Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi

Vibrio harveyi VIB 645, which is very pathogenic towards salmonids and prod uces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated,vhhA and vhhB), whereas the majority of strains examined (11 o f 13) carried only a single hemolysin gene. Both genes from VIE 645 were cl oned and sequenced. The open reading frames (ORFs) of vhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical pol ypeptides comprising 418 amino acid residues. The VHH protein shows homolog y to the lecithinase of V, mimicus and V cholerae. Transformants of Escheri chia coli containing the ORF of either vhhA or vhhB displayed weak hemolyti c activity in rainbow trout blood agar. The hemolytic activity was very hig h when the ORF of vhhB was cloned in E, coli together with the native promo ter. Surprisingly, the level of vhh-specific RNA transcript produced by VIE 635 was found to be very low. We conclude that the hemolytic phenotype of VIE 645 is not due to increased expression of one or both copies of the vhh gene.