New insights into the activation of o‐xylene biodegradation in Pseudomonas stutzeri OX1 by pathway substrates

The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o‐xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set‐up included the prokaryotic enhancer‐binding protein TouR, the σ54‐dependent PToMO promoter and the σ54‐containing RNA polymerase. With this system we prove that direct binding of 2‐methylphenol (o‐cresol) to TouR is the only regulatory step for activation of PToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate PToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o‐cresol addition. The results presented here support the view that TouR‐like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.