FIG 1.

Schematic chart for the construction of the tetH knockout mutant. The suicide plasmid pKIT-KtetH was introduced into A. ferrooxidans ATCC 23270 and integrated into the genome, producing either one of the two types of single recombinants (A and B). Then plasmid pMSD1-I-SceI, carrying the I-SceI gene, was introduced into the single recombinant, resulting in a double-stranded break at the I-SceI site and stimulating the occurrence of the second recombination. As a result, either a tetH gene knockout mutant or a restoration of the wild type was generated, and the kanamycin-resistant gene (KmR) was simultaneously eliminated from the chromosome. The positions of the primers used to identify various recombinants are indicated.