Transient XylR binding to the UAS of the Pseudomonas putida sigma54 promoter Pu revealed with high intensity UV footprinting in vivo.

The binding of the transcriptional regulator XylR to its cognate upstream activating sequences (UAS) of the sigma54-dependent promoter Pu of Pseudomonas putida has been examined in vivo in single copy gene dose and stoichiometry. To this end, we have employed a novel in vivo genomic footprinting procedure that uses short exposures of bacterial cells to diffuse high intensity UV light that causes formation of TT or TC dimers. In contrast to simpler models for activation of sigma54-dependent promoters, our results clearly indicate that the XylR protein is not permanently bound in vivo to its target sites in Pu. On the contrary, the UAS appear to be mostly unoccupied at all growth stages. This is in contrast to the integration host factor (IHF), which binds Pu strongly in vivo at stationary phase, as also revealed by UV footprinting. Only overexpression of XylR altered the photoreactivity of the corresponding DNA region to report stable binding of the regulator to the UAS. However, the presence of aromatic XylR inducers reversed the forced occupation caused by increased levels of the activator. These results are compatible with the notion that XylR interacts very transiently with the UAS and detaches from the promoter during transcriptional activation of Pu.