Figure 2.

Reduced expression of TopoI upon chromosome relaxation. The chromosome relaxation was carried out by ectopic overexpression of M. smegmatis TopoI using tetracycline inducible system. The exponential phase M. smegmatis cells were treated with the tetracycline (25 ng/ml) for 6 h and the expression of TopoI and DNA gyrase was monitored both at protein as well as RNA level. (A) Schematic for the discriminatory qRT PCR. The 5′ UTR of the ectopically expressed topoI (etopoI) and genomic copy of topoI (gtopoI) were different. The primers specific for gtopoI were used to determine the alteration in the expression of gtopoI upon TopoI overexpression (to cause chromosome relaxation). (B) Immunoblot analysis of TopoI and gyrase expression upon induction with the tetracycline. The increased overexpression of GyrA upon TopoI overexpression indicates the chromosome relaxation (RST). (C) Measurement of the genomic topoI transcript abundance in the cells upon overexpression of TopoI by discriminatory qRT PCR analysis. The fold change is expression represents the expression of topoI transcripts upon tetracycline induction normalized with the uninduced RNA samples. topoI (ORF) indicates the expression of total topoI transcripts; gtopoI and etopoI represents the expression of genomic copy and plasmid copy of topoI transcripts, respectively. The error bars represents the SD obtained from three independent experiments. *P < 0.01, **P < 0.001, ns: not significant (P > 0.1).