Cd-specific mutants of mercury-sensing regulatory protein MerR, generated by directed evolution

Abstract

The mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via the mer operator/promoter. An Escherichia coli cell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.

Fig. 1.

Amino acids targeted in each MerR mutant library and their positions in the model of MerR. (a) The schematic shows all six α helices of MerR (colored as described for panel b) and the positions of the restriction enzymes used during library construction. Helices α3, α4, α5, and α6 are underlined in the presented MerR sequence. The locations of the Cd-responsive single mutations and a double mutation (G79S and M106I [in boldface]) described by Caguiat et al. (7) are shown above the MerR sequence. The numbers represent the different mutational strategies used in library construction: 1, randomized amino acids; 2, doped amino acids; and 3, amino acids with limited changes. The parental amino acid sequences are shown as Xs. (b) A model of MerR generated using the crystal structure of CueR (8) as a template. The ribbon diagram shows one protomer in color and the other in gray. The residues targeted in our mutagenesis strategy are shown in red on the colored protomer and in raspberry on the gray protomer. Residues C82, K99, M106, C117, and C126 are presented in stick format in both protomers.

Fig. 2.

Properties of the cells sorted from libraries A, B, and C after the first and second rounds of FACS. Sorted cells were preliminarily tested with a luciferase assay. The letters given after the name of the library (A, B, and C) represent the sorting experiments, D represents the dimmest bacteria that were sorted from Hg-induced cells, and B represents the brightest bacteria that were sorted from Cd-induced cells. The data compare the percentages that each value contributed to the total. The total number of tested colonies is given as a number at the top of each column. The number of mutants sequenced is shown under the bar representing each sorting step. The percentage of mutants having identical sequences is shown in parentheses.

Fig. 3.

Response curves of wt MerR and the Cd-specific MerR mutants found after the first and second mutation rounds. (a) wt MerR. (b) Vector with wt MerR deleted from the reporter gene plasmid. The curve shows continuous expression of the firefly luciferase gene from MerOP. The MerR mutants obtained after the first mutagenesis round were A_D23 (c) and A_D24 (d), and those obtained after the second mutagenesis round were B_D20 (e), B_DB4 (f), B_DB10 (g), B_DB17 (h), B_DB26 (i), B_DB27 (j), B_DB29 (k), and C_DB21 (l). The responses to HgCl₂ and CdCl₂ are shown for wt MerR and the MerR mutants; the detected responses to other metals are shown, while negative results are omitted.

Fig. 4.

Sequencing results from the MerR mutants beginning at residue L67. In the sequence of wt MerR, helices α4, α5, and α6 are underlined. The metal-binding cysteines are marked with black arrows above the wt MerR sequence.