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 glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer 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 glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides 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 biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin‐binding domain that identifies the acceptor.

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

“…We did not detect any Maf-2-dependent glycosylation on any residues on FLAG-FlaA in C. crescentus lacking GlfM. The Maf-2-GlfM-dependent mass modifications are 359 Da or 375 Da, corresponding to the di-acetylated amide and oxidized forms of Pse specific to C. crescentus (also detected in our previous analyses of FlmG-dependent glycosylation) 16 . By contrast, when FLAG-FlaA was extracted from Δ neuB C. crescentus cells that do not produce Pse (derivatives), the 359 Da and 375 Da mass were no longer detected.…”

“…HHpred analyses suggests that Maf1 harbours a C-terminal tetratrico p eptide repeat (TPR) domain (550-820 aa), while Maf-2 has no C-terminal domain after the signature MAF_flag10 domain. Since a TPR domain was recently shown to be critical and sufficient for flagellin binding by the less conserved FlmG-class of fGTs discovered in C. crescentus 4 and B. subvibrioides 16 , we speculated that the TPR could represent as the flagellin binding determinant in Maf-1, while there is no apparent flagellin binding determinant on the polypeptide of Maf-2.…”

“…Yet, we also noted an increase in mobility when cells are unable to produce the Pse glycosylation donor, albeit here the change in migration is less pronounced compared to that seen in the absence of GlfM. As additional control, we also transformed single co-expression plasmid (pP lac - flag-flaA-maf-1 ) encoding FLAG-FlaA and Maf-1 into C. crescentus Δ flmG cells (lacking the native FlmG-type fGT) and Δ neuB cells as a reference point 16 to show the migration of flagellin after Pse-dependent glycosylation by Maf-1 in C. crescentus ( Fig. 5B ).…”

“…Indeed, flagellar filaments are post-translationally modified by sialic acid derivatives called pseudaminic acid (Pse, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L- manno-nonulosonic acid) or its stereoisomer legionaminic acid (Leg) 1,3,5 . Illumination of the mysterious mechanism by which flagellins are specifically selected from other cytoplasmic proteins for covalent modification with Pse or Leg awaited the discovery and reconstitution of glycosylation by the FlmG-class of flagellin glycosyltransferases (fGTs) encoded in the α- proteobacteria Caulobacter crescentus and Brevundimonas subvibroides 4,16 . FlmGs feature an overt and genetically separable two-domain architecture: an N-terminal tetratricopeptide repeat (TPR) that mediates flagellin binding, while a C-terminal glycosyltransferase (GT) domain adopting a GT-B fold executes the glycosylation reaction with preference for Pse or Leg 5,16,17 .…”

“…Illumination of the mysterious mechanism by which flagellins are specifically selected from other cytoplasmic proteins for covalent modification with Pse or Leg awaited the discovery and reconstitution of glycosylation by the FlmG-class of flagellin glycosyltransferases (fGTs) encoded in the α- proteobacteria Caulobacter crescentus and Brevundimonas subvibroides 4,16 . FlmGs feature an overt and genetically separable two-domain architecture: an N-terminal tetratricopeptide repeat (TPR) that mediates flagellin binding, while a C-terminal glycosyltransferase (GT) domain adopting a GT-B fold executes the glycosylation reaction with preference for Pse or Leg 5,16,17 . Importantly, FlmG can glycosylate flagellin when expressed in heterologous hosts synthesizing a suitable donor glycan: a Pse derivative in the case of C. crescentus FlmG and a Leg derivative for B. subvibroides FlmG.…”