Fig 1.

Schematic diagram for construction of the pfkB-knockout mutant. The suicide plasmid pKOT was introduced into A. ferrooxidans ATCC 23270 and integrated into the genome, resulting in either one of the two possible single recombinants. Then plasmid pMSD1-I-SceI, which expresses the I-SceI endonuclease, was introduced into the single recombinant, producing a double-stranded break at the I-SceI site and stimulating the host recombinational repair machinery. This second recombination event generated a mutant or resulted in reversion to wild type. The integrated kanamycin resistance gene was eliminated in any of the types of second recombinants. The annealing sites for the primers used to identify various types of recombinants are indicated.