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Originally published In Press as doi:10.1074/jbc.M108162200 on November 1, 2001

J. Biol. Chem., Vol. 277, Issue 3, 2169-2175, January 18, 2002
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In Vivo UV Laser Footprinting of the Pseudomonas putida sigma 54 Pu Promoter Reveals That Integration Host Factor Couples Transcriptional Activity to Growth Phase*

Marc VallsDagger , Malcolm Buckle§, and Víctor de LorenzoDagger

From the Dagger  Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain and § Enzymologie et Cinétique Structurale, UMR 8532 du CNRS, Institut Gustave Roussy, 94805 Villejuif, France

The occupation of the sigma 54-dependent Pu promoter of Pseudomonas putida by the integration host factor (IHF) under different growth conditions has been monitored in its native state and stoichiometry (i.e. monocopy) with UV laser footprinting technology. We present evidence that an abrupt change in intracellular IHF concentrations occurs when P. putida cells enter stationary phase. This change results in enhanced binding of the factor to the promoter and in the ensuing bending of the target DNA. Since Pu activity depends rigorously on DNA bending, promoter occupation is in turn translated into a much higher transcriptional output when cells leave exponential growth. Inspection of the residual activity of Pu in an IHF- strain reveals that IHF predominantly locks the capacity of the promoter to specific growth stages and also that additional physiological signals are entered in the system through sigma 54-RNA polymerase. The results substantiate the notion that sigma 54 promoters process metabolic co-regulation signals through factor-induced changes in the architecture of the cognate DNA region. Further, they validate UV laser technology as a suitable tool to visualize nondisruptive alterations of DNA shape in vivo.

* This work was supported by European Union Contracts QLK3-CT2000-00170 and QLK3-CT1999-00041, by Grant BIO98-0808 of the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT), and by the Strategic Research Groups Program of the Autonomous Community of Madrid. The support of the Fondation de la Recherche Medicale (FRM) for the award of a grant (to M. B.) for the establishment of a new laboratory is acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The 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.: 34-91-585-4536; Fax: 34-91-585-4506; E-mail: vdlorenzo@cnb.uam.es.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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