Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81–176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81–176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways. Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81–176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81–176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways. The availability of bacterial genomic sequences has provided unprecedented opportunity for comparative studies. The functional analysis of each respective genome is currently under way and often involves an integrative, multidisciplinary approach that combines bioinformatics, mutagenesis, proteomics, and microarray technologies. Most recently, metabolomics-based analyses, although limited in their number, are becoming established as an additional tool to elucidate gene function (1Weckwerth W. Morgenthal K. Drug Discov. Today. 2005; 10: 1551-1558Crossref PubMed Scopus (237) Google Scholar, 2Weckwerth W. Annu. Rev. Plant Biol. 2003; 54: 669-689Crossref PubMed Scopus (537) Google Scholar). Metabolomics is the characterization of all low molecular weight compounds in a defined biological system and differs from classical metabolism studies by its greater breadth and speed of metabolite analysis. Although recent advances in mass spectrometry (MS), 2The abbreviations used are: MS, mass spectrometry; Bac, 2,4-diamino-2,4,6-trideoxy-α-d-glucopyranose (also known as bacillosamine); CE, capillary electrophoresis; ESMS, electrospray ionization-MS; HILIC, hydrophilic interaction liquid chromatography; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; Pse5NAc7NAc, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (also known as pseudaminic acid); Pse5NAc7Am, 5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid; TEAA, triethylammonium acetate; TOCSY, total correlation spectroscopy; UDP-4-keto-4,6-dideoxy-α-d-GlcNAc, UDP-2-acetamido-2,4,6-trideoxy-α-d-glucose-hexos-4-ulose; UDP-6-deoxy-α-d-GlcNAc4NAc, UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-glucose; UDP-4-keto-4,6-dideoxy-β-l-AltNAc, UDP-2-acetamido-2,4,6-trideoxy-β-l-altrose-hexos-4-ulose; UDP-6-deoxy-β-l-AltNAc4N, UDP-2-acetamido-4-amino-2,4,6-trideoxy-β-l-altrose; UDP-6-deoxy-β-l-AltNAc4NAc, UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altrose; J, coupling constant; δ, chemical shift; SPE, solid-phase extraction; LC, liquid chromatography; Am, acetamidino group. such as the ultimate resolving capability of the Fourier-transform ion cyclotron resonance MS, and also in nuclear magnetic resonance spectroscopy (NMR) technologies, such as the development of higher magnetic field spectrometers and cryogenically cooled probes (cold probes), has greatly facilitated metabolomic analysis (2Weckwerth W. Annu. Rev. Plant Biol. 2003; 54: 669-689Crossref PubMed Scopus (537) Google Scholar), such an undertaking still presents numerous technological challenges due to the complexity of the metabolome. Therefore, to facilitate the elucidation of unknown intermediates involved in novel biosynthetic pathways, we felt it might be more useful to employ a focused approach to simplify the metabolite pool and target relevant compounds. Glycosylation of prokaryotic proteins in both N- and O-linkage with novel glycan components is now well established (3Benz I. Schmidt M.A. Mol. Microbiol. 2002; 45: 267-276Crossref PubMed Scopus (175) Google Scholar, 4Upreti R.K. Kumar M. Shankar V. Proteomics. 2003; 3: 363-379Crossref PubMed Scopus (137) Google Scholar, 5Szymanski C.M. Wren B.W. Nat. Rev. Microbiol. 2005; 3: 225-237Crossref PubMed Scopus (338) Google Scholar). Campylobacter jejuni is unique in having both a general protein glycosylation pathway that is responsible for the N-linked addition of a heptasaccharide containing N-acetylgalactosamine, glucose, and 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose (2,4-diacetamido-Bac) to at least 30 different proteins as well as an O-linked system, which is responsible for glycosylation of the flagellin structural protein at up to 19 sites per monomer with the novel 9 carbon sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (Pse5NAc7NAc) and related derivatives including an acetamidino derivative, PseAm (6Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 7Thibault Kelly Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of is by a and genetic that a of biosynthetic The functional characterization of the protein glycosylation N-linked has that the to the N-linked system E. Chem. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, N. M.A. M. Wren B.W. Mol. Microbiol. 2005; PubMed Scopus Google Scholar). In contrast, the O-linked glycosylation is genes the and a large of hypothetical genes as well as genes in glycan biosynthesis M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar, J. Wren B.W. K. C. K. M.A. M.A. PubMed Scopus Google Scholar, J. N. Wren B.W. 2005; PubMed Scopus Google Scholar). Although the of the O-linked is currently the functional characterization of the biosynthetic pathway responsible for the of and related derivatives has E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). and PseAm were shown to be for and Kelly Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Mol. Microbiol. 2003; PubMed Scopus Google Scholar, Kelly Mol. Microbiol. 2002; PubMed Scopus Google Scholar, Kelly Mol. Microbiol. 2003; PubMed Scopus Google and more the of novel moieties the flagellin was shown to a role in M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar, M. J. Mol. Microbiol. 2003; PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). is that which with the flagellin glycosylation and were to the 2002; PubMed Scopus Google Scholar, M. Chem. Biol. 2003; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). the characterization of the genetic as well as the elucidation of glycan biosynthetic is of the flagellin glycosylation of genes from C. jejuni 81–176 was M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar). analysis that a total of genes from the were involved in flagellin of which in a pseC, pseE, pseF, pseG, and In of genes and in flagellin with Although this a role for genes in the biosynthesis or of the the function of genes remains to be In we developed a focused metabolomics approach to examine related to pseudaminic acid biosynthesis within the metabolome of and C. jejuni M. J. Mol. Microbiol. 2003; PubMed Scopus Google Scholar, Kelly N.M. Chem. PubMed Scopus Google Scholar). Using and precursor ion scanning for of we were to identify CMP-Pse5NAc7NAc and as nucleotide-activated related to the biosynthesis of and PseAm in C. jejuni 81–176 Kelly N.M. Chem. PubMed Scopus Google Scholar). In precursor ion scanning for of the the of and intermediates was in defined mutants, and which proteins in the biosynthetic In this we this by including all the by M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google to the role of genes in the and PseAm pathways. The of the C. jejuni 81–176 and isogenic were for nucleotide-activated relevant to biosynthesis the and precursor ion scanning nucleotide-activated of were from cell lysates a novel hydrophilic interaction liquid The of were with NMR at with a cryogenically cooled probe (cold probe) for Bacterial and C. jejuni 81–176 and isogenic by M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google were in under for at of from C. jejuni 81–176 or pseC, from of were analysis of flagellin glycosylation genes from C. jejuni to the gene in C. jejuni and in which the was in C. jejuni are from or of of the was structural characterization of the flagellin protein A in a in glycosylation Wren B.W. 2002; PubMed Scopus Google to the gene in C. jejuni and in which the was in C. jejuni M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google are from M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google or Wren B.W. 2002; PubMed Scopus Google The of the was structural characterization of the flagellin protein M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar). A in a in glycosylation in a and used for method development of and from and and from were from the cell lysates from were a from liquid and were from triethylammonium and acid were from was from a system were from of were and with in a of were and was by for was to the lysates to a of and the was by a The cell lysates were to a in and for analysis. of from C. is of the for the of biological and the of from biological However, nucleotide-activated are have limited such as and which are found in J. M. N. L. J. PubMed Scopus Google of from and bacterial carbon with the triethylammonium in the permitted of and their was by J. M. N. L. J. PubMed Scopus Google Scholar). Using were from the cell lysates of C. jejuni 81–176 and the isogenic an method that of J. M. N. L. J. PubMed Scopus Google Scholar). the solid-phase were of acid by of lysates were the and with of of and with of triethylammonium of from the was of The were to a to the and in either for analysis or for of and C. jejuni 81–176 were to of and for NMR and precursor ion scanning for related to and or were used as a to the of the for the of the and the of the in the was for the and lysates from and isogenic of C. jejuni 81–176 were for intermediates a and precursor ion scanning method Kelly N.M. Chem. PubMed Scopus Google Scholar). The of an system to a mass with a the an system was to the and were at a of were used method and this was to for for the precursor ion all MS, and were the ion at a of in and a time of A in the of of was for a time of and an of was used for was used for and NMR of were in of and by NMR were in and the was the addition of to the of were in NMR and heteronuclear HSQC, HSQC, HMBC, TOCSY, and sequences from were used for general with a and were used for complete and of coupling and nuclear Overhauser J.R. J. NMR of Scholar, Brisson J.R. NMR in and Scholar). NMR were with a with a resonance cryogenically cooled probe (cold probe) and with a with a resonance NMR were at with of the or resonance at and carbon the resonance of was used as an and from in the flagellin glycosylation of 81–176 were with the by for sugar intermediates of the biosynthetic This mutants, of which in the Pse biosynthetic pathway pseC, and E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), that has as CMP-Pse5NAc7NAc to the acid and of unknown function and M. J. Mol. Microbiol. 2003; PubMed Scopus Google Scholar). were that were flagellin modified by PseAm and and the were with no in flagellin glycosylation as by M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar). results that CMP-Pse5NAc7NAc and intermediates were in as Kelly N.M. Chem. PubMed Scopus Google The first of are by and E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), as in A novel was in the and mass spectrometry as in studies Kelly N.M. Chem. PubMed Scopus Google an ion to the sugar at no intermediates were in the in the genes the known in the pathway and and intermediates, as in and that the The accumulate novel intermediates, and in to the both CMP-Pse5NAc7NAc and were This that the gene is involved in biosynthesis of either of the be involved in either of the glycan moieties to the flagellin protein or at a of the biosynthetic for acid and The of and have by E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that of by J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). biosynthetic for which the respective have still to be The of is to from the sialic acid biosynthetic The that PseAm and pseD, a different metabolic CMP-Pse5NAc7NAc was in the with a role for the gene in the biosynthesis of from In contrast, the both CMP-Pse5NAc7NAc and in to the that is involved in the biosynthesis of In an to the gene be involved in glycan to mutants, and Cj1315c, which no in M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar), in addition to CMP-Pse5NAc7NAc and and intermediates. This that genes a more role in the biosynthetic which remains to be The mutants, Cj1334, Cj1341c, and all of which were M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar), accumulate novel intermediates, and the both CMP-Pse5NAc7NAc and in a to the and with the is a in the of the and the for a of In Kelly Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and PseAm as the the flagellin of Campylobacter C. jejuni 81–176 and Campylobacter Although the of from the flagellin of was by that of PseAm remains to of PseAm from flagellin protein for NMR analysis have and the metabolome an opportunity for the structural analysis of the novel PseAm by C. In this was found to be unique in of the of a metabolite to be directly related to biosynthesis functional characterization of that this is a responsible for the of E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this biosynthetic the of UDP-6-deoxy-β-l-AltNAc4NAc, the of a metabolite within and the unknown metabolite were from the cell lysates of the and pseC, for by and as the a recent that in C. jejuni 81–176 of including of CMP-Pse5NAc7NAc and Kelly N.M. Chem. PubMed Scopus Google Scholar). is to the scale of due to the that are involved in the be more for scale purifications of However, because are well it was to employ an of and is an established for analysis. In the use of liquid with triethylammonium as the was shown to J. M. N. L. J. PubMed Scopus Google Scholar). Although it is to use either of for the of the for and be for the of novel in a cell are mass spectrometry is to identify novel unique compounds W. J. 2005; PubMed Scopus Google and that are to mass spectrometry J. 2005; PubMed Scopus Google Scholar, M.A. Chem. 2005; PubMed Scopus Google Scholar). Therefore, it was felt more to employ in this for the and of the for the isolation of and the from cell were focused a of of involved in biosynthesis pathways. A of was that and were the and Although and the of the was of the as their was the mass and of their was by mass spectrometry the the molecular weight of the in the was it was to identify the of their However, novel biosynthesis pathways, the of the be Therefore, it be useful to of the This was in studies precursor ion scanning for a of the and Kelly N.M. Chem. PubMed Scopus Google Scholar). this precursor ion scanning approach with of provided the to for and from the C. jejuni 81–176 and the isogenic were for the of and the analysis of cell lysates from C. jejuni 81–176 and a of different unknown including and Although it was to to and from the total ion a precursor ion scanning approach was to under the and precursor scanning for related to the of within the cell lysates of C. jejuni 81–176 that were to be The mass to the at and the of CMP-Pse5NAc7NAc and which was by mass spectrometry to of cell of the unknown was with to the for isolation of the A and of in the was to of the metabolite the The for the analysis of was a of A to A in to A at A was and was Using the large scale of and from cell lysates of C. jejuni 81–176 and pseC, were The containing or were a and by as a to the of the NMR analysis. CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic analysis that a a was used to target that are unique to such as and The complete system for CMP-Pse5NAc7Am was and NMR to an of known M.A. Chem. 2005; PubMed Scopus Google Scholar), the of CMP-Pse5NAc7Am was to be of the low of the and the of the of the probe was in the of chemical and coupling for CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid and The chemical for and were in at The chemical for and were in at in a The for the and from the of the and at and to unknown were also within the were used to assign the and to coupling of for and that of was used to assign and of for and an additional coupling of was for that was to A this to be a coupling to the of the of the acetamidino group the was in and the was the addition of Although the of the group was at the and were at of an is at the of at and K. E. Brisson J.R. J. PubMed Scopus Google Scholar). the chemical for the and as well as the carbon chemical for the carbon a were to for acetamidino K. E. Brisson J.R. J. PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, I. Brisson J.R. E. J. PubMed Scopus Google Scholar). of the a correlation to of the sugar of the a correlation to the of the group at and the at These were by the results of and that the and and as well as the and and A correlation was also the at and of the at A that permitted the complete of carbon chemical the chemical of and C7 by and with the of J. PubMed Scopus Google Scholar). These are in with the of an acetamidino which was to a of and L. PubMed Scopus Google Scholar). These results established the of the acetamidino group at C7 of the acid chemical and coupling to for Pse in the the of CMP-Pse5NAc7Am was to be J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). The in chemical for and that the with the group the In the for and to be and from to L. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). The chemical for at also that the is at M. PubMed Scopus Google Scholar). These for the and for in with studies that in C. jejuni and C. Kelly Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). results provided the of to be CMP-Pse5NAc7Am. chemical and coupling to in the PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), the metabolite was as The NMR at with the probe of the low of metabolite the of was to be was with to the of at from or were the of for nucleotide-activated sugar In contrast, within the NMR of the at with a probe were as and the was that the probe were used to assign and coupling of and that of for and coupling for and a sugar with an Overhauser for and as well as for and a the The for and at and for of at The at with the probe In contrast, the of the at with a probe was and for and were The metabolite was to have the a recent that is from in C. jejuni E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results established the of as is that a of all low molecular weight is the ultimate way to of However, studies flagellin glycosylation that is in to be from a focused metabolomics approach to target a metabolic a novel metabolomics approach, we have novel biosynthetic gene the structural of unique and for the first time in a unique interaction protein glycosylation pathways. The approach a functional in the glycosylation pathway for a of genes from the Campylobacter glycosylation The recent complete genetic characterization of the glycosylation all the genes from the that were involved in or glycosylation M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar). This and to functional for a of the gene We have now to demonstrate that in addition to pseB, pseC, pseF, and pseI, the of genes, pseA, pseG, and are of the pathway to Although analysis of and in the by M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google no effect we have that and Cj1315c, which are as biosynthetic genes and have an effect the biosynthetic pool of intermediates, an that In contrast, of pseD, which results in an flagellin glycosylation and pseE, which has a no effect the of either CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am nucleotide-activated The role of each of genes, which are of the Wren B.W. 2002; PubMed Scopus Google Scholar), to be it is from this that are biosynthetic of the a recent analysis of flagellin protein from the that the flagellin was no with M. M. Kelly J. Mol. Microbiol. PubMed Scopus Google Scholar). In this we that CMP-Pse5NAc7Am was still in the of the The of a and this analysis that is also involved directly in the biosynthetic in the either of glycan moieties or at a of the all of the a hypothetical biosynthetic pathway is in the functional characterization of and was E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it was in that a was of of E. N.M. Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This also shown that the is an metabolite within the related biosynthetic is an of the N-linked heptasaccharide that proteins in Campylobacter (6Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). was that this that to might be a that from the Pse pathway to the In this we this and that the Pse biosynthetic pathway is in of pseC, the biosynthetic of is to the in the of the metabolite in the metabolome. In contrast, a different metabolic was the Pse biosynthetic pathway was at of the of either pseI, which has shown to the Pse J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and to is to be the CMP-Pse5NAc7NAc a metabolite or of the be that the of and are nucleotide-activated and be the precursor analysis. However, metabolomic analysis of and an of and in both isogenic it that of the pathway at biosynthetic results in the of intermediates from of the Although the of this remains to be are a that results from the of biosynthetic intermediates or the of a higher biosynthetic protein The of a single biosynthetic of a genetic target the We are currently this The of and compounds within the of and pseH the first definitive that the proteins by genes are involved in the biosynthetic pathway in C. metabolomics that an isogenic of the in also an of nucleotide-activated intermediates of mass within the although the structural was M. J. Mol. Microbiol. 2003; PubMed Scopus Google Scholar). Although the biosynthetic function for the gene of and pseH are the that have a functional within the pathway are and the Most this presents the structural characterization of CMP-Pse5NAc7Am found the of C. jejuni 81–176. mass spectroscopy the acetamidino functional group to of Pse in an Kelly Brisson J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We now that it is at the C7 In although we the of CMP-Pse5NAc7Am in a we an of intermediates with an acetamidino group. These that for this the of the pathway to at a the flagellin of C. and C. jejuni have shown to in the PseAm the respective flagellin proteins Kelly Mol. Microbiol. 2002; PubMed Scopus Google Scholar). The of this now a to the of the the

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