The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyl-transferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coil showed that HMWC of NTHi was a general glycosyltransferase capable of glycosylating selected asparagines in proteins other than its HMWA substrate, including Asn78 in E. coli 30S ribosomal protein S5. The equivalent residue in S5 homologues in H. influenzae or other sequenced Pasteurellaceae genomes is not asparagine, and these organisms also showed significantly fewer than expected potential sites of glycosylation in general. Expression of active HMWC in E. coli resulted in growth inhibition compared with expression of inactive enzyme, consistent with glycosylation by HMWC detrimentally affecting the function of some E. coil proteins. Together, this supports the presence of a selective pressure in the Pasteurellaceae against glycosylation sites that would be modified by the general N-glycosyltransferase activity of HMWC. (C) 2014 Elsevier Inc. All rights reserved.