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J. Biol. Chem., Vol. 278, Issue 36, 33763-33773, September 5, 2003

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Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation^*

Chiara Recchi  , Bianca Sclavi ¶, Jean Rauzier , Brigitte Gicquel  and Jean-Marc Reyrat  ||

From the Unité de Génétique Mycobactérienne, Institut Pasteur, 25, Rue du Dr. Roux, 75724 Paris cedex 15 and the ^¶Unité d'Enzymologie et Cinétique Structurale, CNRS/Ecole Normale Supérieure de Cachan, 61, Avenue du President Wilson, 94230 Cachan, France

Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing C-terminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.

Received for publication, June 6, 2003 , and in revised form, June 20, 2003.

^* This work was supported in part by NIAID, National Institutes of Health (support for sequencing the M. smegmatis and M. avium genomes) and by the European TB Vaccine Cluster. 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.

Supported by the Fondation pour la Recherche Médicale.

^|| To whom correspondence should be addressed (present address): Unité Inserm U570, Faculté deMédecine Necker-Enfants Malades, 156 Rue de Vaugirard, 75730 Paris cedex 15, France. Tel.: 33-1-40-61-53-71; Fax: 33-1-40-61-56-77; E-mail: jmreyrat{at}necker.fr.

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