Characterization of Acinetobacter Baumannii Core Oligosaccharide Synthesis Reveals Novel Aspects of Lipooligosaccharide Assembly.

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii. Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, triangle 2903 and triangle lpsB, that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii. Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly.