8220 Nucleic Acids Research, 2010, Vol. 38, No. 22 this function as an interferase, little is known about cellular effects of the other numerous toxins in this pathogen. In contrast to its large number of TA genes, the genome of M. tuberculosis encodes only one single topoisomerase I (MtbTopA) (15). A related but nonpathogenic organism, M. smegmatis, also possesses a single TopA-homolog (MsmTopA) (17) that features a conserved N-terminal domain (NTD) and a highly variable C-terminal domain (CTD) (18). The MtbTopA topoisomerase might be essen- tial for preventing hypernegative supercoiling of DNA during transcription (19), and may also be involved in pathogen survival as attempts to isolate transposon inser- tion mutants in the topA gene have thus far been unsuc- cessful (20). Although both the MazEF system and DNA topoisomerase I appear to function in bacterial stress re- sponses (3,20), interaction between these two proteins remains to be characterized. Recently, the M. tuberculosis MazF homolog, Rv1495, was characterized as a toxin with ribonuclease activity (3,10). In the current study, we report on an interplay between Rv1495 proteins and DNA topoisomerase I that results in mutual inhibition of their activities. An N-terminus fragment of Rv1495, Rv1495-N(29-56), loses its mRNA cleavage activity but retains its ability to physically interact with and inhibit TopA from both M. tuberculosis and M. smegmatis. The Rv1495-N(29-56) fragment is further shown to inhibit mycobacterial growth. These ﬁndings suggest a new mechanism that allows the pathogenic MazEF systems of M. tuberculosis to participate in modulating the activities of DNA topo- isomerase I. MATERIALS AND METHODS DNA primers and peptides All DNA primers for polymerase chain reactions (PCR) were synthesized by Invitrogen and are listed in Supplementary Table S1. Two short peptides Rv1495- N28: MNAPLRGQ VYRCDLGYGAKPWL IVSNNA, and Rv1495-N(29-56): RNRHTADVVAVRLTTTRRT IPTWVAMGP were synthesized by GL Biochem (Shanghai) Ltd. Bacterial strains, plasmids, enzymes and chemicals The host strain E. coli BL21 and pET28a vector (Novagen) were used to express M. tuberculosis proteins. The plasmids pBT, pTRG and E. coli reporter strains for the bacterial two-hybrid assays were purchased from Stratagene. pBluescript and pGEX-4T-1 were purchased from Pharmacia. Restriction enzymes, T4 DNA ligase, DNA polymerase, modiﬁcation enzymes, deoxynucleoside triphosphates (dNTPs) and all antibiotics were purchased from TaKaRa Biotech. DNA puriﬁcation kits were purchased from Watson Biotechnologies. All plasmids constructed in this study are listed in Supplementary Table S2. Cloning, expression and puriﬁcation of M. tuberculosis proteins Rv1495 and the topoisomerase I gene from M. tuberculosis or M. smegmatis genome were ampliﬁed using their PCR primers (Supplementary Table S1) and cloned into the prokaryotic expression vector pET28a or pGEX-4T-1. Escherichia coli BL21 (Novagen) was used as the host strain to express and puriﬁed the proteins according to our previously published procedures (21–23). Both 6 his tagged and GST-fused recombinant proteins were prepared for activity and protein–protein interaction assays. Puriﬁed proteins were >95% pure as determined by SDS–PAGE and subsequent staining by Coomassie blue. Protein concentrations were determined by spectrophotometric absorbance at 260 nm according to Gill and von Hippel (24). Bacterial two-hybrid assay The BacterioMatch II Two-Hybrid System Library Construction Kit (Stratagene) was used to detect protein–protein interactions between Rv1495 and topo- isomerase I proteins. Bacterial two-hybrid system detects protein–protein interactions based on transcriptional activation and analysis was carried out according to pre- viously published procedures (21–23). Positive growth cotransformants were selected on the Selective Screening Medium plate containing 5 mM 3-amino-1,2,4-triazole (3-AT) (Stratagene), 8 mg/ml streptomycin, 15 mg/ml tetracycline, 34 mg/ml chloramphenicol and 50 mg/ml kanamycin. Cotransformants containing pBT-LGF2 and pTRG-Gal11P (Stratagene) were used as positive controls for an expected growth on the Screening Medium. Cotransformants containing empty vector pBT and pTRG were used as negative controls. DNA relaxation assay The assay was carried out according to the previously published procedure (25,26). The supercoiled pBluescript was incubated in the presence of mycobacterial topoisom- erase I (0–0.15 mM) with or without various amounts of Rv1495 or its mutant proteins, and incubated at 37 C for 40 min. After incubation, proteinase K (1 mg/ml) and its buffer (100 mM Tris–HCl, pH 7.8, 5 mM EDTA, 0.5% SDS) were added to the mixture and were further co-incubated for 15 min at 37 C. Samples were resolved by 0.9% agarose gel electrophoresis and then stained with ethidium bromide for analysis. Each analysis was per- formed at least in triplicate and a representative set of data was presented. Electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) was carried out to analyze single-stranded DNA-binding activity of MtbTopA, as described earlier (27). 50-end-labeled 32-mer oligonucleotide (50-CAGTGAGCGAGCTTCCG CTTGACATCCC AATA-30) (27) was used as a speciﬁc single-stranded DNA substrate. The reaction mixtures containing 40 mM Tris–HCl (pH 8.0), 20 mM NaCl, 1 mM EDTA, 150 nM oligonucleotide were incubated