Periplasmic chaperone FkpA is essential for imported colicin M toxicity.
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.
Hullmann
Julia
J
Microbiology/Membrane Physiology, University of Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
Patzer
Silke I
SI
Römer
Christin
C
Hantke
Klaus
K
Braun
Volkmar
V
D001433:Bacteriolysis / Q000235:genetics; D003087:Colicins / Q000378:metabolism* / Q000494:pharmacology; D019286:Endopeptidase K / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006358:Hot Temperature; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D021381:Protein Transport
D003087:Colicins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D019286:Endopeptidase K; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli