et al. (22) and St. Michael, F., Gilbert, M., Syzmanski, C., Chan, K. H., Wakaschuk, W. W., and Monteiro, N., unpublished data. C (AL139077)ATCC 43429O:1 (type strain)VII (GM2) 11Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Moran A.P. Penner J.L. Eur. J. Biochem. 1993; 213: 1017-1027Crossref PubMed Scopus (118) Google Scholar C (AY044156)ATCC 43430O:2 (type strain)VIII (GM3) 12Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Kurjanczyk L.A. Penner J.L. Moran A.P. Eur. J. Biochem. 1993; 213: 1029-1037Crossref PubMed Scopus (72) Google Scholar C (AF400047)1-a See Fig. 1 for the description of the structures.1-b Linton et al. (22Linton D. Gilbert M. Hitchen P.G. Dell A. Morris H.R. Wakarchuk W.W. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 37: 501-514Crossref PubMed Scopus (169) Google Scholar) and St. Michael, F., Gilbert, M., Syzmanski, C., Chan, K. H., Wakaschuk, W. W., and Monteiro, N., unpublished data. Open table in a new tab Aspinall et al. (11Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Moran A.P. Penner J.L. Eur. J. Biochem. 1993; 213: 1017-1027Crossref PubMed Scopus (118) Google Scholar, 12Aspinall G.O. McDonald A.G. Raju T.S. Pang H. Kurjanczyk L.A. Penner J.L. Moran A.P. Eur. J. Biochem. 1993; 213: 1029-1037Crossref PubMed Scopus (72) Google Scholar, 13Aspinall G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Biochemistry. 1994; 33: 241-249Crossref PubMed Scopus (171) Google Scholar, 14Aspinall G.O. Lynch C.M. Pang H. Shaver R.T. Moran A.P. Eur. J. Biochem. 1995; 231: 570-578Crossref PubMed Scopus (76) Google Scholar) and Nam Shin et al. (15Nam Shin J.E. Ackloo S. Mainkar A.S. Monteiro M.A. Pang H. Penner J.L. Aspinall G.O. Carbohydr. Res. 1998; 305: 223-232Crossref Scopus (43) Google Scholar) determined the LOS outer core structures of representative C. jejuni reference strains of the Penner serotyping system. The Penner serotyping system of C. jejuni is based on heat-stable antigens, and it was proposed that the specificity is due to LOS and/or lipopolysaccharide-type molecules (16Penner J.L. Hennessy J.N. Congi R.V. Eur. J. Clin. Microbiol. 1983; 2: 378-383Crossref PubMed Scopus (135) Google Scholar, 17Moran A.P. Penner J.L. J. Appl. Microbiol. 1999; 86: 361-377Crossref PubMed Scopus (50) Google Scholar). However, recent biochemical and genetic studies suggest that capsular polysaccharides account for Penner serotype specificity (6Bacon D.J. Szymanski C.M. Burr D.H. Silver R.P. Alm R.A. Guerry P. Mol. Microbiol. 2001; 40: 769-777Crossref PubMed Scopus (226) Google Scholar, 18Karlyshev A.V. Linton D. Gregson N.A. Lastovica A.J. Wren B.W. Mol. Microbiol. 2000; 35: 529-541Crossref PubMed Scopus (189) Google Scholar). Because the loci responsible for capsule and LOS biosynthesis are distant in the C. jejuni genome (19Parkhill J. Wren B.W. Mungall K. Ketley J.M. Churcher C. Basham D. Chillingworth T. Davies R.M. Feltwell T. Holroyd S. Jagels K. Karlyshev A.V. Moule S. Pallen M.J. Penn C.W. Quail M.A. Rajandream M.-A. Rutherford K.M. van Vliet A.H.M. Whitehead S. Barrell B.G. Nature. 2000; 403: 665-669Crossref PubMed Scopus (1535) Google Scholar) and intraspecies gene transfers are known to be frequent in C. jejuni (20Dingle K.E. Colles F.M. Wareing D.R.A. Ure R. Fox A.J. Bolton F.E. Bootsma H.J. Willems R.J.L. Urwin R. Maiden M.C.J. J. Clin. Microbiol. 2001; 39: 14-23Crossref PubMed Scopus (653) Google Scholar, 21Suerbaum S. Lohrengel M. Sonnevend A. Ruberg F. Kist M. J. Bacteriol. 2001; 183: 2553-2559Crossref PubMed Scopus (118) Google Scholar), it is possible that strains having the same Penner type could express different LOS outer cores. Consequently, we decided to associate the published LOS outer core structures (Fig. 1) with the specific strain identification numbers (ATCC, NCTC, etc.) rather than with the Penner types, although the latter are also provided for convenient reference (Table I). The identification of the genes involved in LOS synthesis and the study of their regulation are of considerable interest for a better understanding of the pathogenesis mechanisms used by these bacteria. The availability of the complete genome sequence of C. jejuni NCTC 11168 (19Parkhill J. Wren B.W. Mungall K. Ketley J.M. Churcher C. Basham D. Chillingworth T. Davies R.M. Feltwell T. Holroyd S. Jagels K. Karlyshev A.V. Moule S. Pallen M.J. Penn C.W. Quail M.A. Rajandream M.-A. Rutherford K.M. van Vliet A.H.M. Whitehead S. Barrell B.G. Nature. 2000; 403: 665-669Crossref PubMed Scopus (1535) Google Scholar) has facilitated the identification of loci involved in the biosynthesis of cell-surface carbohydrates including LOS (22Linton D. Gilbert M. Hitchen P.G. Dell A. Morris H.R. Wakarchuk W.W. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 37: 501-514Crossref PubMed Scopus (169) Google Scholar, 23Linton D. Karlyshev A.V. Hitchen P.G. Morris H.R. Dell A. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 35: 1120-1134Crossref PubMed Scopus (117) Google Scholar). The genome sequence was also used to clone the corresponding LOS biosynthesis locus in other C. jejunistrains (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 25Guerry P. Ewing C.P. Hickey T.E. Prendergast M.M. Moran A.P. Infect. Immun. 2000; 68: 6656-6662Crossref PubMed Scopus (109) Google Scholar), which allowed the identification of genes involved in the transfer of Gal, GalNAc, and N-acetylneuraminic acid (Neu5Ac or sialic acid) to the LOS outer core. Because cell-surface structures such as the LOS are recognized as antigens by the host, it is therefore not surprising that microorganisms will modulate these structures to increase the chances of evading the immune system. The C. jejuni strains used in this study were shown to express a total of 8 different sialylated LOS outer cores (Fig. 1 and see Table I for references). The LOS biosynthesis loci of C. jejuni OH4384 and C. jejuni NCTC 11168 were found to have common genes as well genes unique to each strain (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), which provide a basis for differences in LOS outer cores. However, mechanisms other than differences in gene complement are involved in generating a variety of LOS outer cores. In the strain C. jejuni OH4382, the gene involved in the transfer of the GalNAc residue of the LOS outer core was shown to be inactive (a missing A nucleotide causes a premature translation stop). This results in the expression of a truncated LOS outer core when compared with strain OH4384 (13Aspinall G.O. McDonald A.G. Pang H. Kurjanczyk L.A. Penner J.L. Biochemistry. 1994; 33: 241-249Crossref PubMed Scopus (171) Google Scholar, 24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Parkhill et al. (19Parkhill J. Wren B.W. Mungall K. Ketley J.M. Churcher C. Basham D. Chillingworth T. Davies R.M. Feltwell T. Holroyd S. Jagels K. Karlyshev A.V. Moule S. Pallen M.J. Penn C.W. Quail M.A. Rajandream M.-A. Rutherford K.M. van Vliet A.H.M. Whitehead S. Barrell B.G. Nature. 2000; 403: 665-669Crossref PubMed Scopus (1535) Google Scholar) showed that short homopolymeric nucleotide runs of variable length are commonly found in genes involved in the biosynthesis of C. jejuni carbohydrates, which provides a form of on/off regulation of these genes. Linton et al. (22Linton D. Gilbert M. Hitchen P.G. Dell A. Morris H.R. Wakarchuk W.W. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 37: 501-514Crossref PubMed Scopus (169) Google Scholar) studied in detail a gene encoding a β-1,3-galactosyltransferase that occurs with either an 8- or a 9-G nucleotide tract which results in the expression of either a GM1a or a GM2 ganglioside mimic in C. jejuni NCTC 11168. We reported previously that the cst-II gene occurs as a mono-functional α-2,3-sialyltransferase in C. jejuni ATCC 43446 (O:19 serostrain) and as a bi-functional α-2,3-/α-2,8-sialyltransferase in C. jejuni OH4384 that results in the expression of either a GD1a or GT1a mimic, respectively (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). In this work we describe the mechanisms used by C. jejuni to generate various sialylated outer core structures. In addition to reporting other examples of on/off expression of genes due to variable homopolymeric tracts, we use enzymatic assays to show that amino acid substitutions are responsible for the expression of glycosyltransferases with different substrate specificities, a "strategy" that further expands the ability of C. jejunito express various LOS outer cores. The C. jejuni strains used in this study are listed in Table I. The Penner type strains were obtained from the American Type Culture Collection. C. jejuni OH4382, OH4384 and NCTC 11168 were obtained from the Laboratory Center for Disease Control (Health Canada, Winnipeg, Manitoba, Canada). C. jejuni strains were grown on Mueller-Hinton medium under microaerobic conditions. Escherichia coli AD202 (CGSG 7297) was used to express the different cloned glycosyltransferases and was grown using 2YT agar or broth. The recombinant E. colistrains were incubated at 25 °C for a total of 24 h, with induction with 1 mmisopropyl-1-thio-β-d-galactopyranoside after 6 h forcgtA constructs and with 0.3 mmisopropyl-1-thio-β-d-galactopyranoside after 4.5 h for cst-II constructs. Genomic DNA isolation from theC. jejuni strains was performed using the DNeasy Tissue kit (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 PCRs (>2 kb) were performed using the ExpandTM long template PCR system as described by the manufacturer (Roche Molecular Biochemicals). PCRs to amplify specific ORFs were performed using thePwo DNA polymerase as described by the manufacturer (Roche Molecular Biochemicals). Restriction and DNA modification enzymes were purchased from MBI Fermentas Inc. (Hanover, MD). Site-directed mutagenesis of cst-II was performed using a two stage PCR mutagenesis protocol. Two separate PCR reactions were performed to generate two overlapping gene fragments that both contained the mutation due to either the 5′ or the 3′ primers. The two PCR products were used with the cst-II 5′ and 3′ primers to amplify the full-length mutated version of cst-II. The DNA sequences of the LOS biosynthesis loci of C. jejuni NCTC 11168 (GenBankTM accession number AL139077) and OH4384 (GenBankTM accession number AF130984) were used to design primers to amplify the LOS biosynthesis loci of the other strains described in this work. The primers were designed to obtain overlapping PCR products of 2–5 kb that covered completely each of the LOS locus. The PCR products were sequenced by "primer walking," and new primers were synthesized to amplify and sequence the regions that diverge significantly from the NCTC 11168 and OH4384 sequences. DNA sequencing was performed using an Applied Biosystems (Montreal) model 373 automated DNA sequencer and the manufacturer's cycle sequencing kit. Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce). FCHASE-labeled oligosaccharides were prepared as described previously (26Wakarchuk 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). Extracts were made by sonication, and the enzymatic reactions were performed at 37 °C for 5 min to 2 h. The β-1,4-N-acetylgalactosaminyltransferase was assayed using 0.5 mm Neu5Acα-2,3-Galβ-1,4-Glc-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 Gal-β-1,4-Glc-FCHASE, 0.2 mm CMP-Neu5Ac, 50 mm Hepes, pH 7.5, and 10 mm MgCl2. The α-2,8-sialyltransferase was assayed using 0.5 mm Neu5Acα-2,3-Galβ-1,4-Glc-FCHASE, 0.2 mm CMP-Neu5Ac, 50 mm Hepes, pH 7.5, and 10 mm MgCl2. The CMP-Neu5Ac synthetase was assayed using CTP, Neu5Ac, Gal-β1,4-GlcNAc-FCHASE, and a purified fusion of the N. meningitidis α-2,3-sialyltransferase (MalE-NST) 2M. Gilbert, M.-F. Karwaski, S. Bernatchez, N. M. Young, E. Taboada, J. Michniewicz, A.-M. Cunningham, and W. W. Wakarchuk, unpublished data. in a coupled assay that measured the production of Neu5Acα-2,3-Gal-β1,4-GlcNAc-FCHASE. The reaction mix included 0.5 mm Gal-β-1,4-GlcNAc-FCHASE, 3 mm CTP, 3 mm Neu5Ac, 4 milliunits of α-2,3-sialyltransferase (MalE-NST), 100 mm Tris, pH 7.5, 10 mmMgCl2, and 0.2 mm dithiothreitol. All the reactions were stopped by the addition of acetonitrile (25% final concentration) and were diluted with H2O to get 10–15 μm final concentration of the FCHASE-labeled compounds. The samples were analyzed by capillary electrophoresis performed using the separation and detection conditions as described previously (27Gilbert 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 (144) Google Scholar). The peaks from the electropherograms were analyzed using manual peak integration with the P/ACE Station software. We have compared the LOS biosynthesis loci of 11C. jejuni strains (TableI) that include 7 previously unpublished loci and extend our previous limited comparison of 4C. jejuni strains that included 3 closely related O:19 strains (OH4382, OH4384, and ATCC 43446, the O:19 serostrain) and the genome strain NCTC 11168 (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The LOS outer core structures were published for 10 of the 11 strains included in this study (Fig.1 and Table I). The general organization of the LOS biosynthesis genes allows us to group these C. jejuni strains into three classes "A," "B," and "C" (see Fig. 2). The LOS biosynthesis loci of the six class A strains have 13 ORFs, whereas the LOS biosynthesis loci of the two class B strains and of the three class C strains have 14 ORFs. One gene (orf11) is found only in classes A and B, whereas three genes are unique to class C (orf14, orf15, and orf16). Proposed functions for each ORF are described in TableII.Table IIProposed functions for the LOS biosynthesis locus ORFs (see Fig. 2 for position) and summary of the sequence comparison and experimental evidenceORFGeneProposed functionEvidence by sequence homology and/or experimental evidence1Cj11332-aThe CjXXXX numbers correspond to the gene numbering of the "genome" strain (NCTC 11168, www.sanger.ac.uk/Projects/C_jejuni/). Other gene nomenclature found in the literature is also indicated.waaCHeptosyltransferase IHomology with RfaC and complementation (36Klena J.D. Gray S.A. Konkel M.E. Gene (Amst.). 1998; 222: 177-185Crossref PubMed Scopus (26) Google Scholar)2Cj1134htrBLipid A biosynthesis acyltransferaseHomology with WaaM3Cj1135Two-domain glucosyltransferase: β-1,4-glucosyltransferase (N- terminal domain) and β-1,2- glucosyltransferase (C-terminal domain)The N-terminal domain is homologous with LgtF and the C-terminal domain with various glycosyltransferases. Premature translation stop in the C terminus of all the strains that don't have a β-1,2-glucose residue2-bThis work.4Cj1136β-1,3-GalactosyltransferaseHomology with various galactosyltransferases5Cj1143cgtAβ-1,4-N- Acetylgalactosaminyltransferase (to Gal)In vitro activity (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and knock-out mutant (25Guerry P. Ewing C.P. Hickey T.E. Prendergast M.M. Moran A.P. Infect. Immun. 2000; 68: 6656-6662Crossref PubMed Scopus (109) Google Scholar)6Cj1139c cgtBβ-1,3-Galactosyltransferase (to GalNAc)Homology with various galactosyltransferases In vitro activity (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and knock-out mutant (22Linton D. Gilbert M. Hitchen P.G. Dell A. Morris H.R. Wakarchuk W.W. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 37: 501-514Crossref PubMed Scopus (169) Google Scholar)7Cj1140 cst-II cst-IIIα-2,3 or α-2,3/α-2,8-sialyltransferase (to Gal/Neu5Ac)In vitro activity (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) and knock-out mutant (25Guerry P. Ewing C.P. Hickey T.E. Prendergast M.M. Moran A.P. Infect. Immun. 2000; 68: 6656-6662Crossref PubMed Scopus (109) Google Scholar)8Cj1141 neuB1Sialic acid synthaseHomology with various sialic acid synthases and complementation (23Linton D. Karlyshev A.V. Hitchen P.G. Morris H.R. Dell A. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 35: 1120-1134Crossref PubMed Scopus (117) Google Scholar)9Cj1142neuC1N-Acetylglucosamine-6-phosphate 2-epimeraseHomology with variousN-acetylglucosamine-6- phosphate 2-epimerase and knock-out mutant (25Guerry P. Ewing C.P. Hickey T.E. Prendergast M.M. Moran A.P. Infect. Immun. 2000; 68: 6656-6662Crossref PubMed Scopus (109) Google Scholar)10Cj1143CMP-Neu5Ac synthetaseHomology with various CMP-Neu5Ac synthetase and in vitroactivity2-bThis work.11Putative acetyltransferaseHomology with various acetyltransferases12Cj1146cPutative glycosyltransferaseHomology with various glycosyltransferases13Cj1148waaFHeptosyltransferase IIHomology with RfaF14Cj1137cPutative glycosyltransferaseHomology with various glycosyltransferases15Cj1138Putative glycosyltransferaseHomology with various glycosyltransferases16Cj1145cPutative ORF2-a The CjXXXX numbers correspond to the gene numbering of the "genome" strain (NCTC 11168, www.sanger.ac.uk/Projects/C_jejuni/). Other gene nomenclature found in the literature is also indicated.2-b This work. Open table in a new tab The 11.5-kb DNA sequences of the LOS loci from the six class A strains can be aligned with only minor gaps, the longest being 6 bp. The overall DNA sequence identity is 91% between the six A strains. However, the level of conservation observed in pairwise alignments varies considerably. As reported previously (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) the three O:19 strains (ATCC 43446, OH4382, and OH4384) are closely related. There is only one base difference (a missing A at position 71 oforf5) between the LOS locus of OH4382 and OH4384. There are 68 base differences (20 amino acid differences) between ATCC 43446 (O:19 serostrain) and OH4384. The LOS locus from C. jejuniATCC 43438 (O:10 serostrain) is primarily responsible for decreasing the overall degree of conservation among the A class strains. When the ATCC 43438 strain is excluded from the class A alignment, the overall DNA sequence identity increases to 96.5%. The highest level of divergence between the LOS locus of ATCC 43438 and the other class A strains is found between nt 4500 and 5700 (66% DNA sequence identity), a region that spans both the orf5 and orf6 which encode a β-1,4-N-acetylgalactosaminyltransferase and a β-1,3-galactosyltransferase, respectively. The 12.4-kb LOS biosynthesis locus of the two class B strains (ATCC 43449, the O:23 serostrain, and ATCC 43456, the O:36 serostrain) shows 95.2% DNA sequence identity in a full-length pairwise alignment. However, the sequence identity is only 65.3% in the region from nt 4500 to 5700, whereas it is above 98% in the rest of the locus. It is noticeable that this region corresponds to the same region that was found to diverge considerably between ATCC 43438 and the other class A strains. In fact, ATCC 43438 and ATCC 43449 share 98% DNA sequence identity in the nt 4500–5700 region, whereas the other class A strains and ATCC 43456 share 99% DNA identity in that region. Class B appears to be an evolutionary intermediate between classes A and C because it has two copies of orf5, with one of them (orf5-I) more similar to orf5 from class A (96% DNA sequence identity) and the second copy (orf5-II) more similar to orf5 from class C (85% DNA sequence identity). The orf5-I in the class B is inactive because of premature translational termination after 28 codons in ATCC 43449 and after 86 codons in ATCC 43456. Transcription reinitiation of orf5-Iwould theoretically be possible, but a similar frameshift mutation was described in orf5 of OH4382 and resulted in the expression of a truncated LOS, consistent with the absence of active β-1,4-N-acetylgalactosaminyltransferase (24Gilbert M. Brisson J.-R. Karwaski M.-F. Michniewicz J. Cunningham A.-M. Wu Y. Young N.M. Wakachuk W.W. J. Biol. Chem. 2000; 275: 3896-3906Abstract Full Te