Abstract
InrS is a Ni(II)-responsive, CsoR/RcnR-like, DNA-binding transcriptional repressor of the nrsD gene, but the Ni(II) co-ordination sphere of InrS is unlike Ni(II)-RcnR. We show that copper and Zn(II) also bind tightly to InrS and in vitro these ions also impair InrS binding to the nrsD operator-promoter. InrS does not respond to Zn(II) (or copper) in vivo after 48 h, when Zn(II) sensor ZiaR responds, but InrS transiently responds (1 h) to both metals. InrS conserves only one (of two) second co-ordination shell residues of CsoR (Glu98 in InrS). The allosteric mechanism of InrS is distinct from Cu(I)-CsoR and conservation of deduced second shell residues better predicts metal specificity than do the metal ligands. The allosteric mechanism of InrS permits greater promiscuity in vitro than CsoR. The factors dictating metal-selectivity in vivo are that KNi(II) and ΔGCNi(II)-InrS·DNA are sufficiently high, relative to other metal sensors, for InrS to detect Ni(II), while the equivalent parameters for copper may be insufficient for copper-sensing in S ynechocystis (at 48 h). InrS KZn(II) (5.6 × 10−13 M) is comparable to the sensory sites of ZiaR (and Zur), but ΔGCZn(II)-InrS·DNA is less than ΔGCZn(II)-ZiaR·DNA implying that relative to other sensors, ΔGCZn(II)-Sensor·DNA rather than KZn(II) determines the final detection threshold for Zn(II).
Highlights
As multiple metalloregulators from different structural families are characterized within a single organism it becomes possible to identify facets of metal homeostasis that result from their concerted actions
Orthologous NcrB represses expression of Ni(II)-efflux in Leptospirillum ferriphilum and responds to Ni(II) (Zhu et al, 2011), while InrS itself is a Ni(II)-responsive repressor of the final gene in the nrs Ni(II)-efflux operon in the cyanobacterium Synechocystis (García-Domínguez et al, 2000; Foster et al, 2012)
CsoR and RicR are paralogues that both respond to Cu(I) in M. tuberculosis (Liu et al, 2007; Festa et al, 2011)
Summary
As multiple metalloregulators from different structural families are characterized within a single organism it becomes possible to identify facets of metal homeostasis that result from their concerted actions. As multiple metal sensors from a single family are characterized in different organisms, it becomes possible to confirm (or otherwise) correlations between sequence and structure/ function that were inferred from early work. Both approaches are taken to develop an appreciation of the factors that determine the metal-selectivity of metalloregulators such as InrS. Mycobacterium tuberculosis CsoR and Escherichia coli RcnR are the founder members of the family of metalresponsive repressors that includes Synechocystis PCC 6803 ( referred to as Synechocystis) InrS (Iwig et al, 2006; 2008; Liu et al, 2007; Foster et al, 2012). Cu(I)-sensing CsoR orthologues have been characterized from Bacillus subtilis, Thermus thermophilus, Listeria monocytogenes and Streptomyces lividans (Smaldone and Helmann, 2007; Ma et al, 2009a; Sakamoto et al, 2010; Corbett et al, 2011; Dwarakanath et al, 2012)
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