A general protein glycosylation machinery conserved in species improves bacterial fitness and elicits glycan immunogenicity in humans.

The genus encompasses many Gram-negative bacteria living in the rhizosphere. Some species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. -linked protein glycosylation has been reported, but the chemical structure of the -glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of -glycans, and NMR-based structural analyses, we identified a -glycosylation () gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked -glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the cluster is conserved in the genus, and we confirm the production of glycoproteins with similar glycans in the species: , , and Furthermore, we show that absence of protein glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a larva infection model. Finally, our experiments revealed that patients infected with , , , or develop glycan-specific antibodies. Together, these results highlight the importance of general protein glycosylation in the biology of the genus and its potential as a target for inhibition or immunotherapy approaches to control infections.