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Periplasmic chaperone FkpA is essential for imported colicin M toxicity
Author(s) -
Hullmann Julia,
Patzer Silke I.,
Römer Christin,
Hantke Klaus,
Braun Volkmar
Publication year - 2008
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06327.x
Subject(s) - colicin , periplasmic space , mutant , biology , chaperone (clinical) , escherichia coli , bacterial outer membrane , biochemistry , microbiology and biotechnology , gene , medicine , pathology
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; 10 4 ‐fold dilutions killed fkpA + cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS 1 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.