In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/ mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the -D-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-␣-Lgluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis, a causative agent of child morbidity in underdeveloped countries and an antecedent to the MillerFisher and Guillain-Barré neuropathies (1-5). Furthermore, C. jejuni now surpasses Salmonella, Shigella, and Escherichia in some regions as the primary cause of bacterial gastrointestinal disease (6 -8). Because the number of reported C. jejuni infections is increasing worldwide, there is growing interest to identify virulence mechanisms associated with this mucosal pathogen as a critical step toward the development of control strategies.The capsular polysaccharides (CPS) 5 produced by C. jejuni are known to be important virulence factors that are involved in colonization and invasion (9, 10). CPS expression was shown to be necessary for diarrheal disease in ferrets, mediating mouse and chicken colonization, increasing resistance to human serum, as well as increasing adherence and invasion of human epithelial cells (9). The CPSs produced by C. jejuni are the major antigenic component of Penner's serotyping system (10). There are now more than 60 serostrains described for this bacterium. Although not every strain has been examined, it is thought that each one produces a CPS having a different structure (11, 12). Furthermore, there can be extensive phase-variable structural modifications such as the incorporation of methyl, ethanolamine,...