The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization

Abstract

Complexes between the quorum-sensing regulator TraR and its inducing ligand autoinducer (AAI) are soluble in Escherichia coli, whereas apo-TraR is almost completely insoluble. Here we show that the lack of soluble TraR is due in large part to rapid proteolysis, inasmuch as apo-TraR accumulated to high levels in an E. coli strain deficient in Clp and Lon proteases. In pulse labeling experiments, AAI protected TraR against proteolysis only when it was added before the radiolabel. This observation indicates that TraR proteins can productively bind AAI only during their own synthesis on polysomes, whereas fully synthesized apo-TraR proteins are not functional AAI receptors. Purified apo-TraR was rapidly degraded by trypsin to oligopeptides, whereas TraR-AAI complexes were more resistant to trypsin and were cleaved at discrete interdomain linkers, indicating that TraR requires AAI to attain its mature tertiary structure. TraR-AAI complexes eluted from a gel filtration column as dimers and bound DNA as dimers. In contrast, apo-TraR was monomeric, and incubation with AAI under a variety of conditions did not cause dimerization. We conclude that AAI is critical for the folding of nascent TraR protein into its mature tertiary structure and that full-length apo-TraR cannot productively bind AAI and is consequently targeted for rapid proteolysis.

Figure 1

Apo-TraR accumulation and stability in protease-deficient strains. (A) TraR accumulation assayed with the use of Western immunoblots. Lanes 1 and 2, wild type; lanes 3 and 4, hflB; lanes 5 and 6, hslVU ; lanes 7 and 8, clpP; lanes 9 and 10, lon; lanes 11 and 12, clp, lon; lanes 13 and 14, clp, lon, hslVU. Samples in odd- and even-numbered lanes are from cells cultured in the absence and presence of AAI, respectively. (B) Pulse-labeled TraR in wild-type and protease-deficient E. coli strains. The upper bands are probably due to translation of a particularly stable mRNA from chromosome or a gene containing a T7-like promoter. (C) TraR turnover rates obtained from the data in B. Calculated half-lives are indicated in parentheses. (D) Stabilization of TraR by AAI in wild-type strain and in a clp, lon mutant. (E) TraR turnover rates calculated from data in D.

Figure 2

TraR stability in E. coli in the presence and absence of AAI. Cells expressing TraR from a phage T7 promoter were treated with rifampicin to block host transcription, [³⁵S]methionine, and excess nonlabeled methionine 1 min later to inhibit radiolabeling. AAI was added to a final concentration of 1 μM 20 min (A), 2 min (B), 1 min (C), or 0 min (D) before the addition of the radiolabel, or 1 min (E) or 2 min (F) after the addition of label. In G, AAI was omitted. At various time intervals (0, 2, 4, 8, 16, and 64 min after the addition of nonlabeled methionine in lanes 1–6, respectively), aliquots were frozen at −80°C to terminate proteolysis, lysed, and cleared by ultracentrifugation. Radioactivities of soluble TraR were quantitated with the use of a Storm PhosphorImager. Calculated TraR half-lives are indicated at the right of each panel.

Figure 3

Trypsin-mediated proteolysis of apo-TraR and TraR–AAI complexes. Radiolabeled apo-TraR (1 μM) (lanes 1–7) or TraR–AAI (1 μM) (lanes 8–14) complexes were combined with trypsin at the following concentrations: 0 μM (lanes 1 and 8), 0.125 μM (lanes 2 and 9), 0.25 μM (lanes 3 and 10), 0.5 μM (lanes 4 and 11), 1 μM (lanes 5 and 12), 2 μM (lanes 6 and 13), and 4 μM (lanes 7 and 14); incubated for 30 min at room temperature; and size fractionated by SDS/PAGE.

Figure 4

Gel filtration chromatography of TraR. (A) TraR was radiolabeled in the presence of AAI and purified by ion exchange chromatography. One hundred microliters of an 0.3 μM TraR solution was size fractionated by gel filtration chromatography. Elution of molecular mass standards is indicated (∗). (B) Same as A, but TraR–AAI complexes were preincubated at the indicated concentrations for 4 h at room temperature before size fractionation. (C) Soluble, radiolabeled apo-TraR, purified by ultracentrifugation and ion exchange chromatography. A 100-μl sample of a 0.3 μM solution of apo-TraR was loaded onto the column in the absence of AAI (⧫) or after preincubation with 1 μM AAI for 4 h (■). In the latter case, the elution buffer also contained 1 μM AAI. (D) Gel filtration of TraR–AAI complexes before and after dialysis to remove AAI. ●, TraR–AAI complexes; ▴, TraR after dialysis in the presence of 3% Tween to remove AAI; ■, TraR dialyzed against 3% Tween and then incubated with 1 μM AAI. All three experiments were conducted with 100 μl of a 75 nM TraR solution.

Figure 5

Gel mobility shift assays with MBP-TraR. (A) MBP-TraR–AAI complexes in the following concentrations: 100, 30, 10, 3, 1, 0.3, 0.1, and 0.03 nM. (B) Lane 1, no protein added; lane 2, 1 μM apo-MBP–TraR without AAI; lane 3, 1 μM apo-TraR after incubation with 10 μM AAI for 4 h; lane 4, 1 μM apo-TraR after dialysis in the presence of 3% Tween and 10 μM AAI, followed by dialysis against 0.01% Tween and 10 μM AAI. (C) Gel mobility shifts by mixtures of TraR-AAI and MBP-TraR–AAI. TraR was incubated with MBP-TraR (both complexed with AAI) overnight, combined with DNA fragments, and size-fractionated. Lane 1, no protein; lanes 2–8, TraR and MBP-TraR combined at ratios of 1:0, 1:0.1, 1:0.33, 1:1, 1:3, 1:10, and 0:1, respectively. The total protein concentration was 20 nM. (D) TraR and MBP-TraR (500 nM each) were combined in binding buffer at 28°C for 0, 2, 4, 6, 8, 10, 12, and 14 h (lanes 2–9, respectively) and then stored at −20°C. Samples were thawed and combined with DNA at 0°C for 20 min and size-fractionated. (E and F) Similar to D, except that proteins were combined at 16°C and 4°C, respectively. (G–I) Similar to D–F, respectively, except that proteins were combined at concentrations of 100 nM. Free, unbound DNA; C1, DNA complexed with TraR homodimers; C2, DNA complexed with TraR–MBP-TraR heterodimers; C3, DNA complexed with MBP-TraR homodimers.