Strains, plasmids and DNA manipulation
The strains and plasmids used are described in Table 1. Briefly, the E. coli strain XL-1 blue was used as a host for cloning, and BL21(λDE3) pLEMO was used for TraE and TraD overexpression. The Monarch Plasmid Miniprep kit (NEB) was used to isolate DNA plasmids. All mutations were performed using the QuickChange protocol and the oligonucleotides shown in supplementary Table 1. The mutations were verified by automated Sanger DNA sequencing.
Small-scale TraE and TraD protein expression optimization
TraE and TraD overproduction optimization were performed based on the protocol extensively described by Wang et al.38,. Briefly, for screening overexpression, the E. coli strains BL21(λDE3), BL21 Star(λDE3), C41(λDE3) or C43(λDE3) carrying expression plasmids pHTTraE and pHTTraD were grown under aerobic conditions at 37 °C (co-transformed or not with pLEMO plasmid) in 10 mL LB, 2YT or TB media cultures to exponential phase (OD600 0.6–0.8). The expression was induced by the addition of 0, 0.1–1.0 mM IPTG (with 0.1 mM increments); at temperatures of 18 °C, 25 °C, 30–37 °C and cultures were left shaking for 4, 6, 8–18 h at 220 rpm. Cells were collected in 1.5 mL Eppendorf tubes, the DO600was normalized to 1.0 and centrifuged at 16,000 x g for 10 min. Pellets were kept at −20 °C until further use. Bacterial lysis was performed using the method described by Casu et al.4,. Dot Blot was performed by pipetting and depositing 3 µL of the bacterial lysis supernatant on nitrocellulose membrane and leaving to air dry for 10 min. Blocking and further procedures were the same as for the Western Blot using an anti-His-Tag antibody (1:5000 dilution). Five of the most intense culture conditions visualized by Dot Blot were loaded on SDS-PAGE followed by Western Blot to ensure the protein migrated at the correct molecular weight. For pHTTraE, the best overexpression condition corresponded to the BL21(λDE3) pLEMO induced with 0.5 mM IPTG at 30 °C for 6 h in LB media, while for pHTTraD, the best overexpression condition corresponded to BL21(λDE3) pLEMO induced with 0.5 mM IPTG at 18 °C for 18 h in TB media.
TraD solubilization tests and purification
Five different detergents (Pluronic F-127, Tergitol NP-40, Triton X-100, DDM, OGNG and LMNG) were used to assess TraD extraction and stabilization. Briefly, bacterial cultures were resuspended at 4 °C in Buffer A (50 mM sodium phosphate pH 7.4, 300 mM NaCl, 100 mM imidazole) with cOmplete Mini protease Inhibitor mixture and DNase I at 100 µg/mL and lysed twice using One Shot cell disruptor (Constant Systems, Inc) at 27 kpsi and 4 °C. Ultracentrifugation at 250,000 x g for 1 h at 4 °C was performed, and pellets were collected and solubilized overnight at 4 °C in Buffer A supplemented with 20x CMC of the detergent. Debris were removed by centrifugation at 34,000 x g for 30 min at 4 °C. A fraction of the solution was used for Western Blot analysis to assess the quantity of extracted and solubilized TraD, and the rest was loaded on HisTrap Ni-chelate column (GE Healthcare). After extensive washing in Buffer B (Buffer A plus 3x detergent CMC), the protein was eluted into fractions using Buffer C (50 mM sodium phosphate pH 7.4, 300 mM NaCl, 100 mM arginine, 100 mM glutamate, 10% glycerol, 1 M imidazole and 2x detergent CMC). The fraction with the highest concentration was injected into a Superose 6 10/300 column equilibrated with 50 mM HEPES pH 8.0, 150 mM NaCl, 50 mM arginine, 50 mM glutamate, 50 mM imidazole, and 2x detergent CMC.
TraE purification in OGNG detergent
TraE was purified as described by Casu et al.4,.
Protein molarity quantification and visualization
All protein molar quantifications were based on the hexameric form of TraE and the pentameric form of TraD (as described in3). SDS-PAGE gels were visualized with zinc imidazole39.
Electrophoretic mobility shift assay for TraE and TraD
The EMSA was conducted using 50 nM of 5’ 6-Fluorescein (5’ 6-FAM) pT labelled DNA (hybridized or not with the complementary non-fluorescent DNA strain to form ssDNA and dsDNA) from IDT. LMNG was used as a detergent for TraE, while Triton X-100 was used for TraD. Proteins were diluted in 10 mM Tris pH 9.5, 150 mM NaCl, 10% glycerol, 5x detergent CMC and incubated with DNA for 30 min at 4 °C. Samples were loaded on a 0.5% agarose gel and run in the running buffer: 0.5X Tris-Borate-EDTA (TBE) pH 9.5, 150 mM NaCl, 5% glycerol, 5x detergent CMC at 90 V, 4 °C for 2 h. Agarose gels were prepared by solubilizing agarose with the running buffer, and gel revelation was acquired using a Fluorescein filter from the ChemiDoc MP imaging system. For BAR-072 inhibition experiments, all solutions and gels were supplemented with 10% DMSO.
Fluorescence polarization of TraE and TraD
As for EMSA experiments, 5’ 6-FAM was used but at 10 nM concentration and all fluorescence polarization experiments were performed at least three times. Protein-DNA binding was performed in FP Buffer: 10 mM sodium phosphate (NaPi) pH 7.4, 150 mM NaCl, 10% glycerol and 5x detergent CMC (LMNG for TraE and Triton X-100 for TraD). The probe was incubated with the indicated protein concentration, and measurement was made at room temperature on a Victor3Vplate reader (PerkinElmer). For binding and inhibition, curves were fit with saturation binding, specific binding with Hill slope equations. \(\:Y\:=\:{B}_{min}\:+\:\frac{({B}_{max}\:-\:{B}_{min})\times\:{\left[X\right]}^{h}}{{K}_{d}^{h}+{\left[X\right]}^{h}}\)
For inhibition experiments, 10% DMSO was supplemented with the FP buffer.
Conjugative DNA transfer and Western blot
For conjugative assays, pHT plasmids (kanamycin-resistant) and MG1655 pKM101ΔtraE (donor, spectinomycin-resistant) were used with WL400 cells (recipient cell, chloramphenicol-resistant). E. coli strains MG1655 containing pKM101ΔtraE were transformed with the complementation vector pHT (negative control), pHTTraE (positive control), or pHTTraE expressing TraE variants. The same protocol described by Casu et al.16, was used, except the recipient cell was grown in liquid LB media containing 50 µg/mL kanamycin and 100 µg/mL spectinomycin, and the agar plates for conjugation transfer quantification contained kanamycin and chloramphenicol antibiotics. The bacterial conjugation experiments were performed three times, and a Student’s t-test was conducted.
For the Western Blot, MG1655 pKM101ΔtraEtransformed with pHT, pHTTraE and pHTTraE variants were grown on LB agar plates (with 100 µg/mL spectinomycin and 50 µg/mL kanamycin) at 30 °C overnight. Cells were pelleted, and bacterial lysis was performed4. 50 µL of sample was loaded on SDS-PAGE gel and anti-TraE antibody was used (1:5000 dilution).
