Bacterial infections due to multidrug-resistant bacterial pathogens represent an ever-increasing public health crisis. Gram-negative bacteria in particular present a treatment challenge in part due to their outer membrane, whose outer surface is composed of lipopolysaccharide (LPS). This LPS layer provides a membrane permeability barrier against a variety of insults, and reduces the efficacy of many classes of antibiotics. Scientists exploring alternative targets for new antibacterials have recognized that inhibition of the synthesis and transport of LPS is an effective method for killing Gram-negative bacteria in vitro and in vivo. Our work characterizing the role of LPS biosynthesis and transport has led to the realization that in Pseudomonas aeruginosa LPS phosphorylation by the kinase WaaP is coupled to recognition by the LPS transport machinery. Additionally, our work on Pseudomonas fatty acid biosynthesis has identified a clear connection to LPS biosynthesis and an intact outer membrane permeability barrier. However, there are no compounds nearing regulatory approval that inhibit this pathway, and the discovery of inhibitors of LPS synthesis is complicated by the absence of reliable methods for assaying the biosynthesis and transport of nascent LPS in intact bacteria. Our interdisciplinary group will use advanced chemical and biological techniques to further our understanding of LPS dynamics and support the identification of novel medicines that target this synthetic pathway.