This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cells contain regulatory proteins to detect and respond to deficiency or excess of essential metals to maintain sufficient atoms to satisfy the requirements of metalloproteins avoiding toxicity. The ArsR-SmtB family of transcriptional repressors associate with the promoters of genes encoding proteins involved in the efflux and/or sequestration of excess metal. De-repression occurs when the repressors bind metal effectors coincident with the number of atoms exceeding an optimal cell quota. SmtB-mediated repression is alleviated by Zn(II), ZiaR by Zn(II), ArsR by As(III), Sb(III), and Bi(III), CadC by Cd(II), Pb(II) and Bi(III), and CzrA by Co(II) and Zn(II) and many others. Clearly these sensors discriminate between different metals in vivo, but the factors dictating which inorganic elements elicit responses remain to be defined. 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 also confirmed. KmtR was the second metal sensing repressor protein for nickel and cobalt in M. tuberculosis after the already discovered NmtR protein. The presence of two metal sensing repressor proteins suggesting a special significance of cobalt and nickel ions in this pathogen. KmtR has tighter affinities for nickel and cobalt than NmtR. This result is 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.
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