Genapol x 080

To determine MirA’s subcellular localization (Supplementary Fig. 6c), wild type (BHm170) and P_mirA-mirA^∆AH (BHm174) strains were used. Three x T75 flask of Raw 264.7 cells were infected at an MOI of 2.5 for 3 h. After 48 h, infections were washed with PBS, then scraped and centrifuged at 3200 × g, 5 min, 4 ˚C. Cell pellet was washed once then resuspended in cold 250 µl of K36 buffer (50 mM K₂HPO₄, 100 mM KCl, 15 mM NaCl, pH 7) with protease inhibitors and Dounce homogenized with ~60 strokes. Lysates were then initially spun at 200 × g for 5 min at 4 ˚C to pellet host cell debris. Supernatants were collected and spun at 7000 × g for 1 min at 4 ˚C to pellet bacteria. The supernatant was collected and used as the “host cytosolic” fraction. The pellet was subsequently washed 3× in 1 ml PBS, then resuspended and incubated in 200 µl of K36 buffer with protease inhibitors and 0.5% Genapol X-080 (Sigma; 48750) for 45 min at 37 ˚C. Bacteria were pelleted 7000 × g for 1 min at 4 ˚C and the supernatant was collected and used as the “genapol-extracted” fraction. The bacterial pellet was then washed 3 times in 1 ml PBS. The bacterial pellet was then lysed as described above.

M. marinum cultures were grown to mid-logarithmic phase in 7H9 culture medium supplemented with 0.2% glycerol and 0.2% dextrose. Bacteria were pelleted, washed in PBS and incubated in 0.5% Genapol X-080 (Sigma-Aldrich) for 30 minutes to extract cell wall proteins. Genapol X-080-treated M. marinum cells were disrupted by sonication. Secreted proteins were precipitated from the culture supernatant by 10% trichloroacetic acid (TCA, Sigma-Aldrich). Proteins were separated according to molecular weight on 15% SDS-PAGE gels and subsequently transferred to nitrocellulose membranes (Amersham Hybond ECL, GE Healthcare Life Sciences). Immunostaining was performed with mouse monoclonal antibodies directed against the HA epitope (HA.11, Covance), EsxA (Hyb76-8), or rabbit polyclonal sera recognizing EspE [46 (link)].

Dextran (DEX, M_R ≈70 kDa, PanRecAppliChem, Germany), poly(ethylene glycol) (PEG, M_n ≈6000 Da, Alfa Aesar, Germany), sodium cholate (SC, PanRecAppliChem, Germany), sodium deoxycholate (DOC, Sigma‐Aldrich, USA), Triton X100 (TX100, Thermo Fisher Scientific, USA), Brij 35 (Sigma‐Aldrich, USA), Tween‐20 (VWR, USA), Brij O20 (Sigma‐Aldrich, USA), Brij O10 (Sigma‐Aldrich, USA), Genapol X080 (Sigma‐Aldrich, USA), Brij C10 (Sigma‐Aldrich, USA), Tween‐80 (VWR, USA), and sodium hydroxide (Chempur, Poland) were all of pure p.a. class, so they were employed without any further purification. For this work, Signis SG65i SWCNTs (Sigma‐Aldrich, LOT: MKCK1004) enriched in (6,5) chirality to simplify the interpretation of SWCNT migration between the phases due to the reduced number of species. Wherever specified, HiPco (Nanointegris, Canada; LOT: HP30‐006, purified) and Signis SG76i (Sigma‐Aldrich, LOT: MKBZ1157V) SWCNTs as references were also used to investigate the ATPE mechanics. For all sorting experiments and characterization, double‐distilled water obtained from the Elix Millipore system was used.