Hygromycin b

Escherichia coli DH5α was cultured in Lysogeny broth or onto solid medium (Invitrogen, France). For classical culture conditions, M. smegmatis mc²155^{63 (link)} was grown in Middlebrook 7H9 complete medium containing 0.05% Tween-80 and 0.2% Gly (7H9). M. abscessus CIP104536^T, S and R morphotypes, were cultured in 7H9 medium containing 10% BBL™ Middlebrook OADC Enrichment (7H9^OADC). When required, kanamycin and hygromycin B (Euromedex, France) were added to the medium at a final concentration of 100 µg/mL for mycobacteria and 50 µg/mL and 200 µg/mL, respectively, for E. coli. Bacterial strains used in this study are listed in Table S1.

Escherichia coli DH10β and E. coli C41 (DE3)/pLyS strain were cultured in LB broth or Terrific Broth (Invitrogen, France). M. smegmatis mc² 155 and M. smegmatis mc² 155 groEL1ΔC [31 (link)] strains were cultured in Middlebrook 7H9 broth (BD Difco, Le Pont-de-Claix, France) supplemented with 0.05% (v/v) Tween-80 and 0.2% (v/v) glycerol (Sigma–Aldrich, Saint-Quentin Fallavier, France) (7H9-S). When needed, ampicillin and kanamycin (Euromedex, Souffelweyersheim, France) were added to the medium at final concentrations of 100 and 50 µg/ml for both E. coli and mycobacterial species, respectively. Hygromycin B (Euromedex) was used at a final concentration of 200 and 50 µg/ml for recombinant E. coli and recombinant mycobacteria, respectively.

For intra and extracellular assays, M. tb H37Rv expressing GFP^{20 (link)} was grown for 14 days in 7H9 medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, BD Difco), 0.5% glycerol, 0.05% Tween 80 and 50 µg/mL hygromycin B (Euromedex). For target identification, the experiments were conducted using M. tb mc²6230 (H37Rv ΔRD1 ΔpanCD) a derivative of H37Rv which contains a deletion of the RD1 region and panCD, resulting in a pan(−) phenotype^{64 (link)}. M. tb mc²6230 was grown in 7H9 medium supplemented with 10% OADC (BD Difco), 0.5% glycerol, 0.05% Tween 80 and 24 µg/mL D-panthothenate (Sigma-Aldrich). Cultures were kept at 37 °C without shaking.