Mycobacteria 7h11 agar

Δmdp1 was kindly provided by Dr. John L. Dahl (University of Minnesota Duluth)^{26 (link)}. E. coli DH5α strain was used for all gene manipulation in this study. All M. smegmatis strains were grown in Middlebrook 7H9 broth (BD, Franklin Lakes, NJ) supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween 80 (MP Biomedicals, Santa Ana, CA), and 10% ADC enrichment (5% bovine serum albumin [Wako Pure Chemical Industries, Osaka, Japan], 0.81% NaCl, and 2% D-glucose) (7H9-ADC broth) or on Mycobacteria 7H11 agar (BD) supplemented with 0.5% (v/v) glycerol and 10% OADC enrichment (ADC enrichment supplemented with 0.06% [v/v] oleic acid) (7H11-OADC agar). Appropriate antibiotics were also added to the media to maintain the specific genotypes of each strain. All E. coli strains were cultured in LB broth or on LB agar (both from Sigma-Aldrich, St. Louis, MO). Hygromycin B (Hyg), kanamycin (Km), and RIF were purchased from Wako Pure Chemical Industries (Osaka, Japan). INH and Ace were purchased from Sigma Aldrich. ATc was purchased from Cayman Chemical (Ann Arbor, MI).

All mycobacteria strains were grown in Middlebrook 7H9 broth (BD, Franklin Lakes, NJ) supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween 80 (MP Biomedicals, Santa Ana, CA), and 10% ADC enrichment (5% bovine serum albumin [Wako Pure Chemical Industries, Osaka, Japan], 0.81% NaCl, and 2% D-glucose) (7H9-ADC broth) or on Mycobacteria 7H11 agar (BD) supplemented with 0.5% (v/v) glycerol and 10% OADC enrichment (ADC enrichment supplemented with 0.06% [v/v] oleic acid) (7H11-OADC agar). For biofilm formation experiment mycobacteria strains were grown on liquid.
Hygromycin B (HYG), kanamycin sulfate (KAN), rifampicin (RMP), amikacin (AMK,) and clarithromycin (CLA) were purchased from Wako Pure Chemical Industries [Osaka, Japan]; and INH from SIGMA-ALDRICH [St. Louis, USA]. AMK stock solutions were prepared in water. Stock solutions of all other compounds were prepared in 100% dimethyl sulfoxide (DMSO) and then filter sterilized (pore size 0.45 µm). These components were frozen in aliquots at − 20 °C.
CV-AM and SG were purchased from Invitrogen (Life-Technologies Corporation, California, USA). CV-AM was dissolved in 250 µl (μl) of DMSO. SG was diluted from the manufacture’s 5 mM stock solution to a final concentration of 50 μM by DMSO. The dyes were stored at − 30 °C.
PMAxx dye was purchased from Cosmo Bio Co., LTD (Tokyo, Japan).