Characterization of a chromosomal toxin–antitoxin, Rv1102c–Rv1103c system in Mycobacterium tuberculosis Jeong-Sun Han a , Jae Jin Lee a , Tripti Anandan b , Minghui Zeng b , Srinivas Sripathi c , Wan Jin Jahng c , Sang Hee Lee a , Joo-Won Suh a,⇑ , Choong-Min Kang b,⇑⇑ a Department of Biological Sciences, Myongji University, San 38-2 Namdong, Yongin, Gyeonggido 449-728, Republic of Korea b Department of Biological Science, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202, USA c Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA article info Article history: Received 27 July 2010 Available online 10 August 2010 Keywords: Toxin–antitoxin system Rv1102c Rv1103c Mycobacterium tuberculosis abstract Toxin–antitoxin systems, ubiquitous in prokaryotic genomes, have been proposed to play an important role in several stress responses. While Mycobacterium tuberculosis contains more than 80 putative TA loci, the roles they play in this pathogen are yet to be studied. Here, we characterize a chromosomal Rv1102c– Rv1103c TA system in M. tuberculosis. We found that the Rv1102c toxin interacts with the Rv1103c anti- toxin in a pull-down assay and the yeast two-hybrid system. Rv1102c cleaved the era mRNA in Esche- richia coli, and cleavage was inhibited by co-expression of Rv1103c. Heterologous expression of Rv1102c led to growth arrest in E. coli, which was fully recovered only when Rv1103c was co-expressed in cis with Rv1102c, suggesting that the production and assembly of Rv1102c and Rv1103c are tightly linked. Our additional results indicate that translational coupling of the Rv1102c and Rv1103c genes is important for Rv1102c–Rv1103c binding. Finally, we discovered that the expression of Rv1102c induced growth arrest and increased the level of persister cells in Mycobacterium smegmatis. These results suggest that the Rv1102c–Rv1103c TA system could play a role in M. tuberculosis pathogenesis via generating bacilli that survive in the face of multidrug therapy. Ó 2010 Published by Elsevier Inc. 1. Introduction Toxin–antitoxin (TA) modules have been found in most pro- karyotic genomes including many pathogenic bacteria [1]. TA loci typically consist of an upstream antitoxin gene and a downstream toxin gene [2]. The toxins characterized to date are stable proteins that act as ribonucleases to inhibit translation or as gyrase poisons, leading to cell growth arrest and/or cell death [3]. The antitoxins neutralize their cognate toxins by direct protein–protein interac- tion [2]. Stresses such as starvation have been shown to lead to a reduced level of antitoxin and subsequent toxin activation, result- ing in cell death [4,5]. Alternatively, TA systems, such as the MazEF and RelBE in Escherichia coli and VapBC in Mycobacterium tubercu- losis, have been suggested to induce reversible bacteriostasis that promotes adaptation to adverse stress conditions, or formation of persister cells that exhibit bacterial multidrug tolerance [6–8]. Notably, M. tuberculosis, which must survive under various stress conditions through long-term dormancy, contains more than 80 putative TA systems, many of which are functionally active [9–11]. Since TA systems are known to be required for the forma- tion of persister cells in E. coli, it has been speculated that TA sys- tems in M. tuberculosis may also be important for establishing a latent infection in the human host [12]. Because the majority of current antibiotics kill only actively growing bacterial cells, non- replicating persisters are thought to contribute to the prolonged antibiotic therapy for tuberculosis [13]. In this study, we sought to characterize the Rv1102c–Rv1103c TA system of M. tuberculosis. Recently, Rv1103c has been shown to relieve the toxic effect of Rv1102c in E. coli and M. smegmatis [9,14], but the molecular mechanism by which Rv1103c antago- nizes the toxicity of Rv1102c remains unknown. Here, we show that the Rv1102c toxin forms a complex with the Rv1103c anti- toxin, and that Rv1103c inhibits the ribonuclease activity of Rv1102c in vivo in E. coli. The expression of Rv1102c induced growth arrest in E. coli, which was partially recovered by co- expression of Rv1103c in trans with Rv1102c, but was fully recov- ered when it was co-expressed in cis. This result suggested that the expression and assembly of Rv1102c and Rv1103c are tightly linked, which is further supported by our finding that the expres- sions of Rv1102c and Rv1103c are translationally coupled. Finally, we show that the expression of Rv1102c induces growth arrest and increases the number of persisters in M. smegmatis. 0006-291X/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.bbrc.2010.08.023 ⇑ Corresponding author. Fax: +82 31 330 6190. ⇑⇑ Corresponding author. Fax: +1 313 577 6891. E-mail addresses: jwsuh@mju.ac.kr (J.-W. Suh), cmkang1@wayne.edu (C.-M. Kang). Biochemical and Biophysical Research Communications 400 (2010) 293–298 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc