Structural characterization of a pseudaminic acid-modified lateral flagellar filament from Vibrio alginolyticus

Bacterial flagellar filaments are often modified by glycans, but the structural basis and physiological significance of flagellin glycosylation remain poorly understood in many species. Vibrio alginolyticus produces lateral flagella for surface-associated motility in viscous environments, with LafA forming its filament as flagellin. A maf homolog encoding a putative flagellin glycosylation factor is located immediately downstream of lafA, suggesting that the lateral filament is glycosylated. To investigate this possibility, we purified the lateral flagellar filament from V. alginolyticus and determined the structure at 2.4 Å resolution by electron cryomicroscopy. Upon model building of LafA in the map, we identified additional densities connected to five serine residues, possibly corresponding to O-linked pseudaminic acid modifications. Mass spectrometric analyses identified these modifications as pseudaminic acid attached to Ser148, Ser173, Ser183, Ser189, and Ser197. Deletion of maf abolished lateral flagella formation and motility, and these defects were restored by complementation with maf. These results demonstrate that the Vibrio lateral flagellar filament is extensively modified by pseudaminic acid and that Maf is required for filament formation. Our findings provide the first structural insight into the glycosylation of the Vibrio lateral flagellar filament and establish a framework for understanding the role of flagellin glycosylation in surface-associated motility.IMPORTANCEA gene encoding a putative flagellin glycosylation factor (Maf) is located immediately downstream of lafA, the gene encoding lateral flagellin of Vibrio alginolyticus, suggesting that the lateral flagellar filament is glycosylated. Guided by this assumption, we determined the structure of the lateral flagellar filament by electron cryomicroscopy with mass spectrometry and identified five pseudaminic acid modifications on the filament surface. Genetic analyses further demonstrated that Maf is required for lateral flagellar formation and motility. These results provide the first structural characterization of a glycosylated Vibrio lateral flagellar filament and reveal a close link between flagellin glycosylation and motility organelle assembly.