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Multi‐drug resistant strains of Acinetobacter baumannii are increasingly being isolated in hospitals worldwide. Among the virulence factors identified in this bacterium there is a general O‐glycosylation system that appears to be important for biofilm formation and virulence, and the capsular polysaccharide, which is essential for resistance to complement killing. In this work, we identified a locus that is responsible for the synthesis of the O‐pentasaccharide found on the glycoproteins. Besides the enzymes required for the assembly of the glycan, additional proteins typically involved in polymerization and transport of capsule were identified within or adjacently to the locus. Mutagenesis of PglC, the initiating glycosyltransferase prevented the synthesis of both glycoproteins and capsule, resulting in abnormal biofilm structures and attenuated virulence in mice. These results, together with the structural analysis of A …