FIG 2.

Principle of the counterselection system for targeted gene deletion in T. thermophilus HB27 demonstrated with pLEI271.7. The binding sites of the primers s10068 and s10069 used for analytical PCR of the mutant strains are indicated. The integrative vector pLEI271.7 contains the flanking regions of crtY (encoding lycopene β-cyclase), a kanamycin resistance determinant, and codA (encoding cytosine deaminase) as a counterselectable trait. The plasmid integrates into the genome by homologous recombination with the upstream or downstream flanking region of crtY. The transformants are resistant to Km and sensitive to 5-FC. The cell loses the integrated plasmid by a second homologous recombination. Depending on where the recombination occurs, crtY is cut out, together with the plasmid, or the wild-type situation is restored. The ΔcrtY strain, as well as the reconstituted wild type, is resistant to 5-FC and sensitive to Km.