For complementation in trans, six-well plates separated into two compartments by Millicell Hanging Cell Culture Insert, PET 0.22 μm (Merck) were used. Each well contained 3 ml of MSgg medium. The strain to be complemented (ΔtasA) was inoculated below the filter, whereas the complementing strain was inoculated above. Medium with no bacteria above the filter served as control. After 24 hours, the filter was carefully removed, and the cells from the lower compartment were collected, washed twice in PBS, and analyzed by fluorescence-activated cell sorter (FACS).

For protein complementation, the medium was supplemented with purified TasA protein (final concentration of 20 μg/ml). TasA was expressed and purified as previously described (82) with some changes. BL21(DE3) E. coli competent cells were used for protein expression and purification. Induction of protein expression was performed in 500 ml of LB supplemented with ampicillin. The culture was incubated at 37°C until an OD600 of 0.7 to 0.8 was reached. Protein expression was induced with 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) and incubated overnight at 30°C with shaking to promote the formation of inclusion bodies. The next day, cells were pelleted by centrifugation (5000g for 15 min at 4°C) resuspended in buffer A [50 mM Tris and 150 mM NaCl (pH 8)] and then centrifuged again. Cell pellets were kept at −80°C until purification or processed immediately after 15 min. Cells were resuspended in buffer A, sonicated on ice in a Branson 450 digital sonifier (3 × 45 s, 60% amplitude) and centrifuged (15,000g for 60 min at 4°C). The pellet, mainly consistent of inclusion bodies, was recovered. The pellet was resuspended in buffer A supplemented with 2% Triton X-100 and incubated at 37°C with shaking for 20 min to ensure solubilization of cellular debris. The suspension was then centrifuged (15,000g for 10 min at 4°C), and the supernatant was discarded. The pellet was washed three times with buffer A, resuspended in denaturing buffer (50 mM Tris, 500 mM NaCl, and 6 M GuHCl), and incubated at 60°C with shaking overnight until complete solubilization of the inclusion bodies. The solution was clarified via sonication on ice (3 × 45 s, 60% amplitude) and centrifugation (15,000g for 1 hour at 16°C). Last, the inclusion bodies solution was then passed through a 0.45-μm filter before affinity chromatography. Recombinant TasA was purified using an ÄKTA Start fast protein liquid chromatography system (GE Healthcare). Soluble inclusion bodies were loaded into a HisTrap HP 5-ml column (GE Healthcare) previously equilibrated with binding buffer [50 mM Tris, 0.5 M NaCl, 20 mM imidazole, and 8 M urea (pH 8)]. The elution of the protein was performed isocratically with elution buffer [50 mM Tris, 0.5 M NaCl, 500 mM imidazole, and 8 M urea (pH 8)]. The eluted protein solution was loaded into a HiPrep 26/10 desalting column (GE Healthcare) to remove the urea and the imidazole, and the buffer was exchanged to 20 mM tris and 50 mM NaCl to perform the corresponding experiments.

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