8228 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 7. Activities of Rv1494 and Rv1495-N(29-56) and their effects on the mycobacterial growth. (A) Effects of Rv1495 and Rv1495-N(29-56) on the topoisomerase activities of MsmTopA. The assays were performed as described under ‘Materials and Methods’ section. L, linearized plasmid; R, relaxed plasmid; S, supercoiled plasmid. The protein concentrations were indicated on top of the panels. (B) mRNA cleavage activity assays. Experiments were performed as described under ‘Materials and Methods’ section. A varying concentration of Rv1495 or Rv1495-N(29-56) proteins was incubated with 0.5 mM mRNA substrates in the reactions. The mRNA substrate and products were indicated by arrows. (C) Effects of the expressions of Rv1495 and Rv1495-N(29-56) on the mycobacterial growth. A TetR-controlled expression system was used to analyze the effects of Rv1495 genes on the growth of M. smegmatis mc2 155 as described in the ‘Materials and Methods’ section. The growth of these recombinant mycobacterial strains were examined in the presence (induction) or absence (no induction) of tetracycline (Tc). Aliquots were taken at the indicated times and the OD600 was measured. Each analysis was performed in triplicate. The representative growth curves were plotted. The recombinant mycobacterial strains were indicated above the panels. inhibition of the respective activities of both proteins. Rv1495 physically interacted with MsmTopA, both in vitro and in vivo, and inhibited mycobacterial growth when expressed in M. smegmatis. An N-terminal fragment of Rv1495 was further characterized and shown to retain the capacity for both physical interaction with, and inhib- ition of, TopA, even though it had lost its mRNA cleavage activity. Therefore, in addition to its known RNA interferase function, the bacterial MazF protein also can directly interact with and modulate the activities of topo- isomerase I. This suggests an additional regulatory function of the M. tuberculosis MazEF system on bacterial growth that functions separately from its ribonuclease activity. The MazEF system may be involved in ability of M. tuberculosis to undergo long-term dormancy in human tissues, which is essential for the persistence of the pathogen (31,32). The activity of the sole TopA in this species with respect to relaxation of negative super- coiling might be important during stress responses, since a number of stress genes have to be induced for survival (3,33). In the current study, we found that the function