The defining feature of the Gram-negative cell envelope is the presence of two cellular membranes with the specialized glycolipid lipopolysaccharide (LPS) exclusively found on the surface of the outer membrane. The surface layer of LPS contributes to the stringent permeability properties of the outer membrane which is particularly resistant to permeation of many toxic compounds, including antibiotics. As a common surface antigen, host immune cells recognize LPS and mount defenses to clear pathogenic organisms. To alter properties of the outer membrane or evade the host immune response, Gram-negative bacteria employ a wide variety of chemical modifications to alter LPS. Here we review key features and physiological consequences of LPS biogenesis and modifications.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction.

One contribution of 12 to a theme issue 'The bacterial cell envelope'. The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane.

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.