CO-EVOLUTION OF THE REGULATOR/OPERATOR SELECTIVITY AMONG MerR MONOVALENT METAL-ION TRANSCRIPTION FACTORS

HUMBERT, MARÍA V.; CHECA, SUSANA K.; SONCINI, FERNANDO C.

Rosario, Santa Fe

Congreso; IX Congreso de Microbiología General; 2013

Metal ions are essential cellular constituents but they can be toxic at high concentration. Therefore, all cells possess a battery of highly specific regulatory proteins that control metal ions homeostasis by modulating the balance of intake/efflux of a particular metal ion. This control is fundamental to guarantee the acquisition of the required amount of essential metals and, at the same time, to avoid toxicity caused either by their overload or by the presence of toxic metals. Resistance to excess of the essential copper ion or the presence of toxic silver and gold ions in Salmonella is coordinated by two paralogous transcription factors of the MerR family, CueR and GolS. We previously found that, although highly similar in sequence, and in spite of sharing similarity at their target binding sites, each factor displays in vitro higher affinity for their innate operators than for the binding sites of the non-cognate regulator. Selective promoter recognition depends on the presence of specific bases located at positions -3 and +3 within the operators they interact with. Here, by using fragment swapping and site directed mutagenesis plus reporter-gene expression assays, as well as in vitro protein-DNA interaction assays, we identified the amino acid residues within the N-terminal DNA binding domain of these sensor proteins that are directly involved in operator discrimination. Our studies indicate that the selective operator activation relies on the alpha2-helix. We uncover that a single residue at position 16 within the alpha2-helix, which is an invariant M in all GolS xenologues while in CueR-like proteins varies between a T, an A, or an S, is essential for binding to the cognate target binding sites in vivo, while residue at position 19 finely tunes the regulator/operator interaction.An in silico modeling performed for both CueR and GolS reinforces residues 16 and 19 as candidates for directing selective recognition of target operators. These results highlight the molecular bases of regulator/operator selectivity among paralogous MerR regulators and indicate that co-evolution of a regulator and its cognate operators within the bacterial cell provides the conditions to avoid cross-recognition and guarantees the proper response to metal injury.