AbstractThe gram negative obligate human bacterium N. gonorrhoeae possesses a general O-linked glycosylation system with several target proteins. So far twelve glycoproteins have been identified. In all cases where the glycosylated amino acid could be identified or inferred, the glycan was attached to a serine residue. Moreover, all known gonococcal glycoproteins are membrane attached. Based on these and other commonalities between the gonococcal glycoproteins some topological requirements for protein glycosylation in N. gonorrhoeae have been suggested. However, these assumptions have yet to be experimentally tested.One of the twelve gonococcal glycoproteins is DsbA, a periplasmic protein of the thioredoxin family. N. gonorrhoeae contains two DsbA homologues, Ng1717 is a glycosylated lipoprotein, and Ng1548 which is a soluble periplasmic non-glycosylated protein. By swapping gene segments between Ng1717 and Ng1548 we show here that target proteins need a signal sequence which directs them towards the periplasm, but they do not need to be membrane attached, as has had been previously suggested. Most N. gonorrhoeae glycoproteins have glycosylated serine residues within alanine, serine and proline (A-S-P) rich low complexity regions (LCRs). These are regions that often remain flexible and exposed in folded protein. This indicates that the target selection for glycosylation might require an LCR or some other surface exposed sequence. It also leaves open the possibility that the gonococcal saccharyltransferase may be serine-specific.Identifying the requirements for target selection may ultimately aid our understanding of why N. gonorrhoeae glycosylates proteins, knowledge critical for figuring out how glycosylation impacts the survival and perhaps the pathogenicity of the bacterium.