Outer Membrane Biogenesis

The hallmark of gram-negative bacteria and organelles such as mitochondria and chloroplasts is the presence of an outer membrane. In bacteria such as Escherichia coli, the outer membrane is a unique asymmetric lipid bilayer with lipopolysaccharide in the outer leaflet. Integral transmembrane proteins assume a β-barrel structure, and their assembly is catalyzed by the heteropentameric Bam complex containing the outer membrane protein BamA and four lipoproteins, BamB-E. How the Bam complex assembles a great diversity of outer membrane proteins into a membrane without an obvious energy source is a particularly challenging problem, because folding intermediates are predicted to be unstable in either an aqueous or a hydrophobic environment. Two models have been put forward: the budding model, based largely on structural data, and the BamA assisted model, based on genetic and biochemical studies. Here we offer a critical discussion of the pros and cons of each

Membrane Potential Is Required for MurJ Function

MurJ, the flippase that exports the bacterial cell wall monomer Lipid II to the periplasm, is a target for new antibiotics, which are desperately needed to treat Gram-negative infections. Quantitative methods to monitor MurJ activity are required to characterize inhibitors but are challenging to develop because the lipid-linked substrate is not chemically altered in a flippase reaction. Here we show that MurJ inhibition can be quantified by measuring the accumulation of intracellular Lipid II using a biotin-tagging strategy. We have exploited this assay to show that MurJ is inhibited in the presence of a compound that dissipates the membrane potential. By probing cysteine accessibility we have found that under this condition MurJ relaxes into an inactive, outward-facing conformation reminiscent of that targeted by the peptide antibiotic Lys^M. We conclude that membrane potential is required for MurJ function in <i>E. coli</i>, and we anticipate that the ability to accumulate this inactive conformation will lead to structures useful for inhibitor design

A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery