Southern blot hybridization.

For Southern blot analyses, approximately 1 μg of genomic DNA of WT and mutant M. tuberculosis strains was digested with BamHI and resolved by agarose gel electrophoresis. The DNA samples were next transferred onto an Immobilon membrane (Millipore) by vacuum-based blotting and hybridized with radiolabeled gene-specific probes. The hrcA- and hspR-specific probes were generated by PCR using [α-³²P]dCTP (BRIT, India) and oligonucleotide primers (Table S2), which were used to clone the respective ORFs; prehybridization, hybridization, and washing steps were carried out as described previously (36). The results were developed and digitalized with a Fuji phosphorimager (GE Healthcare).