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A more recent version of this article appeared on January 11, 2002
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M108162200v1
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Papers In Press, published online ahead of print November 1, 2001
J. Biol. Chem, 10.1074/jbc.M108162200
Submitted on August 23, 2001
Revised on November 1, 2001
Accepted on November 1, 2001

In vivo UV-laser footprinting of the Pseudomonas putida s54 Pu promoter reveals that IHF couples transcriptional activity to growth phase

Marc Valls, Malcolm Buckle, and Victor de Lorenzo

Microbial Biotechnology, CSIC, Madrid, Madrid E-28049

Corresponding Author: vdlorenzo{at}cnb.uam.es

The occupation of the s54-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-minus 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 s54-RNAP. The results substantiate the notion that s54 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 non disruptively alterations of DNA shape in vivo.


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