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Journal of Bacteriology, December 2001, p. 6908-6916, Vol. 183, No. 23
Department of Microbiology, Arizona State
University, Tempe, Arizona 85287
Received 11 May 2001/Accepted 5 September 2001
This study describes the isolation and characterization of a unique
class of TolC mutants that, under steady-state growth conditions,
secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the
cell-associated hemolytic activity in these mutants was identical to
that in the parental strain, thus reflecting a normal intracellular
toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or
misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time
course of alpha-hemolysin release showed delayed appearance of toxin in
the culture supernatant of the mutant strain, thus indicating a
possible defect in alpha-hemolysin translocation or release. Eight
different TolC substitutions displaying this toxin secretion defect
were scattered throughout the protein, of which six localized in the
periplasmically exposed
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6908-6916.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Isolation and Characterization of
Escherichia coli tolC Mutants Defective in Secreting
Enzymatically Active Alpha-Hemolysin

-helical domain, while the remaining two
mapped within the outer membrane-embedded
-barrel domain of TolC. A
plausible model for the secretion of inactive alpha-hemolysin in these
TolC mutants is discussed in the context of the recently determined
three-dimensional structure of TolC.
*
Corresponding author. Mailing address: Department of
Microbiology, Arizona State University, Tempe, AZ 85287-2701. Phone: (480) 965-3320. Fax: (480) 965-0098. E-mail:
rajeev.misra{at}asu.edu.
Present address: School of Molecular Biosciences, Washington State
University, Pullman, WA 99163.
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