Protein signatures that promote operator selectivity among paralog MerR monovalent metal ion regulators

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.

Keywords: Bacterial Signal Transduction; Copper; DNA Operators; Helix-Loop-Helix Transcription Factors; MerR Regulators; Metal Homeostasis; Microbiology; Operator Selectivity; Prokaryotic Signal Transduction; Protein-DNA Interaction.

FIGURE 1.

The N-terminal DNA-binding domain is responsible for selective sensor/operator recognition. A, sequences of the Salmonella GolS- and CueR-controlled operators. The predicted −10 and −35 elements are boxed, and the nucleotide bases at 3′- and 3-positions are highlighted. B, schematic representation of GolS, CueR, and the hybrid proteins CueR-N_S and GolS-N_R. The region corresponding to CueR or GolS are colored in black or gray, respectively. C, β-galactosidase activities (Miller units (M.U.)) from pMC1871-derived plasmids carrying lacZ fusions to CueR- or GolS-controlled promoters PgolB or PcopA, respectively. The cells (all derivatives of W3110 ΔlacZ ΔcopA cueR::cat) expressing the indicated CueR or GolS variant from a pSU36 derivative plasmid were grown overnight in LB without (−) or with the addition of 10 μm AuHCl₄ (Au) or 100 μm CuSO₄ (Cu). The data correspond to mean values of four independent experiments performed in duplicate. Error bars, S.D.

FIGURE 2.

The α2-helix determines operator specificity. A, schematic representation of the generated hybrid proteins. The different motifs or regions from the N-terminal DNA binding region of CueR and GolS are indicated. The motifs swapped in each case are shown in either black (CueR) or gray (GolS). Chimeric proteins were constructed as indicated. B, β-galactosidase activity (Miller units (M.U.)) from the PcopA or PgolB reporter fusions as in Fig. 1 expressed on the W3110 ΔlacZ ΔcopA cueR::cat cells, carrying the expression plasmids for the indicated CueR or GolS hybrid proteins, were grown overnight in LB without (−) or with the addition of 100 μm CuSO₄ (Cu). The data correspond to mean values of at least six independent experiments done in duplicate. Error bars, S.D.

FIGURE 3.

The CueR-α2_S and GolS-α2_R proteins change operator preference. A, EMSA using 6 fmol of ³²P-3′-end-labeled PCR fragment from the golB or copA promoter regions and purified CueR, CueR-α2_S, GolS, or GolS-α2_R, as indicated. Wild-type and mutant regulators were used at 0, 0.025, 0.050, 0.075, 0.100, 0.125, 0.250, 0.500, 0.750, and 1.000 μm final concentrations with PcopA and at 0, 0.002, 0.004, 0.012, 0.025, 0.050, 0.100, 0.250, 0.500, and 1.000 μm final concentrations with PgolB. The DNA-protein complex is indicated in each case. B, fluorescence anisotropy titration curves of the fluorescein-labeled PcopA (black circles) or PgolB (white circles) promoter with increasing concentrations of native or mutant transcriptional regulators, as indicated. Binding isotherms were fitted to the equation, r = r_f + (r_b − r_f) × (k_A × x/(1 + k_A × x)) by nonlinear regression. Association equilibrium constants (k_A) for each sensor/operator duplex are indicated. The binding isotherm curves are shown at the bottom. Dissociation equilibrium constants (k_D) were determined, expressing normalized anisotropy values ((k_A × x)/(1 + k_A × x)) against protein concentration at logarithmic scale.

FIGURE 4.

Amino acid residues at positions 16 and 19 of the α2-helix are essential for selective operator recognition. A, consensus motif for the α2-helix region of different metal-binding and non-metal binding MerR proteins. The residues at position 16 and 19 are shaded in black and highlighted (▼), whereas those residues conserved in the majority of the sequences are indicated in boldface type. DNA-contacting residues identified in the crystal structures of MtaN-DNA, BmrR-DNA, and SoxR-DNA complexes are highlighted by an asterisk. B, β-galactosidase activities (Miller units (M.U.)) from the PcopA or PgolB reporter fusions as in Fig. 1 expressed on the W3110 ΔlacZ ΔcopA cueR::cat cells carrying the expression plasmids for the indicated CueR or GolS hybrid or mutant proteins. Bacteria were grown overnight in LB (−) or in LB supplemented with 100 μm CuSO₄ (Cu). The data correspond to mean values of at least three independent experiments done in duplicate. Error bars, S.D. C, structural model for CueR-, CueR-α2_S-, or GolS-DNA complex. The amino acid residues at positions 16 and 19 approached the signature nucleotide base at the operator sequences. The side chain for the residues at positions 16 and 19 in each protein is shown.

FIGURE 5.

The identity of the residues at position 16 and 19 is conserved among CueR-like and GolS-like proteins. Phylogenetic tree obtained by comparison of the full-length CueR-like and GolS-like regulators. The tree was constructed by Bayesian inference as described previously (15). The α2-helix sequence for each CueR or GolS homologue was extracted and listed on the right. Residues 16 and 19 are highlighted.

FIGURE 6.

Operator discrimination among CueR- and GolS-controlled promoters depends on key α2-residues and signature nucleotide bases. A, the sequences of the native and mutant golB and copA promoters as well as the nucleotide bases that have been modified in each case are indicated. B, β-galactosidase activities (Miller units (M.U.)) were determined on ΔcueR ΔcopA ΔlacZ strains carrying each of the reporter plasmids harboring the native (pPgolB or pPcopA) or mutant (pPgolB_CC or pPcopA_AT) versions of the promoters and expression plasmids for the indicated regulator protein. Bacteria were grown overnight in LB (−) or in LB plus 100 μm CuSO₄ (Cu). The data correspond to mean values of at least three independent experiments done in duplicate. Error bars, S.D.