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May, Janine Margaret

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May

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Janine Margaret

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May, Janine Margaret
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    Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane
    (The Royal Society, 2015) Simpson, Brent W.; May, Janine Margaret; Sherman, David; Kahne, Daniel; Ruiz, Natividad
    The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.
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    Lipopolysaccharide transport to the cell surface: periplasmic transport and assembly into the outer membrane
    (The Royal Society, 2015) May, Janine Margaret; Sherman, David; Simpson, Brent W.; Ruiz, Natividad; Kahne, Daniel
    Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.
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    An antibiotic binds to the ATPase that powers lipopolysaccharide transport
    (2016-07-05) May, Janine Margaret; Kahne, Daniel; Gray, Nathanael; Losick, Richard
    The spread of antibiotic resistance has created an urgent need for new antibiotics. The situation is particularly serious for Gram-negative bacteria because they possess an outer membrane (OM) that prevents many antibiotics from entering the cell. The outer leaflet of the OM is composed of lipopolysaccharide (LPS), a complex glycolipid that is critical for creating this permeability barrier. In Escherichia coli, seven LPS transport (Lpt) proteins move LPS from its site of synthesis to the cell surface. Compounds that disrupt LPS transport could either kill Gram-negative bacteria directly or sensitize them to other antibiotics. There are currently no antibiotics that target the Lpt pathway. This work establishes that an antibiotic interacts directly with LptB, the ATPase that powers LPS transport, in addition to its known cellular target. This conclusion is supported by genetic, biochemical, and structural evidence, described below. Mutations in lptB that permeabilize the OM to a wide range of antibiotics, including the antibiotic of interest, can be suppressed by compensatory mutations in lptB. Most of these suppressor mutations confer resistance to all antibiotics tested, suggesting that they correct the permeability defect. One suppressor mutation, however, selectively confers resistance only to the antibiotic of interest, leading to the hypothesis that this compound binds to LptB in vivo. This compound alters the rate of LptB-dependent LPS transport in in vitro reconstitutions, providing evidence that it affects the activity of LptB. A 2.0-Å crystal structure of the compound bound to LptB was obtained. The compound binds in the groove region, which contacts coupling helices from transmembrane-domains LptF/G to form a functional ATP-binding cassette (ABC) system. Lethal mutations in the coupling helices can be suppressed by the compound itself or by mutations in lptB that change the compound-binding site in the protein, suggesting that the binding observed in the crystal structure is physiologically relevant. If this hypothesis is correct, it may be possible to exploit this interaction to generate a new class of antibiotics effective against Gram-negative bacteria.
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    Reconstitution of Peptidoglycan Cross-Linking Leads to Improved Fluorescent Probes of Cell Wall Synthesis
    (American Chemical Society, 2014) Lebar, Matthew D.; May, Janine Margaret; Meeske, Alexander J.; Leiman, Sara; Lupoli, Tania J.; Tsukamoto, Hirokazu; Losick, Richard; Rudner, David; Walker, Suzanne; Kahne, Daniel
    The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.
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    Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
    (American Society for Microbiology, 2016) Simpson, Brent W.; Owens, Tristan; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine Margaret; Trauger, Sunia; Kahne, Daniel; Ruiz, Natividad
    ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction.