The flagellar filaments of Shewanella oneidensis are composed of the major flagellin FlaB and the minor flagellin FlaA.Glycosylation of flagellins is essential to the bacterial motility.An LC-MS analysis demonstrates that the sugars on flagellins are disaccharides,composing of a common 274 Da and one of 236,250,and 264 Da units.As the flagellum consisting of FlaB alone is fully motile,we chose the protein for in-depth analyses.We characterized FlaB by the Site-directed mutagenesis to determine the important residues for glycosylation.Six mutated FlaB in which one of serine or threonine residues was replaced by an alanine caused significant reduction in motility.The S99A and T179A FlaB mutants lost the motility completely,indicating these two sites are essential in secretion and assembly process.Transmission electron microscope(TEM) supported the observation.Four other FlaB mutants (mutations T129A,T135A,T161A,S165A,respectively) showed reduced motility.We then examined the glycosylation pathway for flagellins in S.oneidensis.We found that PseB and PseC are first two enzymes catalyzing the process.