of Rv1495, a toxin protein, was linked to the topoisomer- ase I of the pathogen. Mycobacterium tuberculosis might have evolved a mechanism for survival within its host macrophage cells that operates by perfectly balancing the interactions of a pair of MazEF and the interactions of MazEF with the DNA topoisomerase I. This mechanism would allow the organism to become dormant within the hostile environment of human macro- phage cells. For example, MazE and MazF would normally form a stable complex, which would prevent RNA cleavage by MazF. When the pathogen confronts the stressful environment within human cells, MazE would be degraded by the ATP-dependent ClpPA serine protease (1). This would result in the release of MazF from the MazE–MazF protein complex. As shown earlier (3,16), MazF, on the one hand, could cleave mRNA and inhibit gene expression. On the other hand, as shown in the current study, MazF could inhibit the activity of topoisomerase I through a direct protein– protein interaction, again inhibiting gene expression. These two mechanisms would then contribute to pathogen dormancy. The importance of TA systems in the life cycle of M. tuberculosis is suggested by the presence of the remark- ably large number of these genes (15). In contrast, despite a much larger genome (7 versus 4.4 Mb) M. smegmatis has only two TA systems (2,34), including one MazF homolog. However, although M. tuberculosis and M. smegmatis have multiple groups of MazEF systems, both of their genomes encode a single highly homologous type I topoisomerase. Interestingly, in the current study, we found that Rv1495 interaction was conserved between both MtbTopA and MsmTopA and that it had a similar inhibitory action on topoisomerase activity of both organ- isms. An in vivo interaction between Rv1495 and the MsmTopA of M. smegmatis was also demonstrated. Thus, this interaction between these two proteins might have physiological importance in M. tuberculosis. Mycobacterium smegmatis has been used as a suitable host for the TA growth assays since it contains only two TA systems (2,34). In the current study, we observed that Rv1495 could inhibit the growth of M. smegmatis when Rv1495 was expressed through a pMind recombinant plasmid. Importantly, an N-terminal fragment of Rv1495, Rv1495-N(29-56), was also effective at inhibiting mycobacterial growth (Figure 7C), although it was less effective than the full-length Rv1495 (Figure 7A). Since Rv1495-N(29-56) had lost its mRNA cleavage activity, growth inhibition is most likely attributable to the effects of the MazF protein on the functioning of the topo- isomerase (Figure 7B). Therefore, two different mechan- isms would appear to contribute to the inhibition of mycobacterial growth by Rv1495. On the one hand, its interferase activity would cleave mRNA. On the other hand, its physical interaction with TopA would negatively regulate the relaxation of supercoiled DNA carried out by the topoisomerase. The incidence of tuberculosis has been increasing sub- stantially on a worldwide basis over the past decade. To complicate matters, drug-resistant TB has also become a major public health concern. However, no new Nucleic Acids Research, 2010, Vol. 38, No. 22 8229 tuberculosis-speciﬁc drugs have been discovered for more than 40 years. Bacterial topoisomerase I is required for preventing hypernegative supercoiling of DNA during transcription (35), which has been con- sidered to be especially important for the pathogenic adaptation to stressful environments (33,36). The genome of M. tuberculosis encodes a type I topoisomerase, but all attempts to isolate a transposon insertion mutant in the topA of M. tuberculosis gene have been unsuccessful (20). This suggests that M. tuberculosis topoisomerase I is critical for survival. Recent studies have found that inter- ference with the function of topoisomerase I and stabiliza- tion of its covalent complex with cleaved DNA can lead to bacterial cell death (37–39). Thus, topoisomerase I of pathogens is now becoming recognized as a promising new target for combating bacterial drug-resistance. In the current study, we characterized a N-terminal fragment, Rv1495-N(29-56), which retained the inter- action with TopA and the inhibitory activity on the topo- isomerase function. These speciﬁc protein–protein and protein–peptide interactions provided us with important clues for developing effective antibiotics that could target TB topoisomerase I. Compounds that activate or mimic bacterial toxins could quite feasibly be developed into novel antibiotics. On the one hand, drugs of this type would promote pathogenic programmed cell death by pre- venting or reducing the association between a given MazEF pair. On the other hand, they would also promote an inhibition of the function of the topoisomer- ase I and thus interfere with transcriptional regulation. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. FUNDING National Natural Science Foundation of China (30930003); 973 Program (2006CB504402); National Special Key Project of China on Major Infectious Diseases (2008ZX10003-005). Funding for open access charge: National Special Key Project of China on Major Infectious Diseases (2008ZX10003-005). Conﬂict of interest statement. None declared. REFERENCES 1. Aizenman,E., Engelberg-Kulka,H. and Glaser,G. (1996) An Escherichia coli chromosomal ‘‘addiction module’’ regulated by guanosine 30, 50-bispyrophosphate: a model for programmed bacterial cell death. Proc. Natl Acad. Sci. USA, 93, 6059–6063. 2. Pandey,D.P. and Gerdes,K. (2005) Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res., 33, 966–976. 3. Zhu,L., Zhang,Y., Teh,J.S., Zhang,J., Connell,N., Rubin,H. and Inouye,M. (2006) Characterization of mRNA interferases from Mycobacterium tuberculosis. J. Biol. Chem., 281, 18638–18643. 4. Engelberg-Kulka,H., Hazan,R. and Amitai,S. (2005) mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. J. Cell Sci., 118, 4327–4332.