FIG 2.

Deletion of pyrE. (A) T. kivui was transformed with plasmid pMBTkv002b, which was constructed for the deletion of the pyrE gene via double homologous recombination using 1-kbp upstream and downstream flanking regions (UFR and DFR). After transformation, T. kivui was plated in the presence of 5 mM 5-fluoroorotic acid (5-FOA). (B) The loss of pyrE (573 bp) was verified by PCR using primers binding outside the flanking regions (blue arrows in panel A). Shown is the electrophoretic separation of the DNA fragments from the PCRs using genomic DNA of T. kivui DSM2030 (wild type, WT) and of two 5-FOA-resistant isolates.