Elucidation of the Glycan Structure of the b-type Flagellin of PAO1.

Flagella are essential for motility and pathogenicity in many bacteria. The main component of the flagellar filament, flagellin (FliC), often undergoes post-translational modifications, with glycosylation being a common occurrence. In PAO1, the b-type flagellin is -glycosylated with a structure that includes a deoxyhexose, a phospho-group, and a previous unknown moiety. This structure resembles the well-characterized glycan (Type A) in strain 630, which features an -acetylglucosamine linked to an -methylthreonine via a phosphodiester bond. This study aimed to characterize the b-type glycan structure in PAO1 using a set of mass spectrometry experiments. For this purpose, we used wild-type PAO1 and several gene mutants from the b-type glycan biosynthetic cluster. Moreover, we compared the mass spectrometry characteristics of the b-type glycan with those of modified Type A-peptides from strain 630Δ. Our results demonstrate that the thus far unknown moiety of the b-type glycan in consists of an -dimethylthreonine. These data allowed us to refine our model of the flagellin glycan biosynthetic pathway in both PAO1 and strain 630.