with varying concentrations of MtbtopA (60–240 nM) or Rv1495(240–960 nM) for 40 min on ice. Rv0054 (single-stranded DNA-binding protein) was used as a positive control. The protein–DNA complexes were resolved on 6% non-denaturing PAGE (39.5:0.5) using 0.5 TBE (Tris–borate–EDTA) as the running buffer. Gels were exposed to a storage-phosphor screen overnight at room temperature. The images were acquired by a Typhoon Scanner (GE healthcare). EMSA with plasmid pBluescript DNA was carried out to analyze the DNA-binding activity of Rv1495. The reactions contained 40 mM Tris–HCl (pH 8.0), 20 mM NaCl, 1 mM EDTA and 1.5–6 mM protein. Reactions were performed on ice for 30 min. Samples were resolved by 0.8% TBE agarose gel electrophoresis and then stained with ethidium bromide for analysis. Oligonucleotide cleavage assay The assays for the single-stranded DNA-binding cleavage activity of MtbTopA were performed as described earlier (27). Varying concentrations of MtbTopA (100–400 nM) were co-incubated at 37 C for 30 min with 200 nM 32-mer oligonucleotide, in the same buffer used for EMSA to yield a 50-end-labeled 19-mer cleaved product. The effects of Rv1495 on the cleavage activity of MtbTopA (0.2 mM) were analyzed using varying concentrations of Rv1495 (3–9 mM). Heat-denatured Rv1495 and GST proteins were used as negative controls. The reactions were stopped by adding 10 ml loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue and 0.05% xylene cyanol FF). The samples were incubated at 90 C for 5 min prior to electrophoresis on a 20% poly- acrylamide and 7 M urea gel. The images were acquired by a Typhoon Scanner (GE Healthcare). Assay for toxin growth inhibition A TetR-controlled expression system was used to analyze the effects of Rv1495 or its mutant genes on the growth of M. smegmatis mc2 155 (28). Wild-type or mutant Rv1495 genes were cloned into pMind (29) to produce correspond- ing recombinant plasmids which were then transformed into M. smegmatis. The strain containing the empty pMind plasmid was used as negative control. For all assays, tetracycline was used to induce gene expression (30). The growth of the recombinant mycobacterial strains was examined in the presence (induction) or absence (no induction) of tetracycline (Tc). Cells were grown at 37 C with aeration in 7H9-Kan-Tw (7H9 medium supplemented with 0.5% Tween 80, 30 mg/ml kanamycin and 0.2% glycerol) to an OD600 of 1.5–2.0. Cells were diluted in 7H9 medium to an OD600 of 0.2 and grown at 37 C at 200 rpm for a further 2 h. Aliquots were taken at the indicated times, and the OD600 was measured. Each analysis was performed in triplicate. Representative growth curves were plotted. Co-IP assays The in vivo interactions between Rv1495 and MsmTopA were analyzed by Co-IP according to previously published procedures with some modiﬁcations (20). Exponentially Nucleic Acids Research, 2010, Vol. 38, No. 22 8221 growing cells of M. smegmatis with recombinant plasmid pMind-Rv1495 were harvested, resuspended and lysed in 4 ml of buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40]. Co-IPs were per- formed by incubating and shaking 10 mg of mycobacterial cell extract with 3 ml of MsmTopA antiserum in 100 ml buffer for 3 h at 4 C. A 20-ml slurry of protein A Sepharose was added, and incubation was continued for another hour. Immune complexes were collected, and the beads were washed with the same buffer. Finally, the beads were resuspended in SDS–PAGE sample buffer. After boiling, the samples were analyzed by western blotting using anti-Rv1495 antibody. Cleavage of synthetic RNA by Rv1495 Oligoribonucleotides (11 bases in length) were commer- cially synthesized and 50-labeled with [g-32P]ATP using T4 polynucleotide kinase. Endoribonuclease activity was assayed in 10 ml of the reaction mixture containing 1–5 ml of Rv1495 and 32P-labeled oligonucleotides in 10 mM Tris–HCl (pH 7.8) (3). Reactions were carried out at 37 C for 30 min and stopped by the addition 10 ml of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue). The reaction mixtures were then sub- jected to 20% denaturing PAGE electrophoresis followed by autoradiography. RESULTS Rv1495 physically interacts with MtbTopA To uncover the new and as yet unknown cellular effects of the M. tuberculosis MazF genes, we conducted a screening using a bacterial two-hybrid assay to isolate proteins that interact with Rv1495, a MazF homolog. As shown in Figure 1A, the co-transformants containing Rv1495/MtbTopA grew well on the screening medium (Rv3646 encodes the topoisomerase I (MtbTopA) in the M. tuberculosis genome). Positive co-transformants (CK+) grew on the medium, whereas negative co-transformants (CK ) were incapable of growth on the same screening medium (Figure 1A). No growth was observed for their self-activated controls, or for their co-transformants ex- pressing a non-speciﬁc protein, Rv0054, which could form polymers with itself. A clear interaction between Rv0054 with itself was detected (Figure 1A). These results show that the selective system presented here worked well and that Rv1495 was found to interact with the M. tuberculosis TopA. Further conﬁrmation of this phenomenon was obtained by observing direct physical interaction between Rv1495 and MtbTopA through a GST pull-down assay. As shown in Figure 1B, GST-Rv1495 and His-tagged MtbTopA proteins were co-puriﬁed. One predicted size of His- tagged MtbTopA protein could be readily pulled down by GST-Rv1495, which was further conﬁrmed by a western blotting assay (Figure 1B, lower panel, lane 2). Co-incubation of GST with His-tagged MtbTopA did not produce any speciﬁc bands (Figure 1B, lane 3).