Cd(II)-responsive and constitutive mutants implicate a novel domain in MerR

Abstract

Expression of the Tn21 mercury resistance (mer) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by sigma70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100- and 1, 000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR's metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.

FIG. 1

The Tn21 mer operon. Arrows indicate the direction of transcription.

FIG. 2

LacZ activity of Cd(II)-responsive merR mutants in the absence of metal or when treated with Hg(II), Cd(II), or Zn(II).

FIG. 3

LacZ activity of constitutive merR mutants in the absence of metal or when treated with Hg(II), Cd(II), or Zn(II).

FIG. 4

Alignment of a metal-binding subset of the MerR family of activator-repressor proteins. Residues highlighted in black are identical, and residues highlighted in gray are functionally similar. ZntR contains a V instead of an A at the position corresponding to residue 89 in MerR (A89V). Gray lowercase letters a and d in the heptad repeats identify residues that make contact between coiled strands. The beginning and end of each protein (∼) and gaps in the alignment (.) are indicated.

FIG. 5

Amino acid changes in MerR. Mutations described in this work are shown above the amino acid sequence. Previously described mutations (20, 56, 57, 63, 68) are shown below the sequence. The 32 residues conserved in all known MerR proteins in both gram-positive and gram-negative bacteria are shown in boldface. Symbols: double underline, cysteines involved in Hg(II) binding; single underline, predicted helix; ○○○, turn of predicted helix-turn-helix (DNA binding); dotted underline, predicted coiled-coil region; □, repression deficient only; ▿, Cd(II) responsive; ○, fully constitutive; ◊, activation deficient; ▵, activation and repression deficient; , constitutive mutants constructed in an A89V or S131L background (R62Q and V124A individually combined with S131L, others each with A89V). Superscripted numbers indicate sets of double mutations; e.g., set 2 includes V109E and V124A.

FIG. 6

Coiled-coil predictions for MerR, ZntR, and SoxR, obtained by using the computer program COILS with unweighted MTK parameters (46, 47, 58). The scale for MerR and ZntR probabilities is located on the left y axis, and the scale for SoxR probability is located on the right y axis.

FIG. 7

Antiparallel alignment of two helical wheels representing the predicted coiled-coil region between residues C82 and C117 in a MerR dimer. Symbols are described in the legend to Fig. 5.