Bioconjugate vaccines, consisting of polysaccharides attached to carrier 
proteins, are enzymatically generated using prokaryotic glycosylation systems in 
a process termed bioconjugation. Key to bioconjugation are a group of enzymes 
known as oligosaccharyltransferases (OTases) that transfer polysaccharides to 
engineered carrier proteins containing conserved amino acid sequences known as 
sequons. The most recently discovered OTase, PglS, has been shown to have the 
broadest substrate scope, transferring many different types of bacterial glycans 
including those with glucose at the reducing end. However, PglS is currently the 
least understood in terms of the sequon it recognizes. PglS is a pilin-specific 
O-linking OTase that naturally glycosylates a single protein, ComP. In addition 
to ComP, we previously demonstrated that an engineered carrier protein 
containing a large fragment of ComP is also glycosylated by PglS. Here we sought 
to identify the minimal ComP sequon sufficient for PglS glycosylation. We tested 
>100 different ComP fragments individually fused to Pseudomonas aeruginosa 
exotoxin A (EPA), leading to the identification of an 11-amino acid sequence 
sufficient for robust glycosylation by PglS. We also demonstrate that the 
placement of the ComP sequon on the carrier protein is critical for stability 
and subsequent glycosylation. Moreover, we identify novel sites on the surface 
of EPA that are amenable to ComP sequon insertion and find that Cross-Reactive 
Material 197 fused to a ComP fragment is also glycosylated. These results 
represent a significant expansion of the glycoengineering toolbox as well as our 
understanding of bacterial O-linking sequons.