Cloning and nucleotide sequence analysis of the genes determining verocytotoxin production in a porcine edema disease isolate of Escherichia coli

Abstract

The structural genes determining the edema disease principle were cloned from the total cellular DNA of Escherichia coli strain 412 (O139:K82) isolated from a case of porcine edema disease. An assay for cytotoxicity in Vero cells was used to detect the edema disease principle. A 7.5 kb EcoRI-SalI fragment specifying cytotoxin production was subcloned in pUC18. Sequences which specified production of cytotoxin were localized to a 0.9 kb region by transposon Tn5 mutagenesis. A 2.4 kb EcoRI-BglII fragment encompassing this region was subcloned into pUC18. Using nucleotide sequence analysis, two open reading frames separated by 12 bp were identified. They encoded proteins of 319 (A subunit) and 87 (B subunit) amino acids which both had N-terminal sequences typical of E. coli signal peptides. Comparison of these with the published sequence for the Shiga-like toxin II (SLT-II) showed 91% overall nucleotide sequence similarity. The nucleotide sequence similarity extended to 200 base pairs upstream of the putative A subunit translational start site suggesting a common regulatory mechanism. The deduced amino acid sequences of the processed A and B subunits had 94% and 84% similarity, respectively. These findings confirm the close genetic relationship between SLT-II and edema disease principle.