Genetically Engineered Mycobacterial Strains

Allelic replacement of the genes encoding the alpha (Msmeg_3178) or beta (dnaN, Msmeg_0001) subunits of DNA polymerase III with the alpha-eyfp or dnaN-mCherry fusion genes, as well as replacement of the parB gene with the parB-mNeon green or parB-mCherry fusion genes, was performed as previously described (47 (link), 48 (link)). Replacement of the hupB gene with hupB-egfp was performed as described by Hołówka and coworkers (49 (link), 50 (link)). The mycobacterial strains were cultured in 37°C in 7H9 broth or on 7H10 agar (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD), 0.05% Tween 80, and (when needed) proper additives (kanamycin at 50 μg/ml, X-Gal [5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside], 2% sucrose). Correct allelic replacement and proper incorporation of the integration vectors were confirmed using PCR and Western blotting. Fusion of the functional fluorescent protein was confirmed by seminative SDS-PAGE and was visualized using a Typhoon phosphorimager. Western blotting was performed using standard procedures (77 ) with polyclonal anti-mCherry, monoclonal anti-GFP (Santa Cruz Biotechnology), and monoclonal anti-mNeon (Chromotec) antibodies.