Nucleic Acids Research, 2010, Vol. 38, No. 22 8227 Figure 6. N-terminus of Rv1495 regulates the topoisomerase activities of MtbTopA. Topoisomerase activity and bacterial two-hybrid assays for all mutants were performed as described under ‘Materials and Methods’ section. + represents growth on the screening plate; represents no growth on the screening plate. (A) The N-terminal mutant fragments of Rv1495 containing 56 and 28 amino acid residues (left panel) and their interactions with MtbTopA assayed by the bacterial two-hybrid system (right panel). (B) Two synthesized short peptides and their amino acid sequences. (C) Effect of different amount of Rv1495-N28, Rv1495-N56, and Rv1495-N(29-56) (0–6.0 mM) on topoisomerase activity. and Rv1495-N(29-56), on the topoisomerase activity of MsmTopA. As shown in Figure 7A, the activity of MsmTopA was progressively inhibited as the Rv1495 con- centration was increased, as indicated by the appearance of increasing amounts of supercoiled DNA. Similarly, an inhibition on the function of MsmTopA was also observed in the presence of Rv1495-N(29-56) (Figure 7A, right panel). A further activity assay indicated that Rv1495-N(29-56) lost its mRNA cleavage ability (Figure 7B, lanes 5–9). In contrast, the wild-type protein (Rv1495) demonstrated good activity as increasingly shorter mRNA excision products were observed on the gel as the Rv1495 concen- trations were increased stepwise (Figure 7B, lanes 1–4). Addition of Rv1495 also had clear effects on the myco- bacterial growth, as indicated by an obvious inhibition following induction of Rv1495 expression by tetracycline in M. smegmatis through the pMind recombinant plasmid (Figure 7C, middle panel). An important observation was that Rv1495-N(29-56), although lacking mRNA cleavage activity, was still clearly able to inhibit the growth of M. smegmatis (Figure 7C, right panel). This growth inhib- ition could be rescued by coexpression of the C-terminus of MtbTopA (Supplementary Figure S4). In contrast, the recombinant strain with the pMind alone showed only a slight effect on growth (Figure 7C, left panel). In further experiments, no signiﬁcant effect was observed on the growth of E. coli following induction of Rv1495 expres- sion (Supplementary Figure S5). This lack of effect is most likely because there is no homology between the C-terminus of Mycobacterium and the E. coli TopA enzymes. Therefore, Rv1495 or its N-terminal truncation could negatively regulate the topoisomerase activity of MsmTopA and inhibit mycobacterial growth. DISCUSSION In this study, we have shown that a MazF homolog, Rv1495, from M. tuberculosis could physically interact with TopA and that this interaction resulted in mutual