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J. Biol. Chem., Vol. 279, Issue 9, 7777-7784, February 27, 2004

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Novel Physiological Modulation of the Pu Promoter of TOL Plasmid

NEGATIVE REGULATORY ROLE OF THE TURA PROTEIN OF PSEUDOMONAS PUTIDA IN THE RESPONSE TO SUBOPTIMAL GROWTH TEMPERATURES^*

Emanuela Rescalli, Silvia Saini, Cristina Bartocci, Leszek Rychlewski, Víctor de Lorenzo, and Giovanni Bertoni¶

From the Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita' degli Studi di Milano, via Celoria 26, 20133 Milan, Italy, BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan, Poland, and the Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, 28049 Madrid, Spain

From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression. Ectopic expression of TurA both in P. putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity. turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression. These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures. Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously.

Received for publication, September 24, 2003 , and in revised form, December 3, 2003.

^* This work was supported by QLK3-CT-2000-00170 Contract of the European Union and by "Ministero dell'Istruzione, dell'Università e della Ricerca, Roma" (Fondo per G4 Investimenti della Ricerca di Base Grant RBAU01KHM2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 39-0250315027; Fax: 39-0250315044; E-mail: giovanni.bertoni{at}unimi.it.

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