Generation of Mtb reporter strains

Erdman(smyc′::mCherry) was generated by transforming Erdman WT with the replicating plasmid pCherry3, and selecting for transformants on 7H10 agar containing 50 µg/ml hygromycin B [37] (link). Erdman(rv2390c′::GFP, smyc′::mCherry) and Erdman(hspX′::GFP, smyc′::mCherry) have been previously described [17] (link). To generate Erdman(SSB-GFP, smyc′::mCherry), a 334 bp region immediately upstream of ssb together with the ssb open reading frame (excluding the stop codon) was PCR amplified from Mtb genomic DNA. This was fused to gfpmut2 amplified from the vector pSE100, by overlapping PCR. An 11 amino acid linker region (SAGSAAGSGEF) was included between the ssb and gfpmut2 open reading frames, to help prevent interference of SSB function by GFP [30] (link). This construct was subcloned into pCherry3 and transformed into Erdman Mtb. Selection for transformants was carried out on 7H10 agar containing 50 µg/ml hygromycin B. The reporter Mtb strains were grown to log phase in 7H9 broth supplemented with 50 µg/ml hygromycin B, aliquoted in 10% glycerol, and stored at −80°C until use. For Mtb infections in mice, aliquots were thawed, passed 3–5 times through a tuberculin syringe, and diluted to the required CFUs in sterile phosphate buffered saline (PBS) containing 0.05% Tween 80. For Cl⁻ induction of Erdman(rv2390c′::GFP, smyc′::mCherry), the reporter strain was grown to log phase and seeded at an OD₆₀₀ of 0.05 in 7H9 media containing 250 mM NaCl, buffered at pH 7 with 100 mM MOPS. The induction was allowed to continue for 6 days, after which an aliquot was diluted in PBS containing 0.05% Tween 80 to the necessary CFUs for mice infections.