Mycobacterial Cells Have Dual Nickel-Cobalt Sensors

Abstract

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 His 88 , Glu 101 , His 102 , His 110 , or His 111 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.

Evolution of Metal(loid) Binding Sites in Transcriptional Regulators

“…They respond to a number of transition metals, heavy metals, and metalloids (1-3). Only a few have been examined at the structural level (9,23,24), but a striking observation is that, although they all appear to have evolved from a common winged helix repressor, their metal-(loid) binding appears to have evolved independently from each other and occurs in different spatial locations in their respective structures (3,12,25) (Fig. 8).…”

“…This site, however, is identical to the regulatory Zn(II) site of SmtB from Synechococcus PCC 7942 (10). Another member of the ArsR/SmtB family is the ArsR from Acidithiobacillus ferrooxidans (AfArsR), 3 which has three cysteine residues (Cys 95 , Cys 96 , and Cys 102 ) at the dimer interface rather than in or near the DNA binding domain (11). These three cysteine residues form a three-coordinate or S 3 binding site for trivalent metalloids (12).…”

“…As a consequence, nearly all organisms have genes that confer resistance to arsenic. Environmental arsenic is sensed by members of the ArsR/SmtB family of metalloregulatory proteins (1)(2)(3). These winged helix repressor proteins specifically bind to arsenic and other toxic metals.…”

Nickel and Human Health

“…The importance of metal ion homeostasis for M. tuberculosis is underlined by the observation that its genome encodes an atypically large number (at least twelve) metal sensors of the ArsR-SmtB family [ 144 ], allowing to speculate that these proteins enable the pathogen to respond rapidly to metal fl uxes in the phagosome [ 166 ]. Notably, two of these metal-dependent transcription factors, named NmtR and KmtR, specifi cally respond to Ni 2+ and Co 2+ concentrations and regulate the expression of membrane proteins -an ATPase effl ux pump (NmtA) and a cation diffusion facilitator (CDF), respectively -that control metal ion effl ux from the cell [ 166 ].…”

“…Notably, two of these metal-dependent transcription factors, named NmtR and KmtR, specifi cally respond to Ni 2+ and Co 2+ concentrations and regulate the expression of membrane proteins -an ATPase effl ux pump (NmtA) and a cation diffusion facilitator (CDF), respectively -that control metal ion effl ux from the cell [ 166 ].…”