Characterization of mRNA Interferases from Mycobacterium tuberculosis
2006; Elsevier BV; Volume: 281; Issue: 27 Linguagem: Inglês
10.1074/jbc.m512693200
ISSN1083-351X
AutoresLing Zhu, Yonglong Zhang, Jiah-Shin Teh, Junjie Zhang, Nancy Connell, Harvey Rubin, Masayori Inouye,
Tópico(s)Bacterial Genetics and Biotechnology
ResumomRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazF-mt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues. mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazF-mt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues. Most bacteria contain "suicidal" or "toxic" genes whose expression leads to growth arrest and eventual death upon exposure to stress. These toxin genes are usually coexpressed with their cognate antitoxin genes present in the same operon (1Engelberg-Kulka H. Hazan R. Amitai S. J. Cell Sci. 2005; 118: 4327-4332Crossref PubMed Scopus (138) Google Scholar, 2Gerdes K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (862) Google Scholar). The Escherichia coli chromosome contains six such operons called toxin-antitoxin (TA) 2The abbreviations used are: TA, toxin-antitoxin; ORF, open reading frame. 2The abbreviations used are: TA, toxin-antitoxin; ORF, open reading frame. systems. Of these, the MazE (antitoxin)/MazF (toxin) system is one of the most extensively characterized TA systems, and MazF has been shown to be a sequence-specific endoribonuclease that cleaves at ACA sequences in mRNAs (3Zhang Y. Zhang J. Hoeflich K.P. Ikura M. Qing G. Inouye M. Mol. Cell. 2003; 12: 913-923Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Thus, MazF is an mRNA interferase, and its induction causes the effective inhibition of protein synthesis leading to cell growth arrest. However, MazF-induced cells fully retain cellular metabolic activities, including ATP production and amino acid and nucleotide synthesis, as well as RNA and protein synthesis. Therefore, MazF-induced cells are capable of synthesizing a protein, if the gene encoding this protein is devoid of ACA sequences (4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This metabolically active dormant state caused by mRNA interferase induction is called "quasidormancy" (4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and has important implications in the physiology of various pathogenic bacteria, including persistent multidrug resistance.Notably, Mycobacterium tuberculosis, one of the most devastating human pathogens, contains nearly forty TA systems on its genome, among which nine have been shown to be homologous to E. coli MazF (5Pandey D.P. Gerdes K. Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (713) Google Scholar). It has been proposed that the TA modules play important roles in bacterial survival and formation of stable persisters in adverse conditions. These phenomena are essential for the persistence of M. tuberculosis. Persisters are the few rare pre-existing bacterial cells in a culture at any growth phase that are not growing and are intrinsically resistant to antibiotics by virtue of their subdued metabolism (6Keren I. Kaldalu N. Spoering A. Wang Y. Lewis K. FEMS Microbiol. Lett. 2004; 230: 13-18Crossref PubMed Scopus (762) Google Scholar, 7Spoering A.L. Lewis K. J. Bacteriol. 2001; 183: 6746-6751Crossref PubMed Scopus (676) Google Scholar).In the present paper, we have characterized two of the MazF homologues from this pathogen, which showed high toxicity in E. coli when induced. We have demonstrated that these are indeed mRNA interferases that cleave specific RNA sequences. In view of the quasidormancy caused by MazF induction in E. coli, our results suggest that multiple mRNA interferases in M. tuberculosis may play a role in the multidimensional dormancy of this pathogen in human tissues.EXPERIMENTAL PROCEDURESStrains and Plasmids—E. coli BL21(DE3), BW25113 (ΔaraBAD) were used. Plasmids pET-28a-MazF-mt1 and MazF-mt6 were constructed using pET-28a (Novagen) to express His6MazF-mt1 and His6MazF-mt6. The plasmids pBAD-MazF-mt1 to pBAD-MazF-mt7 were constructed from pBAD vector, in which the protein expression can be tightly regulated by the addition of arabinose (0.2%).Purification of His6-tagged MazF-mt1 and MazF-mt6 Proteins Expressed in E. coli—His6MazF-mt1 and His6MazF-mt6 proteins tagged at the N-terminal end were purified from strain BL21(DE3) carrying pET-28a-MazF-mt1 and MazF-mt6, respectively, using nickel-nitrilotriacetic acid resin (Qiagen). The proteins were stored in 100 mm NaH2PO4, 10 mm Tris-HCl, 0.5% glycerol, 10 mm β-mercaptoethanol buffer, pH 8.0, at -20 °C.Expression and Purification of MazF-mt1 Using Mycobacterium smegmatis MC2155 as a Host—The wild-type MazF-mt1 gene was PCR-amplified and cloned into an E. coli-M. smegmatis shuttle vector downstream of an acetamidase gene promoter (cloned from M. smegmatis genomic DNA) and upstream of a hexahistidine tag. This vector, carrying a hygromycin resistance gene, was constructed in our laboratory. M. smegmatis-competent cells carrying the construct were used to inoculate 2 ml of 7H9 medium, and this starter culture was incubated at 37 °C with shaking for 2 days and then used to inoculate larger volumes of 7H9 medium supplemented with Middlebrook oleic acid-albumindextrose-catalase enrichment and 2% acetamide (Sigma). The culture was grown for three days and cells were then harvested and resuspended in equilibration buffer (25 mm imidazole, 400 mm NaCl, and 50 mm NaPi, pH 8.0). The cells were then lysed with a French press. Cell debris was removed by one round of centrifugation at 25,000 rpm and at 4 °C. The lysate was added to Ni2+-nitrilotriacetic acid-agarose (Qiagen) for 1 h, and the resin was then transferred to a column and washed with 50 ml of 50 mm NaPi, pH 8.0, 400 mm NaCl, and 20 mm imidazole. The His-tagged proteins were eluted with 50 mm NaPi, pH 8.0, 400 mm NaCl, and 250 mm imidazole. Protein fractions were checked at A280 and purity was assessed by SDS-PAGE analysis. The highly purified fractions were collected and dialyzed against 10 mm HEPES buffer containing 5% glycerol, 0.1 mm dithiothreitol, and 500 mm NaCl. The protein aliquots were stored at -80 °C.Primer Extension Analysis in Vivo and in Vitro—For primer extension analysis of mRNA cleavage sites in vivo, pIN-Era plasmid (8Zhang Y. Zhang J. Hara H. Kato I. Inouye M. J. Biol. Chem. 2005; 280: 3143-3150Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) was transformed into E. coli BW25113 cells containing pBAD-MazF-mt1 or MazF-mt6 plasmids. The era mRNA transcription was induced by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside. After a 30-min induction, the toxin protein was induced by the addition of arabinose (a final concentration of 0.2%). Total RNA was isolated at time intervals as indicated in Figs. 3 and 4. Primer extension was carried out using different primers as described previously (3Zhang Y. Zhang J. Hoeflich K.P. Ikura M. Qing G. Inouye M. Mol. Cell. 2003; 12: 913-923Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). For in vitro primer extension analysis, the full-length era mRNAs were synthesized in vitro by T7 RNA polymerase from the DNA fragment containing a T7 promoter sequence and the era open reading frame (ORF) using the RiboMAX™ T7 large scale RNA production system (Promega). The era DNA fragments were amplified using the forward primer (5′-CCCGCGAAATTAATACGACTCACTATAG-3′ T7 promoter) and the reverse primers starting from the 3′-end of the era ORF. RNA substrates were partially digested with purified toxin protein at 37 °C for 15 min. The digestion reaction mixture (10 μl) consisted of 1 μg of RNA substrate, 0.2 μg of E. coli His6MazF-mt1 or 1 μg of M. smegmatis His6MazF-mt1 or E. coli His6MazF-mt6 and 0.5 μl of RNase inhibitor (Roche Applied Science) in 10 mm Tris-HCl (pH 7.8). Primer extension was carried out at 42 °C for 1 h in 20 μl of the reaction mixture as described previously (8Zhang Y. Zhang J. Hara H. Kato I. Inouye M. J. Biol. Chem. 2005; 280: 3143-3150Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The reactions were stopped by adding 12 μl of sequence loading buffer (95% formamide, 20 mm EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol FF (C25H27N2NaO7S2)). The samples were incubated at 90 °C for 5 min prior to electrophoresis on a 6% polyacrylamide and 36% urea gel. The primers E1 (5′-CAGTTCAGCGCCGAGGAAACGCAT-3′), E2 (5′-GATCCCCACAATGCGGTGACGAGT-3′), and E4 (5′-GCGTTCGTCGTCGGCCCAACCGGA-3′) were used for primer extension analysis of the era mRNA. The primers were 5′-labeled with [γ-32P]ATP using T4 polynucleotide kinase.FIGURE 4In vivo and in vitro cleavage of the era mRNA after induction of MazF-mt6. A and B, in vivo and in vitro cleavage of the era mRNA after induction of MazF-mt6 by adding 0.2% arabinose. For in vivo primer extension, total RNA was extracted at each time point after MazF-mt6 induction, and primer extension was carried out using primer E1 (A)orE2(B). For in vitro primer extension, the era mRNA was incubated with (lane 9) or without (lane 10) the purified His6MazF-mt6, and primer extension was carried out using primer E1 (A)orE2 (B). Cleavage sites are shown by arrows on the RNA sequence as determined using the DNA ladder.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cleavage of Synthetic RNA by MazF-mt1—Oligoribonucleotides (11 bases in length; see Fig. 3) were commercially synthesized and 5′-labeled with [γ-32P]ATP using T4 polynucleotide kinase. Endoribonuclease activity was assayed in 10 μl of the reaction mixture containing 0.5 μl (20 units) of ribonuclease inhibitor (Roche Applied Science), 0.2 μg of E. coli MazF-mt1 or 1 μg of M. smegmatis MazF-mt1 and 32P-labeled oligonucleotides in 10 mm Tris-HCl (pH 7.8). Reactions were carried out at 37 °C for 30 min and stopped by the addition of the loading buffer as described above. The reaction mixtures were then subjected to 20% sequence gel electrophoresis followed by autoradiography.RESULTSBlast Search for MazF Homologues in M. tuberculosis—A blast search of all known ORFs in the M. tuberculosis (H37Rv) genome with E. coli MazF identified the gene Rv2801c that encodes a 118-residue protein that has 32.5% identity and 44% homology to E. coli MazF (Fig. 1A). Using this protein MazF-mt1 for further blast search, six more MazF homologues with >20% identity to MazF-mt1 (mt2 to mt7) were found (Fig. 1B). Notably, MazF-mt1, -mt2, and -mt3 have the putative 11-residue S1-S2 loop similar to E. coli MazF (9Kamada K. Hanaoka F. Burley S.K. Mol. Cell. 2003; 11: 875-884Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), whereas the others have much shorter S1-S2 loops (Fig. 1, A and B). The putative helix H1 is longer by five residues in MazF-mt1 and -mt2 and by three residues in -mt3, compared with that of E. coli MazF, whereas it is shorter by one residue in MazF-mt4, -mt5, -mt6, and -mt7 as compared with that of E. coli MazF (Fig. 1, A and B). MazF-mt2 lacks the C-terminal region encompassing β-strand S7 and helix H3. This seems to be due to a 28-bp deletion after the codon for Thr-88, because the 19-residue sequence highly homologous to helix H3 of MazF-mt1 is found immediately downstream of Thr-88 in a different reading frame (68% identity) (Fig. 1B). Interestingly, all MazF homologues appear to be co-translated with a short upstream ORF, most of which overlaps with the MazF ORFs (Fig. 1C). None of the upstream ORFs, however, show homology to E. coli MazE, antitoxin of MazF. As observed with MazE and MazF, most of the pairs consist of acidic and basic proteins, except for the pair of MazF-mt2 and its upstream ORF, both of which are basic proteins (Fig. 1C). It remains to be elucidated whether these upstream ORFs function as antitoxins for their cognate downstream ORFs.FIGURE 1Sequence alignments of MazF homologues from M. tuberculosis with E. coli MazF. A, alignment of MazF-mt1 (Rv2801c) with E. coli MazF. β-strand (S) and helical (H) region are assigned according to Kamada et al. (9Kamada K. Hanaoka F. Burley S.K. Mol. Cell. 2003; 11: 875-884Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Identical and homologous residues are shown in black and shaded backgrounds. B, alignments of MazF-mt2-mt7 to MazF-mt1. The consensus sequence is shown at the bottom. In MazF-mt2, the 19-residue sequence in blue is immediately downstream of the codon for Thr-88 with a -1 frameshift. MazF-mt2-mt7 correspond to Rv0456A, Rv1991c, Rv0659c, Rv1942c, Rv1102c, and Rv1495, respectively. C, upstream ORFs for MazF-mt1-mt7. These overlap with downstream MazF ORFs by 14, 14, 7, 14, 4, 29, and 4 bases for MazF-mt1-mt7, respectively. Total residue numbers and pI values are shown on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Four of the Seven MazF Homologues in M. tuberculosis Show Toxicity to E. coli—All ORFs were tested for their toxicity in E. coli using the pBAD expression plasmid. In the presence of 0.2% arabinose, cells expressing MazF-mt1, -mt3, -mt4, and -mt6 could not grow, whereas cells expressing MazF-mt2, -mt5, and -mt7 proteins were able to grow on an agar plate (Fig. 2A). Inhibitory effects of MazF-mt1, -mt3, -mt4, and -mt6 were also observed in M9-CAA liquid medium in the presence of 0.2% arabinose (Fig. 2B). In the case of cells expressing MazF-mt4 protein, severe growth inhibition was seen only after one generation of cell growth.FIGURE 2Toxicity of M. tuberculosis MazF homologues. A, E. coli BW25113 transformed with pBAD harboring ORFs for MazF-mt1-mt7 were streaked on M9 (glycerol, CAA) plates with (left) and without (right) 0.2% arabinose. The plates were incubated overnight at 37 °C. B, growth curves of E. coli BW25113 harboring pBAD(MazF-mt1), (MazF-mt3), (MazF-mt4), and (MazF-mt6) plasmids in M9 (glycerol, CAA) liquid medium at 37 °C in the presence (blue) and the absence (red) of 0.2% arabinose. Cells harboring pBAD(MazF-mt4) plasmid were diluted 10 times after 150 min. Cell growth was measured by A (absorbance) at 600 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The MazF-mt1 Has Endoribonuclease Activity—We examined whether these MazF homologues have sequence-specific mRNA interferase activity, both in vivo and in vitro. When MazF-mt1 was induced by the addition of 0.2% arabinose, a specific cleavage of the era mRNA between U and A was detected in vivo by primer extension in a time-dependent manner (Fig. 3A). The identical cleavage site was detected with use of the era mRNA synthesized with T7 RNA polymerase and purified MazF-mt1 tagged with His6 at the N-terminal end (Fig. 3B, lane 2). In addition to the same site reported for the in vivo cleavage site (CU ↓ ACC), a weak cleavage site (UU ↓ ACA) was also detected. Using five primers covering almost the entire era mRNA, CU ↓ ACC and UU ↓ ACA were the only major cleavage sites detected, even when era mRNA contained six other UAC sequences (UUACU, AUACU, CUACG, AUACA, GUACU, and UUACG). Importantly, identical sites were cleaved by His6MazF-mt1, which was expressed and purified from M. smegmatis (Fig. 3B, lane 3), indicating that the observed cleavage is due to MazF-mt1. We further confirmed that the cleavage activity was not due to contaminating enzymes, because a purified His6MazF-mt1 (E19A) mutant protein showed a substantially reduced cleavage activity (not shown). This mutation in E. coli MazF has recently been shown to dramatically reduce the MazF mRNA interferase activity (10Li G.Y. Zhang Y. Chan M.C. Mal T.K. Hoeflich K.P. Inouye M. Ikura M. J. Mol. Biol. 2006; 357: 139-150Crossref PubMed Scopus (47) Google Scholar).For further biochemical characterization, purified His6MazF-mt1 was used to cleave a synthetic 15-base RNA (AGAUAU ↓ ACAUAUGAA), which was also shown to be cleaved between U and A of the UAC sequence (Fig. 3C, lane 2 and arrows). A 19-base DNA with a sequence identical to that of the RNA substrate in the center could not be cleaved (lane 4). When the DNA and RNA were mixed and treated with the enzyme, only RNA was cleaved (lane 5), indicating that MazF-mt1 is an endoribonuclease. To further test the cleavage specificity, five 11-base RNA substrates were synthesized, one with the UACA sequence in the center (AUAUACAUAUG) and the others with a G residue in each one of the UACA sequences. The first U and the second A residues could not be replaced with G (Fig. 3D, lanes 3-6). The third C residue also appears to be important, as the cleavage between U and A (lanes 7 and 8) was significantly reduced when this C was replaced with G. On the other hand, the fourth A residue was replaceable with G (lanes 9 and 10), confirming that MazF-mt1 is an endoribonuclease that specifically cleaves UAC sequence.The MazF-mt6 Is an mRNA Interferase—Because MazF-mt6 showed toxicity in E. coli (Fig. 2), we examined whether it, too, is a sequence-specific RNA interferase. We speculated that other MazF homologues are not toxic in E. coli, possibly because they may have highly specific cleavage activities and thus cleave only a very limited number of sequences in E. coli mRNAs or they are not efficiently expressed in E. coli. We carried out in vivo and in vitro primer extension experiments using the era mRNA and the MazF-mt6 protein. As shown in Fig. 4, a number of in vivo cleavage sites were detected in a time-dependent manner after its induction, all of which were also detected in an in vitro experiment with purified His6MazF-mt6 protein. Notably, a new cleavage site was also observed in an in vitro experiment in addition to those found in an in vivo (Fig. 4B, lane 10) experiment. These cleavages preferentially occurred in U-rich regions and after a U residue. Therefore, (U/C)U ↓ (A/U)C(U/C) may be assigned as a consensus cleavage sequence for MazF-mt6 mRNA interferase. The cleavage also occurred after G or A residues in some cases, although all cleavage sites contained UU, UC, or CU residues.DISCUSSIONIn this report, we have demonstrated that M. tuberculosis contains a number of MazF homologues, some of which were toxic to E. coli cells when induced. It has been shown that E. coli MazF causes complete cell growth arrest; however, the cells retain full metabolic activities (4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Therefore, it is tempting to speculate that this novel physiological quasidormant state caused by MazF may be, in some ways, related to the long term dormancy of M. tuberculosis in human tissues. By having a large number of mRNA interferases that may be induced under different environmental conditions, the persistent dormancy of this pathogen in human tissues may be well regulated and may lead to antibiotic resistance.Microarray studies of the stringent response and the starvation response carried out with M. tuberculosis may provide insights into regulation of expression of the toxin genes in this pathogen. In the M. tuberculosis starvation model by Betts et al. (11Betts J.C. Lukey P.T. Robb L.C. McAdam R.A. Duncan K. Mol. Microbiol. 2002; 43: 717-731Crossref PubMed Scopus (1111) Google Scholar), MazF-mt4 (Rv0659c) was found to be down-regulated after 4 h of nutrient starvation. In a microarray study of a strain of H37Rv in which stringent response gene RelMtb is deleted, MazF-mt6 (Rv1102c) expression was shown to increase 3-fold after 6 h of starvation in the knock-out strain, whereas expression was unchanged after 6 h of starvation in wild-type Mtb (12Dahl J.L. Kraus C.N. Boshoff H.I. Doan B. Foley K. Avarbock D. Kaplan G. Mizrahi V. Rubin H. Barry III C.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10026-10031Crossref PubMed Scopus (291) Google Scholar). Both of these observations thereby directly implicate a role for RelMtb and (p)pppGpp ("the stringent response") in the regulation of these toxin genes, namely down-regulation. However, the net effect on cell metabolism or viability of these changes in toxin expression depends on the respective changes in expression of their antitoxin counterparts. These results may be interpreted in the context of regulation of the mazEF operon in E. coli. When the stringent response is triggered in E. coli, the RelA-dependent increase of (p)ppGpp causes a decrease in activity of the P2 promoter of the mazEF operon (13Aizenman E. Engelberg-Kulka H. Glaser G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6059-6063Crossref PubMed Scopus (497) Google Scholar). Subsequently, MazE is degraded rapidly by ClpPA protease with a half-life of ∼30 min, whereas MazF is stable over a period of 4 h. Therefore, down-regulation of the mazEF operon results in the persistence of MazF as the amount of its cognate antitoxin MazE decreases. This ultimately leads to cell growth arrest. In M. tuberculosis, the observations so far are promising in terms of mirroring the mazEF regulatory system of E. coli. However, the challenge will be to identify and confirm the cognate antitoxins for the endoribonuclease toxins reported here, as well as to determine the precise genetic organization and regulation of these sets of genes. Furthermore, the reason why MazF-mt1 was expressed well in M. smegmatis is not known at present. It is possible that M. smegmatis contains enough semicognate antitoxin against MazF-mt1 under the growth condition used or MazF-mt1 is highly specific and thus does not show global effect in M. smegmatis.M. tuberculosis is one of the most devastating human pathogens, as one third of the world's population is infected with this pathogen and eight million people develop active disease each year. Two phenotypic properties of this pathogen account for these statistics: 1) latent infection sometimes referred to as dormancy and 2) persistence of infectious bacteria (14Zhang Y. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 529-564Crossref PubMed Scopus (360) Google Scholar, 15Balaban N.Q. Merrin J. Chait R. Kowalik L. Leibler S. Science. 2004; 305: 1622-1625Crossref PubMed Scopus (1944) Google Scholar). Although the mechanisms of latency and persistence are not well understood, recent studies of the TA systems in this pathogen suggest that these TA systems may be responsible for the dormancy of this pathogen in human tissues (16Arcus V.L. Rainey P.B. Turner S.J. Trends Microbiol. 2005; 13: 360-365Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Our results demonstrating the existence of multiple mRNA interferases in M. tuberculosis support this notion, and further biochemical and genetic characterization of M. tuberculosis toxins will shed light into our understanding of their roles in the pathogenesis of this persistent human pathogen. Most bacteria contain "suicidal" or "toxic" genes whose expression leads to growth arrest and eventual death upon exposure to stress. These toxin genes are usually coexpressed with their cognate antitoxin genes present in the same operon (1Engelberg-Kulka H. Hazan R. Amitai S. J. Cell Sci. 2005; 118: 4327-4332Crossref PubMed Scopus (138) Google Scholar, 2Gerdes K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (862) Google Scholar). The Escherichia coli chromosome contains six such operons called toxin-antitoxin (TA) 2The abbreviations used are: TA, toxin-antitoxin; ORF, open reading frame. 2The abbreviations used are: TA, toxin-antitoxin; ORF, open reading frame. systems. Of these, the MazE (antitoxin)/MazF (toxin) system is one of the most extensively characterized TA systems, and MazF has been shown to be a sequence-specific endoribonuclease that cleaves at ACA sequences in mRNAs (3Zhang Y. Zhang J. Hoeflich K.P. Ikura M. Qing G. Inouye M. Mol. Cell. 2003; 12: 913-923Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Thus, MazF is an mRNA interferase, and its induction causes the effective inhibition of protein synthesis leading to cell growth arrest. However, MazF-induced cells fully retain cellular metabolic activities, including ATP production and amino acid and nucleotide synthesis, as well as RNA and protein synthesis. Therefore, MazF-induced cells are capable of synthesizing a protein, if the gene encoding this protein is devoid of ACA sequences (4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This metabolically active dormant state caused by mRNA interferase induction is called "quasidormancy" (4Suzuki M. Zhang J. Liu M. Woychik N.A. Inouye M. Mol. Cell. 2005; 18: 253-261Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and has important implications in the physiology of various pathogenic bacteria, including persistent multidrug resistance. Notably, Mycobacterium tuberculosis, one of the most devastating human pathogens, contains nearly forty TA systems on its genome, among which nine have been shown to be homologous to E. coli MazF (5Pandey D.P. Gerdes K. Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (713) Google Scholar). It has been proposed that the TA modules play important roles in bacterial survival and formation of stable persisters in adverse conditions. These phenomena are essential for the persistence of M. tuberculosis. Persisters are the few rare pre-existing bacterial cells in a culture at any growth phase that are not growing and are intrinsically resistant to antibiotics by virtue of their subdued metabolism (6Keren I. Kaldalu N. Spoering A. Wang Y. Lewis K. FEMS Microbiol. Lett. 2004; 230: 13-18Crossref PubMed Scopus (762) Google Scholar, 7Spoering A.L. Lewis K. J. Bacteriol. 2001; 183: 6746-6751Crossref PubMed Scopus (676) Google Scholar). In the present paper, we have characterized two of the MazF homologues from this pathogen, which showed high toxicity in E. coli when induced. We have demonstrated that these are indeed mRNA interferases that cleave specific RNA sequences. In view of the quasidormancy caused by MazF induction in E. coli, our results suggest that multiple mRNA interferases in M. tuberculosis may play a role in the multidimensional dormancy of this pathogen in human tissues. EXPERIMENTAL PROCEDURESStrains and Plasmids—E. coli BL21(DE3), BW25113 (ΔaraBAD) were used. Plasmids pET-28a-MazF-mt1 and MazF-mt6 were constructed using pET-28a (Novagen) to express His6MazF-mt1 and His6MazF-mt6. The plasmids pBAD-MazF-mt1 to pBAD-MazF-mt7 were constructed from pBAD vector, in which the protein expression can be tightly regulated by the addition of arabinose (0.2%).Purification of His6-tagged MazF-mt1 and MazF-mt6 Proteins Expressed in E. coli—His6MazF-mt1 and His6MazF-mt6 proteins tagged at the N-terminal end were purified from strain BL21(DE3) carrying pET-28a-MazF-mt1 and MazF-mt6, respectively, using nickel-nitrilotriacetic acid resin (Qiagen). The proteins were stored in 100 mm NaH2PO4, 10 mm Tris-HCl, 0.5% glycerol, 10 mm β-mercaptoethanol buffer, pH 8.0, at -20 °C.Expression and Purification of MazF-mt1 Using Mycobacterium smegmatis MC2155 as a Host—The wild-type MazF-mt1 gene was PCR-amplified and cloned into an E. coli-M. smegmatis shuttle vector downstr
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