Published online 19 August 2010 Nucleic Acids Research, 2010, Vol. 38, No. 22 8219–8230 doi:10.1093/nar/gkq737 Characterization of an interplay between a Mycobacterium tuberculosis MazF homolog, Rv1495 and its sole DNA topoisomerase I Feng Huang and Zheng-Guo He* National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Received May 5, 2010; Revised July 26, 2010; Accepted August 4, 2010 ABSTRACT The MazEF systems are thought to contribute to the capacity for long-term dormancy observed in the human pathogen, Mycobacterium tuberculosis. However, except for their functions as mRNA interferases, little is known regarding any additional cellular functions of these systems in the pathogen. In the present study, we observed a negative inter- play between MazF protein Rv1495 and the sole M. tuberculosis DNA topoisomerase I (MtbTopA) with respect to protein functions. Through its C-terminal domain, MtbTopA physically interacted with and in- hibited the mRNA cleavage activity of Rv1495. Rv1495, in turn, inhibited the DNA cleavage activity of MtbTopA as well as its function of relaxation of supercoiled DNA. An N-terminus fragment of Rv1495, designated Rv1495-N(29-56), lost mRNA cleavage activity, but retained a significant physical interaction and inhibitory effect on TopA proteins from both M. tuberculosis and M. smegmatis. This fragment, although less effective than the full-length protein, was able to inhibit mycobacterial growth when expressed through a recombinant plasmid in M. smegmatis. The Rv1495 physically interacted with the M. smegmatis TopA both in vitro and in vivo. Our findings imply that MazEF systems can affect bacterial survival by a novel mechanism that allows direct modulation of M. tuberculosis topoisomerase I. INTRODUCTION Toxin–antitoxin (TA) gene cassettes are widely found in many free-living prokaryotes including a number of pathogenic bacteria such as Mycobacterium tuberculosis (1,2). These gene cassettes appear to be involved in bac- terial survival as well as in the formation of persistent pathogenic infections in hostile host-cell environments (3). In a typical TA system, the toxin gene is usually co-expressed with its cognate antitoxin gene present in the same operon (4,5). The Escherichia coli chromosome, for example, contains six such operons, two of which [the relBE (6) and the mazEF (1) systems] have been most ex- tensively studied (5). The mazEF system consists of two adjacent genes, mazE and mazF, and has the properties required for an addiction module. MazF is stable and toxic, whereas MazE is a labile and antitoxic protein (1). MazE and MazF interact with each other to form a complex, MazE–MazF, which negatively regulates the ex- pression of mazEF (7–9). Several recent studies have revealed MazF to be a sequence-speciﬁc endoribonuclease that recognizes speciﬁc cleavage sites within single-strand RNA. For this reason, it has been termed an mRNA interferase (8,10,11). Mycobacterium tuberculosis is one of the most devastating human pathogens, causing the deaths of about two million people each year around the globe. As it infects, this pathogen confronts a particularly hostile host cell environment, including a restricted access to nutrients and a reduced oxygen tension within the host macrophage cells (12–14). The presence of TA systems in M. tuberculosis may contribute to its ability to undergo long term dormancy within these inhospitable human tissues, and consequently might prove to be essential for the persistence of the pathogen (3). This potential importance of TA systems in the life cycle of M. tuberculosis is suggested by the presence of a remark- ably large number of these TA genes in its genome (15). Of the nine MazF homologs discovered thus far in M. tuberculosis (2,3,10,16), four have been shown to cause cell growth arrest when induced in E. coli. The bacterial MazF also shows an mRNA interferase activity both in vivo and in vitro (3,10,16). However, except for *To whom correspondence should be addressed. Tel: +86 27 87284300; Fax: +86 27 87280670; Email: he.zhengguo@hotmail.com; hezhengguo@ mail.hzau.edu.cn The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.