Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures

- LasR was purified from Escherichia coli cultures grown in the absence of added homerine lactones.
- Biophysical analyses indicate that the purified LasR is functional.
- Purified protein binds to promoter DNA.
- Purified protein showed significant thermal stability and is present as a mixture of monomers and dimers.

Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.