Perfringolysin O expression in Clostridium perfringens is independent of the upstream pfoR gene

Abstract

The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C. perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA(+) shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C. perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C. perfringens. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C. perfringens.

FIG. 1.

Schematic representation of the pfoR-pfoA gene region. Fragments contained within pTS302 and pJIR1972 (A), pJIR1807 and pJIR1973 (B), and pJIR2058 (C) are shown. The dotted line represents a deletion of 0.8 kb within the pfoR gene. Fragment sizes are indicated in kilobases.

FIG. 2.

Construction of a pfoR mutant by homologous recombination. A single crossover event between the pfoR gene on the JIR325 chromosome and an internal pfoR gene region located on the suicide vector pJIR2058 resulted in the construction of the pfoR mutant JIR4583 as shown.

FIG. 3.

Southern hybridization analysis. Chromosomal DNA was prepared from the wild-type strain JIR325 (WT) and the pfoR mutant JIR4583 (M), digested with EcoRI, separated by agarose gel electrophoresis, and probed with digoxigenin-labeled DNA fragments specific for the pfoR, tet(M), pfoA, plc, and virR genes as indicated. Molecular size standards are as indicated (S).