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J. Biol. Chem., Vol. 282, Issue 44, 32298-32310, November 2, 2007

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Mycobacterial Cells Have Dual Nickel-Cobalt Sensors

SEQUENCE RELATIONSHIPS AND METAL SITES OF METAL-RESPONSIVE REPRESSORS ARE NOT CONGRUENT^*

Duncan R. Campbell¹, Kaye E. Chapman¹, Kevin J. Waldron, Stephen Tottey, Sharon Kendall^¶, Gabriele Cavallaro^||, Claudia Andreini^||, Jason Hinds^**, Neil G. Stoker^¶, Nigel J. Robinson², and Jennifer S. Cavet³

From the Life Sciences, University of Manchester, 1.800 Stopford Building, Manchester M13 9PT, United Kingdom, the Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle NE2 4HH, United Kingdom, ^¶The Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom, the ^||Magnetic Resonance Centre, University of Florence, and Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine Paramagnetiche, 50019 Sesto Fiorentino, Florence, Italy, and the ^**Bacterial Microarray Group, St. George's University of London, London SW17 0RE, United Kingdom

A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterized from mycobacteria. Mutants of Mycobacterium tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays and fluorescence anisotropy show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. Incubation of KmtR with cobalt inhibits DNA complex assembly and metal-protein binding was confirmed. KmtR is the second, to NmtR, characterized ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, whereas NmtR retains a response to nickel and cobalt under these conditions. KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB-related proteins are listed in databases. KmtR has none of the previously defined metal-sensing sites. Substitution of His⁸⁸, Glu¹⁰¹, His¹⁰², His¹¹⁰, or His¹¹¹ with Gln generated KmtR variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory motifs revealing that only the latter should be used to predict metal sensing.

Received for publication, April 25, 2007 , and in revised form, August 22, 2007.

^* This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) Plant and Microbial Sciences committee and Agri-Food committee and BBSRC studentships (to D. R. C. and K. E. C.). The Wellcome Trust for funding the multicollaborative microbial pathogen microarray facility under its Functional Genomics Resources Initiative. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4 and Table S1.

¹ These authors have contributed equally to this work.

² To whom correspondence may be addressed: Tel. 44-191-222-7695; Fax: 44-191-222-7424; E-mail: n.j.robinson{at}newcastle.ac.uk.

³ To whom correspondence may be addressed: Tel. 44-161-275-51543; Fax: 44-161-275-5656; E-mail: jennifer.s.cavet{at}manchester.ac.uk.

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