Burkholderia PglL enzymes are serine preferring oligosaccharidetransferases which target conserved proteins across the Burkholderia genus

Glycosylation is increasingly recognised as a common protein modification within bacterial proteomes. While great strides have been made in identifying species that contain glycosylation systems, our understanding of the proteins and sites targeted by these enzymes is far more limited. Within this work we explore the conservation of glycoproteins and O-linked glycosylation sites across the pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach we generate high-confidence glycoproteomes and associated glycosylation sites in two widely utilized B. cenocepacia strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively at serine residues and that glycoproteins/glycosylation sites are highly conserved across 294 publicly available B. cenocepacia genomes. Consistent with this we demonstrate that the substitution of Serine for Threonine residues in a model protein results in a dramatic decrease in glycosylation efficiency by the oligosaccharidetransferase pglLBC even when pglLBC is overexpressed. This preference for glycosylation at Serine residues is observed across at least 9 Burkholderia glycoproteomes supporting that Serine is the dominant residue targeted by pglL-mediated glycosylation across the Burkholderia genus. Using population genomics we observe that pglL targeted glycosylated proteins are common across Burkholderia species. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharidetransferases that target conserved and shared protein substrates across the Burkholderia genus.