The cell envelope of Gram-negative bacteria is composed of the inner and outer membranes (IM and OM, respectively), an aqueous compartment known as the periplasm, and a mesh-like layer known as peptidoglycan (PG).  Proper envelope biogenesis is crucial for the bacterium and it requires the coordinated synthesis, transport and assemblage of all its components.  In our laboratory, we use genetic and biochemical approaches to understand envelope biogenesis in the Gram-negative bacterium Escherichia coli.
 

PG is a glycopeptide polymer composed of glycan chains that are cross-linked through their stem peptides.  PG is conserved among most bacteria and protects such cells from osmotic lysis.  In addition, the PG mesh determines cell shape and serves as a scaffold for other envelope structures.

 

In order to build the PG layer, the cell first synthesizes the PG precursor lipid II in the cytoplasm.  Then, lipid II is transported across the IM so that its disaccharide-peptide moiety is incorporated into the preexisting periplasmic PG structure through the actions of transglycosylases and transpeptidases.  MurJ is the flippase that translocates lipid II across the IM, and one of the main projects in our laboratory is to understand how MurJ functions.
 

In E. coli, the OM serves as the main protective barrier against toxic molecules present in the environment.  Because of the impermeability of its OM, E. coli is naturally resistant to many antibiotics.  The main component of the OM responsible for providing this barrier-like quality to the OM is LPS, a lipopolysaccharide that is located at the cell surface.

 

LPS is synthesized in the cytoplasmic leaflet of the IM.  Therefore, it must be transported across the cell envelope to be assembled at the cell surface.  We know that the ABC transporter MsbA flips LPS across the IM and that once it is in the periplasmic leaflet of the IM, LPS is transported to the cell surface by the Lpt trans-envelope complex.  This Lpt complex is composed of seven different proteins that are essential for LPS transport and viability of E. coli.  In our laboratory, we are interested in understanding how the Lpt transport machine assembles and functions.