Stereoisomer‐specific reprogramming of a bacterial flagellin sialyltransferase

Abstract

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as gly-cosyl donors, particularly pseudaminic acid (Pse) and its stereoiso-mer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosyla-tion and motility in Caulobacter crescentus and Brevundimonas sub-vibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthe-sis, and validated it by reconstitution experiments in a heterolo-gous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glyco-syltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.