title: Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin.
abstract: 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 alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-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.
authors.affiliation: Department of Microbiology, Arizona State University, Tempe, AZ 85287, USA.
authors.forename: H
authors.initials: H
authors.lastname: Vakharia
authors.forename: G J
authors.initials: GJ
authors.lastname: German
authors.forename: R
authors.initials: R
authors.lastname: Misra
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000737:chemistry; D006461:Hemolysis; D026901:Membrane Transport Proteins; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli