Gel shift competition assays. (A) Graphic representation of the pht region. Each arrow represents an individual gene, with the direction of the arrow indicating the direction of transcription. Red arrows indicate genes whose function have been previously reported (B) Detailed view of the phtD operon upstream region indicating the P
fragments used as unlabeled DNA competitors. The blue bars represent the probes able to compete the DNA-protein complex, while the red bars represent probes unable to compete the complex. The fragment "I" corresponding to the region of 104 bp defined as the binding site for protein. (C) An example of gel shift competition assays used in this case, fragment "I" as competitor. These assays were carried out using crude protein extracts of P. syringae pv. phaseolicola NPS3121 grown at 18°C in M9 minimal medium and increasing concentrations of different unlabeled DNA fragments indicated in (B) as competitors. We show the gel shift competition assay performed with the 104 bp probe, which was identified as the minimum region necessary to bind a putative transcription factor. The concentration of unlabeled DNA competitors was as follows: lanes 1 and 2, no competitor DNA; lane 3, 25 ng (0.36 pmol); lane 4, 50 ng (0.73 pmol); lane 5, 60 ng (0.87 pmol); lane 6, 100 ng (1.46 pmol); lane 7, 150 ng (2.18 pmol); and lane 8, 200 ng (2.9 pmol).