Molecular genetics of Clostridium perfringens epsilon-toxin

Hunter, Sophie Emma Clare (1992) Molecular genetics of Clostridium perfringens epsilon-toxin. University of Southampton, Doctoral Thesis.

Abstract

The sequence of 20 amino acids from the N-terminus of Clostridium perfringens ϵ-toxin was determined. A degenerate 23 base oligonucleotide probe was designed from the amino acid sequence data and used to isolate a fragment of the ϵ-toxin gene (etx) from a library of HindIII digested DNA from C. perfringens type B strain NCTC 8533. Subsequently, the complete gene was isolated on a 1.65kb Sau3A fragment, which was cloned into pUC18 to form the recombinant plasmid pSH109. A 1.25kb Sau3A fragment located downstream of the etx gene was also cloned. The sequence of 1862 nucleotides containing the etx gene and its flanking regions was determined. The amino acid sequence derived from the gene contained a region of 20 residues identical to the N-terminal sequence of ϵ-toxin. The first 32 amino acids encoded by the open reading frame contained features characteristic of a signal peptide. The predicted molecular weight of the mature exported protein was 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli igma^70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product accumulated in the periplasmic space. Cloned ϵ-toxin had a molecular weight and isoelectric point similar to those of the native protein, and reacted with ϵ-toxin-specific monoclonal antibodies. Southern hybridisation experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce ϵ-toxin. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus insertion sequence IS256.

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