The contribution of two unrelated -haemolytic toxins to virulence was assessed in a suckling mouse model. The first haemolysin gene, isolated from an A6 cosmid bank, encoded a potential gene product of 621 amino acids and a predicted molecular size of 69.0 kDa. The inferred amino acid sequence showed 89% identity to the AHH1 haemolysin of ATCC 7966, and 51% identity to the HlyA haemolysin of El Tor strain O17. The second haemolysin gene (designated ), which encodes aerolysin, a pore-forming toxin, was partially cloned by PCR for the purpose of mutant construction. This PCR product was a 1040 bp fragment from the C-terminal region of It is proposed that the 69.0 kDa -HlyA-like haemolysin gene be termed to contrast with the terminology for the aerolysin. A suicide vector was used to inactivate both the and genes in A6. When assessed in the suckling mouse model, only the double mutant showed a statistically significant reduction in virulence - a 20-fold change in LD (Scheffe test, <0.05). Cytotoxicity to buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the double mutants. This is the first report of cloning and mutagenesis of two unrelated haemolytic toxin genes in the same strain of a mesophilic aeromonad. For , a two-toxin model provides a more complete explanation of virulence.


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