Construction of Deletion Mutants of Shiga (-Like) Toxin Genes (stx-1 and/or stx-2) on Enterohemorrhagic Escherichia coli (O157:H7)

We constructed isogenic Shiga (-like) toxin (Stx-1 and/or Stx-2) gene-deletion mutants of enterohemorrhagic Escherichia coli (EHEC) GPU96MM (O157:H7). A vector with temperature-sensitive replication origin was used for the construction. The parts of stx-1 and stx-2 on the GPU96MM genome were replaced with kanamycin and chloramphenicol resistance genes, respectively. The mutants deficient in stx-1, stx-2, and both of them were named GPU993, GPU994, and GPU995, respectively. Each mutation was confirmed by the polymerase chain reaction, enzyme-linked immunosorbent assay using antibodies to B subunits of Stx-1 and Stx-2, and the cytotoxic activity of the bacterial culture supernatants toward HeLa cells was detected for GPU96MM and the mutants except for GPU995. These results indicate that GPU993 and GPU994 lack productivity for the respective toxins and GPU995, for both of them.