A Novel in vivo Cell-Wall Labeling Approach Sheds New Light on Peptidoglycan Synthesis in Escherichia coli

Authors

  • Dr. Nick K. Olrichs,

    1. Department of Chemical Biology and Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands), Fax: (+31) 30-2533969
    2. Present address: Department of Biochemistry and Cell Biology, University of Utrecht, Yalelaan 2, 3508 TD Utrecht (The Netherlands)
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  • Mirjam E. G. Aarsman,

    1. Molecular Cytology, Swammerdam Institute for life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam (The Netherlands)
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  • Jolanda Verheul,

    1. Molecular Cytology, Swammerdam Institute for life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam (The Netherlands)
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  • Dr. Christopher J. Arnusch,

    1. Department of Chemical Biology and Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands), Fax: (+31) 30-2533969
    2. Present address: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 (Israel)
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  • Dr. Nathaniel I. Martin,

    1. Department of Chemical Biology and Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands), Fax: (+31) 30-2533969
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  • Dr. Mireille Hervé,

    1. CNRS Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS, Université Paris-Sud, 91405 Orsay Cedex (France)
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  • Prof. Waldemar Vollmer,

    1. Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH (UK)
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  • Prof. Ben de Kruijff,

    1. Department of Chemical Biology and Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands), Fax: (+31) 30-2533969
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  • Dr. Eefjan Breukink,

    Corresponding author
    1. Department of Chemical Biology and Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands), Fax: (+31) 30-2533969
    • Department of Chemical Biology and Organic Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht (The Netherlands), Fax: (+31) 30-2533969
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  • Dr. Tanneke den Blaauwen

    1. Molecular Cytology, Swammerdam Institute for life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam (The Netherlands)
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Abstract

Peptidoglycan synthesis and turnover in relation to cell growth and division has been studied by using a new labeling method. This method involves the incorporation of fluorescently labeled peptidoglycan precursors into the cell wall by means of the cell-wall recycling pathway. We show that Escherichia coli is able to import exogenous added murein tripeptide labeled with N-7-nitro-2,1,3-benzoxadiazol-4-yl (AeK–NBD) into the cytoplasm where it enters the peptidoglycan biosynthesis route, resulting in fluorescent labels specifically located in the cell wall. When wild-type cells were grown in the presence of the fluorescent peptide, peptidoglycan was uniformly labeled in cells undergoing elongation. Cells in the process of division displayed a lack of labeled peptidoglycan at mid-cell. Analysis of labeling patterns in cell division mutants showed that the occurrence of unlabeled peptidoglycan is dependent on the presence of FtsZ, but independent of FtsQ and FtsI. Accumulation of fluorescence at the division sites of a triple amidase mutant (ΔamiABC) revealed that AeK–NBD is incorporated into septal peptidoglycan. AmiC was shown to be involved in the rapid removal of labeled peptidoglycan side chains at division sites in wild-type cells. Because septal localization of AmiC is dependent on FtsQ and FtsI, this points to the presence of another peptidoglycan hydrolase activity directly dependent on FtsZ.

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