Gram-negative bacteria are characterized by a peculiar envelope architecture comprising an outer membrane (OM) in addition to the cytoplasmic, inner (IM) membrane. Gram-negative infections are especially difficult to treat, as there has not been a new class of antibiotics targeting Gram-negative organisms in the last fifty years. A major reason that Gram-negative pathogens are so resilient is that they have an asymmetric OM with, lipopolysaccharide (LPS) in the outer leaflet. In our laboratory, we are dissecting LPS biogenesis in the model organism Escherichia coli and in the opportunistic pathogen Pseudomonas aeruginosa. We have established that the transport and assembly of LPS involve seven essential proteins that proteins form a transenvelope complex and transport LPS molecules from the IM to the outer leaflet of the OM. We use genetic and biochemical approaches to understand how this molecular machine works. A long-term goal of our research is to discover molecules that interfere with the assembly of the OM that could be developed as antibiotics or antibiotics adjuvants.