Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda

Abstract

Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional β-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed by in situ hemolysin activity analysis. The cloned fragment containing the β-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the β-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436-bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804 Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5α(pETH3E) is very likely the β-hemolysin encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5α(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli.