The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.The cell envelope of gram-negative bacteria consists of an inner (IM) and an outer (OM) membrane separated by the periplasmic space, which contains the peptidoglycan layer. The OM is an asymmetric bilayer, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) facing outward (28, 32). LPS, a molecule unique to gram-negative bacteria, consists of the lipid A moiety (a glucosamine-based phospholipid) linked to a short-core oligosaccharide and the distal O-antigen polysaccharide chain. The core oligosaccharide can be further divided into the inner core, composed of 3-deoxy-D-mannooctulosanate (KDO) and heptose, and the outer core, with a somewhat variable structure. Escherichia coli K-12 synthesizes a shorter LPS consisting of a lipid A moiety linked to a short-core oligosaccharide but missing the O-antigen chain (28). LPS is essential in most gram-negative bacteria, with the notable exception of Neisseria meningitidis. In E. coli, the minimal part required for viability consists of the KDO 2 lipid IV A moiety (28).LPS is synthesized in the cytoplasmic face of the IM and must traverse the IM and the periplasm to reach its final destination in the outer face of the OM (6, 28). Translocation across the IM requires the ABC transporter MsbA, which mediates the flipping from the inner leaflet (the site of the synthesis) to the outer leaflet of the IM (14, 27, 44). MsbA has also been implicated in phospholipid transport across the IM of E. coli (13,14). Interestingly, MsbA in N. meningitidis is not essential and seems not to be required for phospholipid transport (39). How LPS traverses the periplasm and is inserted into the OM is much less understood.Because of its hydrophobic lipid A moiety, transport of LPS through the aqueous periplasm is thermodynamically un...