[Cloning and sequencing of hemolysin gene from clinical Aeromonas hydrophila]

Aerolysin is one of the important virulence factors for Aeromonas hydrophila infection. To understand the characteristics of the aerolysin gene in clinical A. hydrophila, a genomic library was constructed by using pUC19 as cloning vector. The positive clone containing the aerolysin gene was selected by the colony hybridization method, using the aerolysin probe (an 48 mer oligonucleotide) of aquatic A. hydrophila. Four positive out of 1,200 transformants were obtained from the primarily screened genomic library, and were further identified for specific binding capability by the dot blot hybridization method. One of three positive clones, designated as pAH-1, was further analyzed by the restriction mapping technique. For further sequencing the aerolysin gene nucleotides, the 3.0 kb fragment of A. hydrophila isolated from pAH-1 was cloned into pBluescriptII, pKS+, resulting in plasmid pKAH-1. Subclones of pKAH-1 were constructed and determined for the presence of 48 mer aerolysin gene sequence by dot blot hybridization method. These (subclones pKS+SS and pSK+SS) were selected for nucleotide sequencing by single-stranded dideoxy sequencing method. The nucleotide sequence similarity coefficient between clinical and aquatic strains of A. hydrophila was 76%; whereas between clinical strains of A. hydrophila and A. sobria was 71%. Since the length of the A. hydrophila DNA fragment cloned into pAH-1 is 3.0 kb, a larger size than the aerolysin gene of aquatic A. hydrophila (1.45 kb) and, also, includes 3' region of aerolysin gene in aquatic A. hydrophila, the indication is that the clone, pKAH-1, contains an entire aerolysin gene of A. hydrophila. The similarity between a small region (containing 8 amino acids) of the aerolysin in the clinical A. hydrophila and the alpha toxin of Staphylococcus aureus suggests that there is functional significance in this region.