is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of isolates. Among the nine main O-serotypes of , O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of → 3)-β-d-Gal-(1 → 3)-α-d-Gal-(1 → repeating units) capped by d-galactan-II (built of [ → 3)-α-d-Gal-(1 → 3)-β-d-Gal-(1 →] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→ 3)-β-d-Gal-(1 → 3)-[α-d-Gal-(1 → 4)]-α-d-Gal-(1 →) was reported. Substitution of → 3)-α-d-Gal by a branching terminal α-d-Gal was dependent on the presence of the operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by , as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.