• Open Access

Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment

Authors

  • Lakshmiprasad Potluri,

    1. Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, US.
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  • Aneta Karczmarek,

    1. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, P.O. Box 94215, 1090 GE Amsterdam, the Netherlands.
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  • Jolanda Verheul,

    1. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, P.O. Box 94215, 1090 GE Amsterdam, the Netherlands.
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  • Andre Piette,

    1. Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, B-4000 Sart Tilman, Belgium.
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  • Jean-Marc Wilkin,

    1. Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, B-4000 Sart Tilman, Belgium.
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    • Present addresses: GSK. Biologicals, rue de l'Institut 39, B-1330 Rixensart, Belgium;

  • Nadine Werth,

    1. Mikrobielle Genetik, Eberhard Karls Universität Tübingen, Tübingen, Germany.
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    • Institut für Medizinische Mikrobiologie und Hygiene, Eberhard Karls Universität Tübingen, Tübingen, Germany.

  • Manuel Banzhaf,

    1. Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
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  • Waldemar Vollmer,

    1. Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
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  • Kevin D. Young,

    1. Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205-7199, US.
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  • Martine Nguyen-Distèche,

    1. Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, B-4000 Sart Tilman, Belgium.
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  • Tanneke Den Blaauwen

    Corresponding author
    1. Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, P.O. Box 94215, 1090 GE Amsterdam, the Netherlands.
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E-mail t.denblaauwen@uva.nl; Tel. (+31) 205 255196; Fax (+31) 205 257934.

Summary

The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.

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