Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila

Abstract

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage λEMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli[pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [ 35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56°C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and λ bacteriophage libraries. We localized this heat-stable (56°C/20 min) enterotoxin to a 4.8-kb SalI- BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI- BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.