Primer extension and S1 protection assay identified the transcription start site of P. aeruginosa PA01 gene lasB, and comparison of the wild-type extension product with the (non-existent) product of the lasR-mutant strain validated lasB up-regulation by lasR. Inspection of the promoter region identified a putative site (OP2) upstream of and similar to one published previously (OP1), both comparable to the V. fischeri lux operator. Beta-gal results of site-directed mutagenesis at positions 3, 5, and 18 in OP1 demonstrated reduced expression (98%, 27%, and ~100% reduction, respectively) with positions 3 (cytosine) and 18 (guanine) critical for expression. Double-point mutation of OP2 resulted in 20% loss of expression, though partial and total deletion assays resulted in 66% and 93% expression loss, respectively.
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|