Enhancer-like activity of AlgR1-binding site in alginate gene activation: Positional, orientational, and sequence specificity

Significant activation of promoters of alginate genes such as algD or algC occurs in mucoid Pseudomonas aeruginosa during its proliferation in the lungs of cystic fibrosis patients. These promoters have been shown to be responsive to environmental signals such as high osmolarity. The signaling is mediated by a so-called two-component signal transduction system, in which a soluble protein, AlgR2, undergoes autophosphorylation and transfers the phosphate to a DNA-binding response regulator protein, AlgR1. The phosphorylated form of AlgR1 has a high affinity for binding at upstream sequences of both the algC and algD promoters. Two AlgR1-binding sites (ABS) have been reported upstream of the algC gene. One of the two ABSs (algC-ABS1, located at -94 to -81) is critical for the algC activation process, while the second ABS (algC-ABS2, located at +161 to +174) is only weakly active. We now report the presence of a third ABS within the structural gene of algC, and this ABS (algC-ABS3) is also important for algC promoter activation. algC-ABS1 can be replaced functionally by algC-ABS2, algD-ABS1, or algD-ABS2 and somewhat weakly by algD-ABS3. Introduction of a half-integral turn in the DNA helix between the algC site of transcription initiation and algC-ABS1 allowed only slight reduction of promoter activity, suggesting that the binding site could be appreciably functional even when present in the opposite face of the helix. Activation of the algC promoter is independent of the relative location (upstream or downstream of the mRNA start site), the number of copies, or the orientation of algC-ABS1, suggesting that it behaves like a eukaryotic enhancer element in promoting transcription from the algC promoter.