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Journal of Bacteriology, January 2005, p. 125-134, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.125-134.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

m-Xylene-Responsive Pu-PnifH Hybrid {sigma}54 Promoters That Overcome Physiological Control in Pseudomonas putida KT2442

Manuel Carmona,1 Silvia Fernández,2 María J. Rodríguez,3 and Víctor de Lorenzo3*

Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco,3 Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9,1 Instituto de Neurobiología Ramón y Cajal CSIC, Madrid, Spain2

Received 29 July 2004/ Accepted 17 September 2004

The sequences surrounding the –12/–24 motif of the m-xylene-responsive {sigma}54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH {sigma}54 promoter variants known to have various degrees of efficacy and affinity for {sigma}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 (XylR) fused to the –12/–24 region of the wild-type PnifH and its higher {sigma}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 XylR 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.

* Corresponding author. Mailing address: Centro Nacional de Biotecnología del CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 585 4536. Fax: 43 91 585 4506. E-mail: vdlorenzo{at}cnb.uam.es.

Journal of Bacteriology, January 2005, p. 125-134, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.125-134.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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