Interconversion of tripartite and bipartite systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria

Abstract

Many bacteria decorate flagellin with sialic acid-like sugars such as pseudaminic acid (Pse) by O-glycosylation on serine or threonine residues. Evidence for sufficiency of sialylation by a conserved flagellin glycosyltransferase (fGT) system is lacking, presumably because of (a) missing component(s). Here, we reconstituted two Maf-type fGTs from the Gram-negative bacterium Shewanella oneidensis MR-1 in a heterologous host producing a Pse donor sugar. While Maf-1 is sufficient for flagellin glycosylation, Maf-2 reconstitution requires a newly identified, cis-encoded and conserved specificity factor GlfM, predicted to form a four-helix bundle. While GlfM binds Maf-2 to form a ternary complex with flagellin, the C-terminal tetratricopeptide repeat (TPR) domain of Maf-1 confers flagellin acceptor and O-glycosylation specificity at preferred serine residues. GlfM from Gram-negative and Gram-positive bacteria are functional, providing evidence for convergent evolution of specialized flagellin modification systems with acceptor serine selectivity, while also shaping the interconversion of bacterial tripartite and bipartite O-glycosylation systems.