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Periplasmic chaperone FkpA is essential for imported colicin M toxicity

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Patzer,  SI
Department Protein Evolution, Max Planck Institute for Developmental Biology, Max Planck Society;

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Römer,  C
Department Protein Evolution, Max Planck Institute for Developmental Biology, Max Planck Society;

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Braun,  V
Department Protein Evolution, Max Planck Institute for Developmental Biology, Max Planck Society;

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Citation

Hullmann, J., Patzer, S., Römer, C., Hantke, K., & Braun, V. (2008). Periplasmic chaperone FkpA is essential for imported colicin M toxicity. Molecular Microbiology, 69(4), 926-937. doi:10.1111/j.1365-2958.2008.06327.x.


Cite as: https://hdl.handle.net/21.11116/0000-0010-353B-1
Abstract
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; 10(4)-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.