E. coli strain MG1655 with chromosomally integrated YpTop1 WT (BWYTOP) or YpTop1-D117N (BW117N) bearing an N-terminal TRX tag was a gift from Prof Yuk-Ching Tse-Dinh (Florida International University, USA). Cells were cultured at 37°C in LB medium containing 25 μg/ml chloramphenicol. Top1 expression was induced in exponentially growing cultures (OD600=0.3) by addition of arabinose (Sigma Aldrich). Cell division was monitored by measuring OD600.
M. smegmatis (mc2155) strain was cultured in 7H9 medium with ADC supplement (HiMedia) as described (28 (link)). M. smegmatis was transformed using a MicroPulser™ Electroporator Bio-Rad), and transformed cells selected and propagated in medium containing 200 μg/ml hygromycin B (Thermo-Fisher). MtTop1 expression was induced in exponentially growing (OD600=0.5) cultures of M. smegmatis by addition of 50 ng/ml anhydrotetracycline (ATc; Takara-Clontech) (29 (link)). Cell survival was quantified by culturing cells on LB plates containing 200 μg/ml hygromycin B for 3 days at 37°C.
M. tuberculosis H37Rv (ATCC 25618) strain was grown in 7H9 medium with ADC supplement (HiMedia) as described (28 (link)). Up to 2×109 Mtb bacilli were incubated in LSB (see below) for 15 min at 65°C with occasional vortexing, washed once in water, plated, and then incubated for four weeks at 37°C.
M. smegmatis (mc2155) strain was cultured in 7H9 medium with ADC supplement (HiMedia) as described (28 (link)). M. smegmatis was transformed using a MicroPulser™ Electroporator Bio-Rad), and transformed cells selected and propagated in medium containing 200 μg/ml hygromycin B (Thermo-Fisher). MtTop1 expression was induced in exponentially growing (OD600=0.5) cultures of M. smegmatis by addition of 50 ng/ml anhydrotetracycline (ATc; Takara-Clontech) (29 (link)). Cell survival was quantified by culturing cells on LB plates containing 200 μg/ml hygromycin B for 3 days at 37°C.
M. tuberculosis H37Rv (ATCC 25618) strain was grown in 7H9 medium with ADC supplement (HiMedia) as described (28 (link)). Up to 2×109 Mtb bacilli were incubated in LSB (see below) for 15 min at 65°C with occasional vortexing, washed once in water, plated, and then incubated for four weeks at 37°C.