Antitoxin autoregulation of M. tuberculosis toxin-antitoxin expression through negative cooperativity arising from multiple inverted repeat sequences

Abstract

Toxin-antitoxin systems play key roles in bacterial adaptation, including protection from antibiotic assault and infection by bacteriophages. The type IV toxin-antitoxin system AbiE encodes a DUF1814 nucleotidyltransferase-like toxin, and a two-domain antitoxin. In Streptococcus agalactiae, the antitoxin AbiEi negatively autoregulates abiE expression through positively co-operative binding to inverted repeats within the promoter. The human pathogen Mycobacterium tuberculosis encodes four DUF1814 putative toxins, two of which have antitoxins homologous to AbiEi. One such M. tuberculosis antitoxin, named Rv2827c, is required for growth and whilst the structure has previously been solved, the mode of regulation is unknown. To complete the gaps in our understanding, we first solved the structure of S. agalactiae AbiEi to 1.83 Å resolution for comparison with M. tuberculosis Rv2827c. AbiEi contains an N-terminal DNA binding domain and C-terminal antitoxicity domain, with bilateral faces of opposing charge. The overall AbiEi fold is similar to Rv2827c, though smaller, and with a 65° difference in C-terminal domain orientation. We further demonstrate that, like AbiEi, Rv2827c can autoregulate toxin-antitoxin operon expression. In contrast with AbiEi, the Prv2827c promoter contains two sets of inverted repeats, which bind Rv2827c with differing affinities depending on the sequence consensus. Surprisingly, Rv2827c bound with negative co-operativity to the full Prv2827c promoter, demonstrating an unexpectedly complex form of transcriptional regulation.

Keywords: Mycobacterium tuberculosis; co-operativity; regulation; structural biology; toxin-antitoxin system; transcription.

Figure 1.. Antitoxin AbiEi is a two-domain protein with bilateral opposingly charged faces.

(A) Scaled representation of the four M. tuberculosis TA systems containing NTase-like toxin genes and AbiE from S. agalactiae. Numbers in parentheses indicate amino acid length. All five toxins are DUF1814 proteins; Rv1044, Rv2827c and AbiEi are COG5340-containing antitoxins. Putative antitoxin Rv0837c is a COG4861 protein and the significantly shorter putative antitoxin Rv0078B is unclassified. The four M. tuberculosis systems were re-named as shown. (B) AbiEi antitoxin structure shown in pink cartoon representation, in two views rotated 180°. (C) Electrostatic potential of AbiEi, posed as per (B), with electropositive charge in blue and electronegative charge in red. (D) Previously solved Rv2827c structure shown in blue cartoon representation, in two views rotated 180° (PDB: 1ZEL). (E) Electrostatic potential of Rv2827c, posed as per (D), colored as per (C).

Figure 2.. AbiEi and Rv2827c are structurally similar, but have been captured in different positions with differing predicted protein interaction interfaces.

(A) AbiEi (pink) and Rv2827c (blue) in cartoon representation, aligned via the N-terminal winged helix-turn-helix domains, shown as two orthogonal views. The positions of the C-terminal domains diverge at a 65° angle. (B) Close-up structural superposition of the isolated N-terminal helices of AbiEi and Rv2827c, colored as per (A). The three helices (H1–3) of the N-terminal winged helix-turn-helix domains align well. (C) Close-up structural superposition of the isolated C-terminal domains of AbiEi and Rv2827c, colored as per (A). The core secondary structural features of the C-terminal domains approximate to the same positions, but the Rv2827c C-terminal domain has additional features at the C-terminus. (D) AbiEi has C-terminal residues predicted to be involved in making protein–protein interactions, which might allow positive co-operativity in AbiEi monomer binding. AbiEi is in pink cartoon representation with identified interacting residues in red, and is shown in orthogonal views. (E) Rv2827c does not have an equivalent patch of C-terminal interacting residues. Rv2827c is in blue cartoon representation, with identified interacting residues in cyan, and is shown in 180° rotation. Residues were identified using the cons-PPISP server. Rv2827c PDB code: 1ZEL.

Figure 3.. The rv2827c–rv2826c promoter has similar features but is more complex than the abiE promoter.

(A) Cartoon of the abiE and rv2827c–rv2826c promoters (pink and blue, respectively), showing the relative positions of the 23 bp inverted repeats (IRs). Putative transcriptional −35, −10 and start sites, along with ribosome binding sites (RBS), are indicated where possible. (B) Alignment of the six, 23 bp, IR sequences shows consensus sequences between the abiE and rv2827c–rv2826c promoters. The alignment was made using MView and the consensus was made using WebLogo.

Figure 4.. Rv2827c binds non-co-operatively to the IR3–IR4 region of the rv2827c–rv2826c promoter.

(A) Sequence level cartoon of the fluorescently labeled probe containing IR3–IR4, with −35, −10 and transcriptional start indicated. (B) Electrophoretic mobility shift assay (EMSA) of titrated Rv2827c with the probe in (A). (C) EMSA of titrated Rv2827c with the probe in (A) altered by replacing IR4 with polyC. (D) EMSA of titrated Rv2827c with the probe in (A) altered by replacing IR3 with polyC. (E) EMSA of titrated Rv2827c with the probe in (A) altered by replacing both IR3 and IR4 with polyC. For (B–E); protein concentrations are shown on each panel together with the binding events (0, 1 or 2); S — each experiment contained 100-fold excess of the specific unlabeled probe; NS — each experiment contained 100-fold excess of non-specific unlabeled probe; numbering −1 to −71 denotes the promoter region included in the probe, upstream of the translational start site in order to include all of IR4. (F) Fractional saturation curve plotted using the EMSA data of (B). (G) Hill plot using the EMSA data from (B). For (F) and (G), points are plotted from triplicate data and display mean values with standard error of the mean.

Figure 5.. Rv2827c binds with weak negative co-operativity to the IR1–IR2 region of the rv2827c–rv2826c promoter.

(A) Sequence level cartoon of the fluorescently labeled probe containing IR1–IR2. (B) Electrophoretic mobility shift assay (EMSA) of titrated Rv2827c with the probe in (A). (C) EMSA of titrated Rv2827c with the probe in (A) altered by replacing IR2 with polyC. (D) EMSA of titrated Rv2827c with the probe in (A) altered by replacing IR1 with polyC. (E) EMSA of titrated Rv2827c with the probe in (A) altered by replacing both IR1 and IR2 with polyC. For (B–E); protein concentrations are shown on each panel together with the binding events (0, 1 or 2); S — each experiment contained 100-fold excess of the specific unlabeled probe; NS — each experiment contained 100-fold excess of non-specific unlabeled probe; numbering −60 to −131 denotes the promoter region included in the probe. (F) Fractional saturation curve plotted using the EMSA data of (B). (G) Hill plot using the EMSA data from (B). For (F) and (G), points are plotted from triplicate data and display mean values with standard error of the mean.

Figure 6.. Rv2827c binds with negative co-operativity to the full rv2827c–rv2826c promoter.

(A) EMSA of titrated Rv2827c with a probe covering from −1 to −131 of the rv2827c–rv2826c promoter, covering IR1 to IR4. The titration was performed across two EMSA gels, with an additional zero protein lane included in the second gel for normalization. Protein concentrations are shown below each gel together with the binding events (0, 1, 2, 3 or 4); S — each experiment contained 100-fold excess of the specific unlabeled probe; NS — each experiment contained 100-fold excess of non-specific unlabeled probe. (B) Fractional saturation curve plotted using the EMSA data of (A). (C) Hill plot using the EMSA data from (A). (D) Semi-log saturation curve plotted using the EMSA data of (A), showing distinct breaks in the binding curve, in accordance with the multiple binding sites contained in the probe. (E) Sequence level cartoon of the fluorescently labeled probe containing rv2827c–rv2826c −1 to −131. (F–M) Saturation curves (F,H,J,L) and Hill plots (G,I,K,M) for each IR calculated using individual IR data gathered using mutant probes (Figures 4C, D and 5C,D). For (B–D) and (F–M), points are plotted from triplicate data and display mean values with standard error of the mean.

Figure 7.. Rv2827c–Rv2826c is a negatively autoregulating system in E. coli.

(A) Promoter activity from upstream promoter regions of abiE (99 bp), and rv2827c–rv2826c and rv1044–rv1045 (500 bp for both) detected using lacZ transcriptional fusions. Both the abiE and rv2827c–rv2826c constructs are active, but the rv1044–rv1045 construct is not. Plotted data are normalized to the vector-only control. (B) Autoregulation of promoter activity by antitoxins. LacZ activity was measured from the abiE and rv2827c–rv2826c constructs with or without induction of the cognate antitoxin (AT, ±IPTG). Both AbiE and Rv2827c negatively autoregulate expression. Plotted data are normalized to the uninduced vector-only control. All data (A–B) are plotted as the means of triplicate data, and error bars show standard deviations from the mean.

Figure 8.. Proposed model for negative autoregulation caused by Rv2827c binding to the four rv2827c–rv2826c promoter inverted repeats.

(A) Schematic representation of the putative rv2827c–rv2826c type IV toxin-antitoxin system. Model shows both rv2827c and rv2826c being translated into the antagonistic antitoxin and toxin protein pair, respectively. The antitoxin, Rv2827c, has a second function and binds to the rv2827c–rv2826c promoter, negatively autoregulating the operon. (B) An order of binding is created by the distinct affinity values for the inverted repeats represented in the sequence level cartoon, calculated from individual IR data gathered using mutant probes (Figures 4C,D and 5C, D). Rv2827c binds negatively co-operatively, initially to IR1 (0.0205 µM) followed by IR4 (0.121 µM), IR2 (0.862 µM) and finally IR3 (11.0 µM).