The Escherichia coli toxin MqsR destabilizes the transcriptional repression complex formed between the antitoxin MqsA and the mqsRA operon promoter

Abstract

Bacterial biofilms are complex communities of cells containing an increased prevalence of dormant cells known as persisters, which are characterized by an up-regulation of genes known as toxin-antitoxin (TA) modules. The association of toxins with their cognate antitoxins neutralizes toxin activity, allowing for normal cell growth. Additionally, protein antitoxins bind their own promoters and repress transcription, whereas the toxins serve as co-repressors. Recently, TA pairs have been shown to regulate their own transcription through a phenomenon known as conditional cooperativity, where the TA complexes bind operator DNA and repress transcription only when present in the proper stoichiometric amounts. The most differentially up-regulated gene in persister cells is mqsR, a gene that, with the antitoxin mqsA, constitutes a TA module. Here, we reveal that, unlike other TA systems, MqsR is not a transcription co-repressor but instead functions to destabilize the MqsA-DNA complex. We further show that DNA binding is not regulated by conditional cooperativity. Finally, using biophysical studies, we show that complex formation between MqsR and MqsA results in an exceptionally stable interaction, resulting in a subnanomolar dissociation constant that is similar to that observed between MqsA and DNA. In combination with crystallographic studies, this work reveals that MqsA binding to DNA and MqsR is mutually exclusive. To our knowledge, this is the first TA system in which the toxin does not function as a transcriptional co-repressor, but instead functions to destabilize the antitoxin-operator complex under all conditions, and thus defines another unique feature of the mqsRA TA module.

FIGURE 1.

MqsR readily cleaves mqsRA mRNA. A, size exclusion chromatogram of free MqsR, the MqsR-MqsA complex which was co-expressed and co-purified (CoExp-MqsR-MqsA), and the MqsR-MqsA complex which was produced by incubating free MqsA with refolded MqsR prior to SEC (Rec-MqsR-MqsA). B, CD wavelength scan of refolded MqsR. C, EMSA with increasing amounts of either MqsA alone (lanes 2 and 3) or MqsR alone (lanes 5 and 6) with biotin-labeled PmqsRA. D, MqsR toxin readily degrades both mqsA and mqsR transcripts (lanes 4 and 9). MqsR activity was inhibited when bound to MqsA, independent of whether the MqsR-MqsA complex was formed in vitro (lanes 2 and 7) or co-expressed and co-purified (lanes 3 and 8). Full-length mqsA mRNA (415 nucleotides, lane 1) and full-length mqsR mRNA (316 nucleotides, lane 6) were incubated with buffer under the same reaction conditions as a negative control. Lane 5 contains a single-stranded RNA ladder.

FIGURE 2.

MqsR forms an extremely stable complex with MqsA. A, raw isothermal titration calorimetry data (upper) and derived binding isotherm plotted versus the molar ratio of titrant which was fit using a one-site model (lower) for MqsA (titrant) into MqsR (sample). Due to the high affinity, the volume of injections 8–27 (of 38 total) was decreased from 10 to 5 μl to better monitor the binding transition. The solid line in the lower panel is the best fit to the data using the nonlinear least squares regression algorithm (ORIGIN). B, thermal denaturation at 208 nm of MqsR-MqsA (2.5 μm, solid line). The complex unfolds with a single-state transition and a T_m of 83.4 ± 0.3 °C. The T_m for MqsR (5 μm, dashed line) and MqsA (2.5 μm, crossed line) is 48.1 ± 0.3 °C and 61.1 ± 3.3 °C, respectively.

FIGURE 3.

The MqsR-MqsA complex does not bind to the mqsRA promoter in vitro. A, sequence of the mqsRA promoter. DNA constructs used in this study are illustrated. Palindromes 1 and 2 are boxed (nucleotides that interact specifically with MqsA, as determined from the MqsA-DNA crystal structure, are in blue). The PmqsRA DNA is highlighted in green, and the primers used to amplify it are underlined. B, EMSA with increasing amounts of either MqsA alone (lanes 2–6) or the untagged MqsR-MqsA complex (lanes 8–12) incubated with biotin-labeled palindrome 1 (Pal1) of PmqsRA. C, same as B, except proteins were incubated with biotin-labeled Palindrome 2 (Pal2). D, same as B, except proteins were incubated with biotin-labeled PmqsRA promoter DNA. The two shifted bands represent protein bound to either one (middle arrow; 1× MqsA-PmqsRA) or both palindromes (top arrow; 2× MqsA-PmqsRA). DNA binding in the presence of the MqsR-MqsA complex is due to trace amounts of free MqsA, as the observed migration positions are identical to that seen with MqsA alone. For all gels, the negative control (100 fmol of labeled DNA lacking protein) is shown in lanes indicated as 0.

FIGURE 4.

MqsR destabilizes the interaction of MqsA with DNA. A, EMSA of MqsA binding to PmqsRA DNA in the presence of increasing amounts of MqsR. The two shifted bands represent protein bound to either one palindrome (middle arrow; 1× MqsA-PmqsRA) or both palindromes (top arrow; 2× MqsA-PmqsRA) simultaneously. Lanes 2–6 contain a constant amount of MqsA (20 nm) whereas lanes 3–6 contain 10, 15, 20, or 40 nm MqsR. Biotin-labeled PmqsRA in the absence of protein (50 fmol, lane 1) was used as a negative control. B, preformed MqsR-MqsA samples. MqsA was incubated with MqsR at the indicated ratios for 10 min at room temperature. Biotin-labeled PmqsRA DNA was then added to the MqsR-MqsA complex and the entire reaction incubated at room temperature for an additional 20 min followed by electrophoresis. Preformed MqsA-PmqsRA samples: a constant amount of MqsA (400 fmol) was first incubated with PmqsRA DNA (50 fmol; 10 min, room temperature) then increasing amounts of MqsR added at the indicated ratios. MqsR amounts were 0, 50, 100, 150, 200, or 400 fmol. The control reaction (50 fmol of biotin-labeled PmqsRA only) is shown in the first lane.

FIGURE 5.

MqsR and DNA have overlapping binding sites on MqsA. A, model of the hypothetical MqsR-MqsA-PmqsRA ternary complex. The model was generated by superimposing the MqsR-MqsA-N crystal structure (PDB 3HI2; colored blue/brown, respectively) with the MqsA-DNA crystal structure (PDB 3O9X; colored orange/gray, respectively) using the respective MqsA N-terminal domains; for clarity, only one monomer of MqsA from the MqsA-DNA crystal structure is shown. As modeled, there are structural clashes between MqsR and the DNA. The MqsA binding sites for MqsR and the PmqsRA DNA partially overlap as MqsA residue Arg-61 provides electrostatic interactions in both the toxin- and DNA-bound states; Arg-61 from both structures shown as sticks. Asp-33 (MqsR, blue) and the phosphate backbone from nucleotides thymine 14 and 15 (pink) are also shown; nitrogen atoms in dark blue, oxygen atoms in red. B, zoom-in view of overlay in A of just the MqsR-MqsA-N structure. MqsA residue Arg-61 forms a salt bridge with MqsR Asp-33. C, zoom-in view of overlay in A of just the MqsA-DNA structure. MqsA residue Arg-61 forms a salt bridge with the phosphate backbone of nucleotides thymine 14 and 15 from one strand. Electrostatic interactions in B and C are indicated by black dashed lines. D, EMSA with increasing amounts of WT MqsA (lanes 1–4), MqsA R61A (lanes 5–8), or MqsA R61D (lanes 9–12) incubated with biotin-labeled palindrome 1 (Pal1) of PmqsRA.