Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum

A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of lambda gt10. The DNA insert of a hemolysin-positive (Hly+) lambda-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly+ phenotype on E. coli. Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an alpha toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the alpha toxin secreted by C. septicum. Taken together, the data indicated that plasmid pCS21 indeed encoded an alpha toxin gene of C. septicum.