Outer membrane biogenesis in Escherichia coli: genetic and physiological cell response to lipopolysaccharide transport defects

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer formed by phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, with a large number of embedded or associated proteins. The primary function of this structure essential for Gram-negative viability is to establish an additional selective permeability barrier that enables the cell to maintain favourable intracellular conditions even in harsh environments and the LPS layer greatly contributes to this peculiar property. The transport of LPS to the cell surface is an essential process for OM biogenesis; the LPS transport (Lpt) system, originally identified in E. coli, is the protein machine responsible for LPS delivery from the periplasmic side of the inner membrane (IM) to the OM. It is composed of seven proteins forming a complex which spans from IM to OM. At the IM the ABC transporter LptBFG, associated to the membrane-bound protein LptC interacts with the periplasmic protein LptA that connects, through structurally conserved domains, the IM ABC transporter with the OM translocon LptDE, responsible for LPS assembly at the cell surface. In order to gain more insight in the mechanism of LPS transport and more in general in OM homeostasis we used both a genetic and a proteomic approach. The former was based on the selection of suppressors of LPS transport defects obtained with two different types of mutants. i) a quadruple nonlethal lptA mutant (lptA41) that displayed increased sensitivity to toxic compounds, and ii) a lethal deletion mutant of lptC. Genome sequencing analysis of spontaneous suppressors of lptA41 phenotype revealed two different mechanism of suppression: one mechanism involves the Mla system, a protein machinery which contributes to maintain OM asymmetry; the second mechanism involves both an intragenic mutation improving LptA41 protein stability and an extragenic mutation affecting osmoregulated periplasmic glucans (OPGs) synthesis. Viable mutants lacking lptC were obtained using a plasmid shuffling technique. Genome sequencing of such mutants revealed single amino acid substitutions at the R212 residue of the IM component LptF (lptFSupmutants). Our results suggest that LptC may serve as a chaperon of the Lpt machine assembly and/or activity rather than an essential structural component and the periplasmic domain of LptF might be implicated in the formation of the Lpt bridge. The latter approach consisted of the analysis of differential envelope proteins content of an E. coli lptC conditional expression mutant upon depletion of LptC and thus impairment of LPS transport. By Outer membrane biogenesis in Escherichia coli: genetic and physiological cell response to lipopolysaccharide transport defects 2 two-dimensional chromatography coupled to tandem mass spectrometry (Multidimensional Protein Identification Technology, MudPIT) we identified 123 proteins whose level is significantly modulated upon LptC depletion. Most of these proteins belong to pathways that contribute to repair OM and restore its permeability barrier properties, including protein involved in maintaining OM asymmetry, in the synthesis of phospholipids and exopolysaccharides as substrate for lipid A modification enzymes, and in peptidoglycan synthesis/remodelling. Overall these data contribute to our understanding of the multiple strategies that E. coli cells may adopt to respond to perturbations of the OM permeability barrier and to restore OM functionality.