CONICET | Buscador de Institutos y Recursos Humanos

Protein glycosylation, the covalent attachment of carbohydrates to specific amino acid residues, is a common posttranslational modification in bacteria. Glycosylation of flagellin, the structural subunit of the flagellar filament, has been reported in many Gram-negative bacteria, playing roles in filament assembly, stability and function, adhesion, host recognition, immune evasion and virulence. Ralstonia solanacearum is a soil-borne β-proteobacterium that causes the bacterial wilt disease on more than 200 plant species, including economically important crops. Genomic analysis aimed at identifying genes involved in glycosylation systems of R. solanacearum revealed the presence of two open reading frames (RSp0387 and RSp0388) encoding for putative glycosyltransferases, in close proximity to the flagellar genes. In the present work, monomeric flagellin was purified from the wild-type R. solanacearum GMI1000 strain and subjected to HLPC-ESI to confirm its identity. Glycosylation of the protein was corroborated by PAS stain and then the glycosyl moiety was characterized by mass spectrometry and HPAEC-PAD, revealing the presence of N- and O-glycans of 3 to 14 hexose units. To initiate the functional study of the RSp0387 and RSp0388 genes, two deficient mutant strains were generated and physiologically characterized. The mutant strains exhibited similar growth kinetics than the wild-type strain in liquid BG medium and normal colony morphology on triphenyltetrazolium-supplemented agar plates. The presence of flagellar filaments in both strains was evidenced by transmission electron microscopy; however, the swimming ability in liquid medium was impaired in both mutants, suggesting that the flagellar function is affected by the deletion of the RSp0387 and RSp0388 genes.