In vitro properties and in vivo responses of CoaR, ZiaR & Zur (trans-family metal-sensing)

Abstract

In prokaryotic organisms metal-sensing transcriptional regulators must respond selectively to metal ions. The factors which govern selectivity across different sensor families from Synechocystis sp. PCC 6803 have been investigated using three sensor proteins; ZiaR, an ArsR-SmtB Zn2+-de-repressor; Zur, a Fur-like Zn2+ co-repressor and CoaR, a MerR-like Co2+-dependent activator. Zn2+ bound and allosterically regulated recombinant ZiaR and Zur in vitro. Cu+ failed to allosterically regulate recombinant Zur and inhibited Zn2+-mediated allostery in vitro. In Δatx1ΔgshB Synechocystis cells, proposed to be defective in normal cytosolic Cu+ trafficking, copper toxicity and inhibition of Zn2+-mediated repression of Zur-regulated gene expression was detected, consistent with inhibition of Zn2+-sensing in vivo by Cu+ normally bound to Atx1. Cu+ bound in preference to Zn2+ at the α3N sites of recombinant ZiaR and was allosterically ineffective; however, divalent metal binding and allostery were retained at the α5 sites in vitro. In Δatx1 Synechocystis cells, enhanced ZiaR-dependent expression was observed with no copper-dependency suggesting Atx1 also sequestered Zn2+. Hence, Atx1 is proposed to restrict access to Zn2+ and Cu+ ions in vivo. Co2+ bound to recombinant Zur and ZiaR, impaired ZiaR binding to DNA in vitro but ZiaR does not respond to Co2+ in vivo. Unexpectedly, the Co2+ affinity of recombinant CoaR was weaker than ZiaR and Zur, and Zur acquired Co2+ in preference to CoaR following direct competition. ZiaR and Zur are soluble however CoaR is membrane associated with predicted hydrophobic regions and requires a non-ionic detergent for preparation in vitro. Membrane-localised CoaR is proposed to exist in an intracellular niche in which [Co2+] >> CoaR KCo and is thus sufficient to activate the protein. Zn2+ binds to CoaR more tightly than Co2+ but ZiaR and Zur out-compete CoaR for Zn2+ Thus, in vivo responses to Zn2+ correlate with relative affinities for this group of sensors.