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Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate

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      Abstract

      One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. 32P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo2-[4′-32P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.

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      Most cited references 53

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        Measurement of protein using bicinchoninic acid.

        Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
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          Lipopolysaccharide endotoxins.

          Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating "core" oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.
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            Author and article information

            Affiliations
            [1 ]simpleLaboratoire des Enveloppes Bactériennes et Antibiotiques, Unité Mixte de Recherche 8619 CNRS, Université Paris-Sud 91405 Orsay, France.
            [2 ]simpleDepartment of Microbiology, James H. Quillen College of Medicine, East Tennessee State University Johnson City, TN 37614, USA.
            [3 ]simpleDepartment of Biochemistry and Molecular Biology, Medical College of Georgia Augusta, GA 30912, USA.
            Author notes
            *For correspondence. E-mail strent@ 123456mcg.edu ; Tel. (+1) 706 721 1407; Fax (+1) 706 721 6608;
            [**]

            E-mail dominique.mengin-lecreulx@ 123456u-psud.fr ; Tel. (+33) 1 69 15 48 41; Fax (+33) 1 69 85 37 15.

            [†]

            Authors contributed equally.

            Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

            Journal
            Mol Microbiol
            mmi
            Molecular Microbiology
            Blackwell Publishing Ltd
            0950-382X
            1365-2958
            January 2008
            02 November 2007
            : 67
            : 2
            : 264-277
            2229476
            18047581
            10.1111/j.1365-2958.2007.06044.x
            © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd
            Categories
            Research Articles

            Microbiology & Virology

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