Involvement of σ54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter



The σ54-dependent Pu promoter of the TOL plasmid pWWO of Pseudomonas putida becomes activated by the prokaryotic enhancer-binding XyIR protein when cells encounter m-xylene in the medium. However, even in the presence of the aromatic inducer, Pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium. Various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expression of Pu is limited during the exponential phase of growth. A truncated and fully constitutive XyIR derivative deleted of its signal reception N-terminal domain was found to be subjected to the same exponential silencing as the wild-type XyIR when exposed to m-xylene. This indicated that the phenomenon is not due to a late activation of XyIR by the aromatic effector. A Pu variant in which the integration host factor (IHF)-binding site had been functionally replaced by a statically curved DNA segment showed the same induction pattern, thus ruling out variations in the intracellular levels of IHF changes during growth as the element responsible for the inactivity of Pu in rapidly growing cells. On the contrary, overproduction of the σ54 factor allowed Pu expression during exponential phase. As σ54 protein levels remained approximately constant during growth, the exponential silencing of Pu could be caused ultimately by changes in the activity of the factor itself. This effect may not be exclusive to Pu, but could be a general co-regulation mechanism in σ54-dependent promoters that connects transcription of a specific set of genes with the general physiological status of the cells.