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Periplasmic chaperone FkpA is essential for imported colicin M toxicity

Authors

  • Julia Hullmann,

    1. Microbiology/Membrane Physiology, University of Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
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  • Silke I. Patzer,

    1. Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstr. 35, 72076 Tubingen, Germany.
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  • Christin Römer,

    1. Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstr. 35, 72076 Tubingen, Germany.
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  • Klaus Hantke,

    1. Microbiology/Membrane Physiology, University of Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
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  • Volkmar Braun

    Corresponding author
    1. Microbiology/Membrane Physiology, University of Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
    2. Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstr. 35, 72076 Tubingen, Germany.
      *E-mail volkmar.braun@tuebingen.mpg.de; Tel. (+49) 7071 601 343, Fax (+49) 7071 601 349.
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  • Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

*E-mail volkmar.braun@tuebingen.mpg.de; Tel. (+49) 7071 601 343, Fax (+49) 7071 601 349.

Summary

Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 104-fold dilutions killed fkpA+ cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.

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