IDENTIFICATION AND MOLECULAR ANALYSIS OF A LOCUS THAT REGULATES EXTRACELLULAR TOXIN PRODUCTION IN CLOSTRIDIUM-PERFRINGENS

The anaerobic bacterium Clostridium perfringens mediates clostridial m yonecrosis, or gas gangrene, by producing a number of extracellular to xins and enzymes. Transposon mutagenesis with Tn916 was used to isolat e a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any de tectable perfringolysin 0 activity. Southern hybridization revealed th at a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens thi s fragment was able to complement the Tn916-derived mutation. Transfor mation of the mutant with plasmids containing the 2.7kb HindIII fragme nt, or the 4.3kb PstI fragment, resulted in toxin and enzyme levels gr eater than or equal to those of the wild-type strain. The PstI fragmen t was sequenced and found to potentially encode seven open reading fra mes, two of which appeared to be arranged in an operon and shared sequ ence similarity with members of two-component signal transduction syst ems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the re sponse regulator family of proteins. The next gene, virS, into which T n916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51274. The putative VirS protein h ad sequence similarity to sensor proteins and also contained a histidi ne residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model imp licated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator sy stem that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A mo del that described the regulation of extracellular toxin production in C. perfringens was constructed.