Abstract
Two paralog transcriptional regulators of the MerR family, CueR and GolS, are responsible for monovalent metal ion sensing and resistance in Salmonella enterica. Although similar in sequence and also in their target binding sites, these proteins differ in signal detection and in the set of target genes they control. Recently, we demonstrated that selective promoter recognition depends on the presence of specific bases located at positions 3' and 3 within the operators they interact with. Here, we identify the amino acid residues within the N-terminal DNA-binding domain of these sensor proteins that are directly involved in operator discrimination. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins but absent in all CueR-like xenologs, is the key to selectively recognize operators that harbor the distinctive GolS-operator signature, whereas the residue at position 19 finely tunes the regulator/operator interaction. Furthermore, swapping these residues switches the set of genes recognized by these transcription factors. These results 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.
Highlights
Two nucleotide bases distinguish promoters controlled by paralog MerR monovalent metalloregulators, avoiding cross-activation
Selective Regulator/Operator Recognition Is Directed by Amino Acids within the ␣2-Helix of the Sensor Protein—Salmonella CueR and GolS can distinguish their target operators from those of the paralog regulator by selectively recognizing two nucleotide bases located at the 3Ј- and 3-positions from the center of the operator (Fig. 1A) [15]
The encoding mutant alleles were cloned in plasmids and introduced either into an S. enterica serovar Typhimurium ⌬gol ⌬cueP ⌬cueR-copA or into an E. coli W3110 ⌬cueR-copA ⌬lacZ strain harboring lacZ reporter fusions to copA or golB promoters. (The use of strains deleted in copA helped to minimize differences in intracellular copper levels derived from partial activation of the copper transporter by the regulator variants.) The cells were grown in LuriaBertani broth (LB) in the absence or presence of either 10 M AuHCl4 or 100 M CuSO4, concentrations required to attain the maximal induction of the reporter genes [7, 14]
Summary
Two nucleotide bases distinguish promoters controlled by paralog MerR monovalent metalloregulators, avoiding cross-activation. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins but absent in all CueR-like xenologs, is the key to selectively recognize operators that harbor the distinctive GolS-operator signature, whereas the residue at position 19 finely tunes the regulator/operator interaction Swapping these residues switches the set of genes recognized by these transcription factors. Transcriptional regulators of the MerR family modulate transcription in response to different environmental signals, including heavy metal ions, organic compounds, or oxidative stress [1] These proteins have a structurally conserved Nterminal DNA-binding domain with two helix-turn-helix. To identify the amino acid residues involved in operator discrimination, here we constructed a series of GolS and CueR hybrid proteins replacing different portions of the DNA-binding domain of each regulator by the same region of the paralogous protein and tested their ability to direct expression from the golB or copA promoter. Our results provide additional evidence of the co-evolution of both the MerR regulators and the regulated genes to avoid cross-recognition and guarantee a proper response to metal injury
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