Noncanonical lipooligosaccharide assembly in Acinetobacter baumannii is mediated by the glycosyltransferases KdoT and GnaT

The asymmetric outer membrane is a defining feature of Gram-negative bacteria that provides essential barrier function. The inner leaflet contains glycerophospholipids whereas the outer leaflet is composed of lipopolysaccharide or lipooligosaccharide (LOS). Lipopolysaccharide is comprised of a lipid A anchor, core oligosaccharide (core OS), and O-antigen, while LOS lacks the O-antigen component. Modifications to any of these elements alter barrier permeability. Acinetobacter baumannii demonstrates an unusual ability to survive in the absence of LOS, which offers resistance against select antibiotics but forfeits the outer membrane integrity afforded by LOS. Despite this important relationship, the steps involved in building the core OS component of A. baumannii LOS remain incompletely described. Here, we complete the elucidation of this pathway by establishing a unique method of KdoIII addition via the glycosyltransferase KdoT followed by GlcNAcA addition via GnaT-a clear departure from the typical WaaA-only model of consecutive Kdo transfer. We reconstituted in vitro a two-step sequence in which KdoT transfers the final Kdo residue (KdoIII) and GnaT subsequently transfers GlcNAcA. Heterologous expression confirmed the presence of KdoT homologs across several Gram-negative species, indicating that this split Kdo pathway is not unique to A. baumannii. Structural modeling and targeted mutagenesis further examined the glycosyltransferase assignments of KdoT and GnaT and probed the potential mechanisms employed by each. Together, these data complete the early core OS synthesis pathway in A. baumannii by establishing a noncanonical two-enzyme mechanism for inner core Kdo transfer followed by GlcNAcA addition.