Open AccessMolecular Cloning and Characterization of Bacillus alvei Thiol-dependent Cytolytic Toxin Expressed in Escherichia coli
Author(s) -
C Geoffroy,
Joseph E. Alouf
Publication year - 1988
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.019
H-Index - 179
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-134-7-1961
Subject(s) - escherichia coli , cytolysin , biology , microbiology and biotechnology , hemolysin , haemolysis , plasmid , pbr322 , isoelectric focusing , cytolysis , biochemistry , molecular cloning , bacillus cereus , dna , bacteria , peptide sequence , cytotoxicity , enzyme , gene , virulence , immunology , in vitro , genetics
A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (Mr 60,000, pI 5.0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64 kDa polypeptide of pI 8.2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.