Bacterial swimming is mediated by the rotation of a flagellar filament. Many bacteria are now known to be able to O ‐glycosylate their flagellins, the proteins that make up the flagellar filament. For bacteria that use nonulosonic acid sugars such as pseudaminic acid, this glycosylation process is essential for the formation of a functional flagellum. However, the specific role of glycosylation remains elusive. Aeromonas caviae is a model for this process as it has a genetically simple glycosylation system. Here, we investigated the localization of the glycans on the A. caviae flagellum filament. Using mass spectrometry it was revealed that pseudaminic acid O‐glycosylation was heterogeneous with no serine or threonine sites that were constantly glycosylated. Site‐directed mutagenesis of particular glycosylation sites in most cases resulted in strains that had reduced motility and produced less detectable flagellin on Western blots. For flagellin O ‐linked glycosylation, there is no known consensus sequence, although hydrophobic amino acids have been suggested to play a role. We, therefore, performed site‐directed mutagenesis of isoleucine or leucine residues flanking the sites of glycosylation and demonstrated a reduction in motility and the amount of flagellin present in the cells, indicating a role for these hydrophobic amino acids in the flagellin glycosylation process.

Aeromonas are Gram-negative facultative anaerobic rods, which inhabit various aquatic environments and are pathogens of both warm and cold-blooded animals. In humans they cause gastro-enteritis and wound infections. They are motile in liquid environments by a single polar type of flagellum. The flagellum plays an important role for the bacterial colonisation and the adhesion to the host cells. The Aeromonaspolar flagella filament is a polymer composed of two flagellins, FlaA and FlaB. The flagellins are O-linked glycosylated through the addition of the unusual bacterial sugar pseudaminic acid to serine and threonine residues within the flagellins D2/D3 domain. The addition of this sugar is essential for flagella filament assembly and bacterial motility. The flagellin’s are modified by between 6 – 8 sugar residues that occupy the potential 14 sites of attachment. Motility accessory factors (Maf proteins) are candidate enzymes for transferring glycan molecules to the flagellin (glycosyltransferases transferring sugar to flagellin) due to their genetic location and motility phenotypes associated with disruption mutants. This study utilised site-directed mutagenesis to change the potential sites of flagellin glycosylation to assess the effect of these mutations on motility by swimming assays and flagella filament formation by electron microscopy. The analysis of different numbers of site-directed mutants suggest that some sites are more important than others and that the removal of 4 sites results in greatly reduced motility.