The effect of ANR on the expression of certain P. putida oxidases was first investigated by RT-PCR, performed on one gene for each oxidase. Data indicated up-regulation of gene ccoO1 (Cbb3-1 oxidase) and down-regulation of genes cyoA and cioA (Cyo and CIO oxidases, respectively) with no noticeable effect on other tested genes. A comparable decrease in the cytochrome b and c content of P. putida membranes in Cbb3-1-/anr+ and Cbb3-1+/anr- strains confirmed Cbb3-1 up-regulation by ANR. S1 nuclease protection assays were performed with ANR mutant strain RNA to identify the transcription start sites of P. putida cioAB and ccoN1O1Q1P1 genes. Once identified, visual inspection (based on the P. aeruginosa ANR consensus binding site) yielded two putative ANR binding sites for PcioA and one for PccoN1 in P. putida. DNase I footprinting identified two binding sites upstream of the PcyoA promoter, and confirmed the two binding sites at PcioA and the one at PccoN1. EMSA further confirmed the two binding sites at PcyoA and PcioA.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|
|TTGATCCAGATCAA||cyoB, cyoA, cyoC, cyoD, cyoE-2,||
|TTGAGGCCTATCAA||cyoA, cyoB, cyoC, cyoD, cyoE-2,||
|TTGATCCCCGTCAA||ccoN-1, ccoO-1, ccoQ-1, ccoP-1,||