m-Xylene-responsive Pu-PnifH hybrid [[sigma].sup.54] promoters that overcome physiological control in Pseudomonas putida KT2442

The sequences surrounding the -12/-24 motif of the m-xylene-responsive [[sigma].sup.54] promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH [[sigma].sup.54] promoter variants known to have various degrees of efficacy and affinity for [[sigma].sup.54]-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XyIR) fused to the -12/-24 region of the wild-type PnifH and its higher [[sigma].sup.54]-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XyIR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.