8226 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 5. Interactions of Rv1495 with MsmTopA in vitro and in vivo. (A) Bacterial two-hybrid assays (Stratagene). Experiments were performed as described under ‘Materials and Methods’ section. Left panel, plate minus streptomycin (str) and 5 mM 3-amino-1,2,4-triazole (3-AT), Middle panel, plate plus str and 5 mM 3AT. Right panel, an outline of the plates. (B) Pull-down assays for examining the interaction between Rv1495 and MsmTopA. Equimolar amounts of 6 His-MsmTopA combined with GST-Rv1495 were used for pull-down assays as described in the ‘Materials and Methods’ section. GST was used as the negative control. One predicted size of his-tagged MsmTopA protein, pulled down by GST-tagged Rv1495 protein, was further examined by a western blotting assay. (C) Co-IP assays. Exponentially growing cells of the recombinant M. smegmatis containing Rv1495-expression plasmid were harvested, resuspended and lysed. Co-IPs were performed by incubating 10 ml of M. smegmatis cell extracts with 3 ml of MsmTopA antiserum 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 buffer. Finally, the beads were resuspended in SDS–PAGE sample buffer. After boiling, the samples were analyzed by western blotting using anti-Rv1495 antibody. extracts (Figure 5C, lane 1). In contrast, no obvious speciﬁc signal was detected for the association if the anti-MsmTopA antibody was absent from the reactions (Figure 5C, lane 3). Therefore, M. tuberculosis Rv1495 could interact with the homolog of M. smegmatis topo- isomerase I both in vitro and in vivo. Characterization of an inhibitory peptide fragment from Rv1495 To map the interactive domain of Rv1495 with MtbTopA, we produced several mutant fragments of the Rv1495 gene (Figure 6A). Using bacterial two-hybrid assays, we found that the N-terminal 56 amino acid fragment of Rv1495 interacted with MtbTopA (Figure 6A, right panel). We designed and synthesized two shorter peptides, Rv1495- N28 and Rv1495-N(29-56), as shown in Figure 6B, and tested these for inhibitory action on MtbTopA activity. Rv1495-N(29-56) retained an inhibitory effect on the topoisomerase I activity (Figure 6C, lower right panel). Rv1495-N(29-56) loses the mRNA cleavage activity but retains its ability to inhibit MsmTopA activity and mycobacterial growth As shown in Figure 6, Rv1495-N(29-56) retained its ability to interact with MtbTopA and to inhibit topoisomerase activity. We compared the effects of two proteins, Rv1495