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Journal of Bacteriology, September 2003, p. 5357-5362, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5357-5362.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis FurA Autoregulates Its Own Expression

Claudia Sala,1 Francesca Forti,1 Elisabetta Di Florio,1 Fabio Canneva,2 Anna Milano,2 Giovanna Riccardi,2 and Daniela Ghisotti1*

Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Milan,1 Dipartimento di Genetica e di Microbiologia A. Buzzati Traverso, Università di Pavia, Pavia, Italy2

Received 11 April 2003/ Accepted 25 June 2003

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.

* Corresponding author. Mailing address: Università degli Studi di Milano, Dipartimento di Genetica e di Biologia dei Microrganismi, Via Celoria 26, 20133 Milan, Italy. Phone: 39-02-50315020. Fax: 39-02-50315044. E-mail: Daniela.Ghisotti{at}unimi.it.

Journal of Bacteriology, September 2003, p. 5357-5362, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5357-5362.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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