Envelope biogenesis in Escherichia coli and Pseudomonas aeruginosa

Harvard Medical School, Boston MA

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Abstract

SUMMARY A major goal of bacterial cell biology is to understand the mechanisms underlying the assembly and growth of the cell envelope. In addition to addressing a fundamental biological question, studies in this area have significant consequences for human health because the envelope serves as both a major target for antibiotics and, in the case of Gram-negative bacteria, a formidable barrier that prevents drugs from reaching their target. Thus, understanding of the mechanisms controlling the construction of the Gram-negative envelope will help identify new vulnerabilities in the process to target for antibiotic development. The Gram-negative envelope consists of two membranes, an inner membrane (IM) and outer membrane (OM), with a thin layer of peptidoglycan (PG) cell wall sandwiched between them. The IM is a typical phospholipid (PL) bilayer whereas the OM has an asymmetric structure with PL in the inner leaflet and an outer leaflet composed of lipopolysaccharide (LPS) glycolipids. Lateral interactions between LPS molecules provide the barrier function to the OM, preventing entry of both large polar molecules and small hydrophobic molecules. The PG layer fortifies the IM and prevents osmotic lysis. Studies of envelope biogenesis in many labs over the last several decades have identified and characterized most proteins essential for cell surface assembly in proteobacterial cells. The next major challenge in the field is to elucidate the mechanisms by which biogenesis of the different envelope layers is coordinated to promote uniform surface growth and maintain the OM permeability barrier. Therefore, the focus of this proposal is on defining the mechanisms that balance the supply of envelope subunits to ensure that each layer of the surface can be built at a similar rate. Balance in the supply of envelope components is critical because the overaccumulation of one component can induce toxic alterations to the envelope. Coordinating synthesis of envelope building blocks is also important because their synthesis pathways share common precursors such as: (i) acyl-ACP (acyl-carrier protein), the source of acyl chains for LPS and PLs, (ii) UDP-N-acetylglucosamine (UDP-GlcNAc), the source of sugar moieties for LPS and PG, and (iii) the lipid carrier undecaprenol-phosphate (C55P) used for surface glycan polymer biogenesis. Thus, if flux through these pathways is not balanced, the overproduction of one envelope component will indirectly impair the biogenesis of another, causing a damaging precursor deficit. Our work has recently revealed several novel mechanisms responsible for balancing the synthesis of envelope building blocks in Escherichia coli and Pseudomonas aeruginosa. We have identified a conserved feedback mechanism governing C55P utilization by the cell wall synthesis pathway, a potential regulatory system connecting PL and LPS synthesis in E. coli, and a regulatory interaction between LPS and PG synthesis enzymes in P. aeruginosa likely used to efficiently distribute UDP-GlcNAc between the pathways. The experiments described in this proposal will enhance our understanding of the molecular nature of these controls and reveal new regulatory pathways for further study.