The Gram-negative cell envelope is defined by its asymmetric outer membrane, which provides a critical barrier against environmental stressors and antibiotics. This protection is largely mediated by the surface-exposed molecule lipopolysaccharide (LPS), a negatively charged glycolipid consisting of a hydrophobic lipid A anchor, a short chain of sugars called the core oligosaccharide, and an extended series of repeating sugars termed the O-antigen. Many organisms possess lipooligosaccharide (LOS), a truncated chemotype consisting of only lipid A and core oligosaccharide. LPS or LOS is widely regarded as essential for Gram-negative survival, but a rare exception can be found in Acinetobacter baumannii, a high-priority pathogen able to survive in the absence of LOS. This unique quality along with its clinical relevance makes this organism a valuable tool to study Gram-negative cell envelope biogenesis and maintenance. While much work has examined the synthesis and modification of lipid A in A. baumannii, considerably less attention has been paid to the core oligosaccharide. The studies presented in this dissertation address this gap by combining genetic and biochemical approaches to define how the A. baumannii core oligosaccharide is synthesized. As in other Gram-negative bacteria, synthesis is initiated by the transfer of 3-deoxy-D-manno-octulosonic acid (Kdo) sugars, of which there are three in most A. baumannii strains. This transfer would traditionally be mediated exclusively by the enzyme WaaA, which adds all consecutive inner core Kdo residues. Our work, however, reveals a striking departure from this model. A. baumannii instead employs a two-enzyme mechanism in which the glycosyltransferases KdoT and GnaT are required in tandem for final Kdo and subsequent N-acetylglucosaminuronic acid (GlcNAcA) additions, linking Kdo trisaccharide completion with the remainder of the inner core. This represents a fundamental divergence from long-standing assumptions about how Gram-negative bacteria construct their core oligosaccharides.
Together, our findings complete the core biosynthetic pathway in A. baumannii, provide an explanation for its surprising strategy of LOS synthesis, and illuminate how LOS composition and assembly intersect with bacterial physiology. This work expands the framework of Gram-negative envelope biology beyond canonical models and highlights the unique strategies A. baumannii employs to build and adapt its cell envelope.
