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• W2024398218 abstract "The role of chromosomal toxin-antitoxin (TA) modules in bacterial physiology remains enigmatic despite their abundance in the genomes of many bacteria. Mycobacterium smegmatis contains three putative TA systems, VapBC, MazEF, and Phd/Doc, and previous work from our group has shown VapBC to be a bona fide TA system. In this study, we show that MazEF and Phd/Doc are also TA systems that are constitutively expressed, transcribed as leaderless transcripts, and subject to autoregulation, and expression of the toxin component leads to growth inhibition that can be rescued by the cognate antitoxin. No phenotype was identified for deletions of the individual TA systems, but a triple deletion strain (ΔvapBC, mazEF, phd/doc), designated ΔTAtriple, exhibited a survival defect in complex growth medium demonstrating an essential role for these TA modules in mycobacterial survival. Transcriptomic analysis revealed no significant differences in gene expression between wild type and the ΔTAtriple mutant under these conditions suggesting that the growth defect was not at a transcriptional level. Metabolomic analysis demonstrated that in response to starvation in complex medium, both the wild type and ΔTAtriple mutant consumed a wide range of amino acids from the external milieu. Analysis of intracellular metabolites revealed a significant difference in the levels of branched-chain amino acids between the wild type and ΔTAtriple mutant, which are proposed to play essential roles in monitoring the nutritional supply and physiological state of the cell and linking catabolic with anabolic reactions. Disruption of this balance in the ΔTAtriple mutant may explain the survival defect in complex growth medium. The role of chromosomal toxin-antitoxin (TA) modules in bacterial physiology remains enigmatic despite their abundance in the genomes of many bacteria. Mycobacterium smegmatis contains three putative TA systems, VapBC, MazEF, and Phd/Doc, and previous work from our group has shown VapBC to be a bona fide TA system. In this study, we show that MazEF and Phd/Doc are also TA systems that are constitutively expressed, transcribed as leaderless transcripts, and subject to autoregulation, and expression of the toxin component leads to growth inhibition that can be rescued by the cognate antitoxin. No phenotype was identified for deletions of the individual TA systems, but a triple deletion strain (ΔvapBC, mazEF, phd/doc), designated ΔTAtriple, exhibited a survival defect in complex growth medium demonstrating an essential role for these TA modules in mycobacterial survival. Transcriptomic analysis revealed no significant differences in gene expression between wild type and the ΔTAtriple mutant under these conditions suggesting that the growth defect was not at a transcriptional level. Metabolomic analysis demonstrated that in response to starvation in complex medium, both the wild type and ΔTAtriple mutant consumed a wide range of amino acids from the external milieu. Analysis of intracellular metabolites revealed a significant difference in the levels of branched-chain amino acids between the wild type and ΔTAtriple mutant, which are proposed to play essential roles in monitoring the nutritional supply and physiological state of the cell and linking catabolic with anabolic reactions. Disruption of this balance in the ΔTAtriple mutant may explain the survival defect in complex growth medium. Toxin-antitoxin (TA) 3The abbreviations used are: TAtoxin-antitoxinHdBHartmans de Bont mediumMUMiller unitsqPCRquantitative real time PCRTPP+methyltriphenylphosphonium iodideTSStranscriptional start siteADCalbumin/glucose/catalase enrichmentFforwardRreverseTctetracyclineTSStranscriptional start siteRACErapid amplification of cDNA endsDocdeath-on-curing protein. systems were first discovered as plasmid stability systems that rely on the constant production of the labile antitoxin to prevent the release of active toxin from the benign protein complex that is normally found in the cell (1.Ogura T. Hiraga S. Mini-F plasmid genes that couple host cell division to plasmid proliferation.Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 4784-4788Crossref PubMed Scopus (405) Google Scholar, 2.Jaffé A. Ogura T. Hiraga S. Effects of the ccd function of the F plasmid on bacterial growth.J. Bacteriol. 1985; 163: 841-849Crossref PubMed Google Scholar). Active toxin may cause cell death to prevent loss of the plasmid within the population, or it may inhibit cell growth to allow the levels of the plasmid to return to normal. TA systems have also been found in bacterial chromosomes, and at present there has been considerable debate about the role of these systems in cell physiology with several hypotheses being presented (3.Magnuson R.D. Hypothetical functions of toxin-antitoxin systems.J. Bacteriol. 2007; 189: 6089-6092Crossref PubMed Scopus (213) Google Scholar). There are three types of TA systems, and they differ primarily in the interactions between the toxin and the antitoxin to form the benign complex. In type I TA systems, such as the Hok/Sok system of plasmid R1, the antitoxin is a small RNA that inhibits the mRNA of the toxin. The complex in type II systems is composed of two proteins, and the toxic protein of the type III system is inhibited by the antitoxic RNA (4.Fineran P.C. Blower T.R. Foulds I.J. Humphreys D.P. Lilley K.S. Salmond G.P. The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 894-899Crossref PubMed Scopus (363) Google Scholar). toxin-antitoxin Hartmans de Bont medium Miller units quantitative real time PCR methyltriphenylphosphonium iodide transcriptional start site albumin/glucose/catalase enrichment forward reverse tetracycline transcriptional start site rapid amplification of cDNA ends death-on-curing protein. Nine families of toxins have been characterized for the type II TA systems (5.Gerdes K. Christensen S.K. Løbner-Olesen A. Prokaryotic toxin-antitoxin stress-response loci.Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (864) Google Scholar, 6.Van Melderen L. Saavedra De Bast M. Bacterial toxin-antitoxin systems. More than selfish entities?.PLoS Genet. 2009; 5: e1000437Crossref PubMed Scopus (381) Google Scholar). Of these, the mazEF, relBE, and vapBC families have been studied in detail. All three of these toxins cleave mRNA; RelE requires the ribosome for RNA cleavage, whereas MazF, HigB, and VapC do not (7.Zhang Y. Zhang J. Hoeflich K.P. Ikura M. Qing G. Inouye M. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli.Mol. Cell. 2003; 12: 913-923Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 8.Pedersen K. Zavialov A.V. Pavlov M.Y. Elf J. Gerdes K. Ehrenberg M. The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site.Cell. 2003; 112: 131-140Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 9.Arcus V.L. Bäckbro K. Roos A. Daniel E.L. Baker E.N. Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease.J. Biol. Chem. 2004; 279: 16471-16478Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10.Christensen-Dalsgaard M. Gerdes K. Two higBA loci in the Vibrio cholerae superintegron encode mRNA cleaving enzymes and can stabilize plasmids.Mol. Microbiol. 2006; 62: 397-411Crossref PubMed Scopus (141) Google Scholar). The CcdB and ParE toxins inhibit chromosome replication through DNA gyrase (11.Jiang Y. Pogliano J. Helinski D.R. Konieczny I. ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase.Mol. Microbiol. 2002; 44: 971-979Crossref PubMed Scopus (211) Google Scholar, 12.Bernard P. Couturier M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.J. Mol. Biol. 1992; 226: 735-745Crossref PubMed Scopus (369) Google Scholar, 13.Maki S. Takiguchi S. Miki T. Horiuchi T. Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins.J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar); the Doc toxin inhibits the 30 S subunit of the ribosome (14.Liu M. Zhang Y. Inouye M. Woychik N.A. Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30 S ribosomal subunit.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5885-5890Crossref PubMed Scopus (105) Google Scholar), and HipA phosphorylates the translation factor EF-Tu (15.Schumacher M.A. Piro K.M. Xu W. Hansen S. Lewis K. Brennan R.G. Molecular mechanisms of HipA-mediated multidrug tolerance and its neutralization by HipB.Science. 2009; 323: 396-401Crossref PubMed Scopus (260) Google Scholar). Even though the toxins target important components of the cell, the role of chromosomally encoded TA systems has been difficult to determine as the phenotypes appear to be pleiotropic. A five TA deletion strain has been created in Escherichia coli where the mazEF, relBE, yefM-yoeB, chpB, and dinJ-yafQ TA systems have been deleted (16.Tsilibaris V. Maenhaut-Michel G. Mine N. Van Melderen L. What is the benefit to Escherichia coli of having multiple toxin-antitoxin systems in its genome?.J. Bacteriol. 2007; 189: 6101-6108Crossref PubMed Scopus (169) Google Scholar). No phenotypic difference was found in response to stress and recovery responses. However, when Kim et al. (17.Kim Y. Wang X. Ma Q. Zhang X.S. Wood T.K. Toxin-antitoxin systems in Escherichia coli influence biofilm formation through YjgK (TabA) and fimbriae.J. Bacteriol. 2009; 191: 1258-1267Crossref PubMed Scopus (144) Google Scholar) investigated the role of TA systems in biofilms, they found that this five TA deletion strain had reduced initial biofilm formation and reduced biofilm dispersal. Each of the five TA systems was found to play a different role in the process of forming biofilms, with MazF appearing to be the main effector in the network (18.Kolodkin-Gal I. Verdiger R. Shlosberg-Fedida A. Engelberg-Kulka H. A differential effect of E. coli toxin-antitoxin systems on cell death in liquid media and biofilm formation.PLoS One. 2009; 4: e6785Crossref PubMed Scopus (79) Google Scholar). Several studies have been performed to identify TA systems in the chromosomes of sequenced bacteria and archaea (19.Pandey D.P. Gerdes K. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (723) Google Scholar, 20.Anantharaman V. Aravind L. New connections in the prokaryotic toxin-antitoxin network. Relationship with the eukaryotic nonsense-mediated RNA decay system.Genome Biol. 2003; 4: R81Crossref PubMed Google Scholar). These findings suggest a large amount of diversity in the number of TA systems in each genome. One group that is of interest is the genus Mycobacterium. The number of TA systems in the genome of Mycobacterium tuberculosis has been greatly expanded with 88 putative TA systems present (19.Pandey D.P. Gerdes K. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (723) Google Scholar, 21.Ramage H.R. Connolly L.E. Cox J.S. Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems. Implications for pathogenesis, stress responses, and evolution.PLoS Genet. 2009; 5: e1000767Crossref PubMed Scopus (367) Google Scholar). In contrast, the chromosome of Mycobacterium leprae contains only toxin pseudogenes, whereas Mycobacterium smegmatis contains only three TA systems (19.Pandey D.P. Gerdes K. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.Nucleic Acids Res. 2005; 33: 966-976Crossref PubMed Scopus (723) Google Scholar, 22.Robson J. McKenzie J.L. Cursons R. Cook G.M. Arcus V.L. The vapBC operon from Mycobacterium smegmatis is an autoregulated toxin-antitoxin module that controls growth via inhibition of translation.J. Mol. Biol. 2009; 390: 353-367Crossref PubMed Scopus (82) Google Scholar). The greatest number of TA modules in M. tuberculosis belongs to VapBC with 47 in total. The vapBC family of TA systems consists of the antitoxin VapB and the toxin VapC, which is proposed to be an RNase. This family has recently been reviewed (23.Arcus V.L. McKenzie J.L. Robson J. Cook G.M. The PIN-domain ribonucleases and the prokaryotic VapBC toxin-antitoxin array.Protein Eng. Des. Sel. 2011; 24: 33-40Crossref PubMed Scopus (126) Google Scholar). There are examples where VapBC systems have been shown to play a role in the adaptation of the bacteria to an environmental niche (e.g. epithelial cells and root nodules) (24.Hopper S. Wilbur J.S. Vasquez B.L. Larson J. Clary S. Mehr I.J. Seifert H.S. So M. Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers.Infect. Immun. 2000; 68: 896-905Crossref PubMed Scopus (56) Google Scholar). To provide a molecular model for understanding the role of TA systems in mycobacteria, we have characterized all three TA systems in M. smegmatis at a molecular level. We show that all three TA systems are bona fide TA modules, and deleting all three (vapBC-mazEF-phd/doc) in M. smegmatis leads to a survival defect in complex growth medium. All strains and plasmids used in this study are listed in Table 1. E. coli strains were grown in Luria-Bertani (LB) medium at 37 °C with agitation (200 rpm). M. smegmatis strain mc2155 and derived strains were routinely grown at 37 °C, 200 rpm in LB containing 0.05% (w/v) Tween 80 (LBT), in Middlebrook 7H9 medium (Difco) supplemented with 10% albumin/glucose/catalase enrichment (ADC; BD Biosciences) and 0.05% (w/v) Tween 80, or in modified Hartmans de Bont medium (HdB) (25.Smeulders M.J. Keer J. Speight R.A. Williams H.D. Adaptation of Mycobacterium smegmatis to stationary phase.J. Bacteriol. 1999; 181: 270-283Crossref PubMed Google Scholar). The composition of this medium per liter was as follows: 10 ml of trace metals, 27.4 mm glycerol, 15 mm ammonium sulfate, 21 mm Na2HPO4, and 11 mm KH2PO4 and 0.05% Tween 80. For nutrient limitation studies, glycerol was reduced to 11 mm for carbon limitation, ammonium sulfate reduced 100-fold for nitrogen limitation, and Na2HPO4 and K2HPO4 reduced 100-fold for phosphate limitation. During phosphate limitation, the buffering capacity of the medium was increased by the addition of 50 mm MOPS.TABLE 1Bacterial strains and plasmids used in this studyDescriptionaThe following abbreviations are used: Gmr, gentamicin resistance; Hygr, hygromycin B resistance; Kmr, kanamycin resistance; Apr, ampicillin resistance; Strepr, streptomycin resistance; Specr, spectomycin resistance; Sacs, sucrose sensitivity; ts, temperature sensitivity.Source or ref.Bacterial strains E. coliDH10BF− mcrAΔ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74deoRrecA1 araD139Δ(araleu)7697galUgalKrpsLendA1nupG77.Hanahan D. Jessee J. Bloom F.R. Plasmid transformation of Escherichia coli and other bacteria.Methods Enzymol. 1991; 204: 63-113Crossref PubMed Scopus (451) Google ScholarM. smegmatismc2155Electrocompetent wild-type strain of M. smegmatis78.Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.Mol. Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (1009) Google ScholarRF100mc2155ΔmazEFThis studyRF101mc2155Δphd/doc::aphA-3; KmrThis studyRF102mc2155ΔmazEFΔvapBC::aphA-3; KmrThis studyRF103mc2155ΔmazEFΔphd/doc::aphA-3; KmrThis studyRF104mc2155ΔvapBC::aphA-3Δphd/doc::aphA-3; KmrThis studyRF105mc2155ΔmazEFΔvapBC::aphA-3Δphd/doc::aphA-3; ΔTAtriple; KmrThis studyRF121mc2155 with pRF211 integrated in attB; StreprThis studyRF122RF101 with pRF211 integrated in attB; Kmr StreprThis studyPlasmidspBluescript II KSCloning vector; AprStratagenepUC18KE. coli plasmid containing an excisable, nonpolar kanamycin resistance cassette; Kmr Apr33.Ménard R. Sansonetti P.J. Parsot C. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells.J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (615) Google ScholarpX33E. coli-mycobacteria shuttle vector for allelic exchange mutagenesis in mycobacteria, pPR23, carrying a constitutive xylE marker; Gmr Sacs ts30.Gebhard S. Tran S.L. Cook G.M. The Phn system of Mycobacterium smegmatis. A second high-affinity ABC transporter for phosphate.Microbiology. 2006; 152: 3453-3465Crossref PubMed Scopus (56) Google ScholarpSM128lacZ transcriptional fusion vector derived from pYUB, with mycobacteriophage L5 integrase and attP for integration into attB of mycobacteria; Specr Strepr35.Dussurget O. Timm J. Gomez M. Gold B. Yu S. Sabol S.Z. Holmes R.K. Jacobs Jr., W.R. Smith I. Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR.J. Bacteriol. 1999; 181: 3402-3408Crossref PubMed Google ScholarpJEM15E. coli-mycobacteria shuttle vector for the creation of transcriptional promoter fusions to lacZ; Kmr34.Timm J. Lim E.M. Gicquel B. Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ. The pJEM series.J. Bacteriol. 1994; 176: 6749-6753Crossref PubMed Google ScholarpMindTetracycline-inducible expression vector; Kmr Hygr29.Blokpoel M.C. Murphy H.N. O'Toole R. Wiles S. Runn E.S. Stewart G.R. Young D.B. Robertson B.D. Tetracycline-inducible gene regulation in mycobacteria.Nucleic Acids Res. 2005; 33: e22Crossref PubMed Scopus (143) Google ScholarpSE100Tetracycline-inducible expression vector; Hygr27.Guo X.V. Monteleone M. Klotzsche M. Kamionka A. Hillen W. Braunstein M. Ehrt S. Schnappinger D. Silencing Mycobacterium smegmatis by using tetracycline repressors.J. Bacteriol. 2007; 189: 4614-4623Crossref PubMed Scopus (93) Google Scholar; Addgene 17972pMC1sTetracycline repressor under strong mycobacterial promoter; Kmr28.Ehrt S. Guo X.V. Hickey C.M. Ryou M. Monteleone M. Riley L.W. Schnappinger D. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.Nucleic Acids Res. 2005; 33: e21Crossref PubMed Scopus (271) Google Scholar; Addgene 17969pJR113pX33 harboring ΔvapBC::aphA-3; Kmr Gmr Sacs ts22.Robson J. McKenzie J.L. Cursons R. Cook G.M. Arcus V.L. The vapBC operon from Mycobacterium smegmatis is an autoregulated toxin-antitoxin module that controls growth via inhibition of translation.J. Mol. Biol. 2009; 390: 353-367Crossref PubMed Scopus (82) Google ScholarpRF202pSE100 harboring mazEF with RBS from kanamycin marker; HygrThis studypRF203pSE100 harboring mazF with RBS from kanamycin cassette; HygrThis studypRF200pMind harboring doc with RBS from kanamycin marker; Kmr (Hygr)This studypRF201pMind harboring phd/doc with RBS from kanamycin marker; Kmr (Hygr)This studypRF210pJEM15 harboring a 663-bp PmazE-lacZ fusion; KmrThis studypRF211pSM128 harboring a 576-bp Pphd-lacZ fusion; Specr StreprThis studypRF220pX33 harboring ΔmazEF; Gmr Sacs tsThis studypRF221pX33 harboring Δphd/doc::aphA-3; Kmr Gmr Sacs tsThis studya The following abbreviations are used: Gmr, gentamicin resistance; Hygr, hygromycin B resistance; Kmr, kanamycin resistance; Apr, ampicillin resistance; Strepr, streptomycin resistance; Specr, spectomycin resistance; Sacs, sucrose sensitivity; ts, temperature sensitivity. Open table in a new tab M. smegmatis transformants were grown at 28 °C for propagation of temperature-sensitive vectors and at 40 °C for allelic exchange mutagenesis. Selective media contained kanamycin (50 μg ml−1 for E. coli; 20 μg ml−1 for M. smegmatis), gentamicin (20 μg ml−1 for E. coli; 5 μg ml−1 for M. smegmatis), hygromycin B (200 μg ml−1 for E. coli; 50 μg ml−1 for M. smegmatis), spectinomycin (50 μg ml−1 for E. coli), streptomycin (50 μg ml−1 for E. coli, 20 μg ml−1 for M. smegmatis), and ampicillin (100 μg ml−1 for E. coli). Solid media contained 1.5% agar. Growth curves were performed in triplicate in LBT and HdB. Bacterial cell viability was monitored by cell counts based on cfu ml−1 where serial dilutions of bacterial cell culture in phosphate-buffered saline with 0.05% (w/v) Tween 80 (PBS-T) were spread onto LBT agar plates supplemented with appropriate antibiotics. For adaptation to hypoxia experiments, 100-ml serum vials were used, containing 30 ml of LBT or HdB. For iron limitation the iron chelator, ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid), was added to media at a final concentration of 10 μm, and to supplement the media with extra iron, iron sulfate was added to a final concentration of 100 μm. External pH of cultures was measured using pH indicator strips (4.0–7.0 or 7.5–14.0; Merck). Absorbance was measured at 600 nm (A600) using culture samples diluted in saline to bring A600 to below 0.5 when measuring in cuvettes of 1-cm light path length in a Jenway 6300 spectrophotometer. Genomic DNA from M. smegmatis was isolated using the cetyltrimethylammonium bromide method (26.Bull T.J. Hermon-Taylor J. Pavlik I. El-Zaatari F. Tizard M. Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing.Microbiology. 2000; 146: 2185-2197Crossref PubMed Scopus (115) Google Scholar). Restriction or DNA-modifying enzymes and other molecular biology reagents were obtained from Roche Diagnostics or New England Biolabs. All primers used in this study are shown in supplemental Table S1. The tetracycline-inducible expression construct for mazF was made by amplifying the gene using primers mazFpSE100 F and mazFpSE100 R. This product was ligated into the BamHI/PstI sites of pSE100 (27.Guo X.V. Monteleone M. Klotzsche M. Kamionka A. Hillen W. Braunstein M. Ehrt S. Schnappinger D. Silencing Mycobacterium smegmatis by using tetracycline repressors.J. Bacteriol. 2007; 189: 4614-4623Crossref PubMed Scopus (93) Google Scholar) (Addgene plasmid 17972), creating plasmid pRF202. This was also done for the mazEF operon using primers mazEFpSE100 F and mazFpSE100 R, and this created plasmid pRF203. Plasmids pRF202 and pRF203 were electroporated into mc2155 with pMC1s (28.Ehrt S. Guo X.V. Hickey C.M. Ryou M. Monteleone M. Riley L.W. Schnappinger D. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.Nucleic Acids Res. 2005; 33: e21Crossref PubMed Scopus (271) Google Scholar), which integrates into the attB site. The primers docpMind F and docpMind R were used to amplify the doc gene, and this product was ligated into the BamHI/SpeI sites of pMind (29.Blokpoel M.C. Murphy H.N. O'Toole R. Wiles S. Runn E.S. Stewart G.R. Young D.B. Robertson B.D. Tetracycline-inducible gene regulation in mycobacteria.Nucleic Acids Res. 2005; 33: e22Crossref PubMed Scopus (143) Google Scholar), creating pRF200. The phd/doc operon was amplified using primers phd/docpMind F and docpMind R. This product was also ligated into the BamHI/SpeI sites of pMind, creating pRF201. To create a construct for the deletion of MSMEG_4447 and MSMEG_4448 (mazEF), PCR products of ∼850 bp flanking the mazEF genes of M. smegmatis were amplified using the primer mazEFKOLF F with primer mazEFKOLF R (left flank) and primer mazEFKORF F with primer mazEFKO RF R (right flank). Primer mazEFKOLF R and primer mazEFKORF F contain 7-bp overlapping regions. The left flank and right flank PCR products were used in a second PCR with primer mazEFKOLF F and primer mazEFKORF R to generate an overlapping PCR product of the mazEF flanking regions with 88% of the genes deleted. This PCR product was digested with SpeI and ligated into the SpeI site of the pBluescript II KS plasmid. The insert was then subcloned into pX33 (30.Gebhard S. Tran S.L. Cook G.M. The Phn system of Mycobacterium smegmatis. A second high-affinity ABC transporter for phosphate.Microbiology. 2006; 152: 3453-3465Crossref PubMed Scopus (56) Google Scholar) generating the pRF220 plasmid. Allelic replacement was carried out essentially as described previously (31.Pelicic V. Reyrat J.M. Gicquel B. Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors.Mol. Microbiol. 1996; 20: 919-925Crossref PubMed Scopus (135) Google Scholar, 32.Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10955-10960Crossref PubMed Scopus (386) Google Scholar); it was achieved by growing a culture of M. smegmatis carrying pRF220 in Middlebrook 7H9-ADC medium with gentamicin at 28 °C with agitation (200 rpm) to an A600 of ∼0.5, followed by plating on LBT plates with gentamicin and incubating at 40 °C. Incubation at 40 °C selected for integration of the entire deletion construct into the chromosome of M. smegmatis via a single crossover event at either the left or right flank. Colonies that formed a yellow product when exposed to 250 mm catechol because of the presence of the xylE marker were screened by Southern hybridization analysis for correct integration of the construct. One integrant was chosen and grown in LBT with gentamicin at 37 °C, 200 rpm to an A600 of ∼0.5, followed by plating onto low salt LBT plates (2 g of NaCl liter−1) containing 10% sucrose and incubated at 40 °C to select for a second crossover event resulting in loss of the plasmid and replacement of mazEF with the overlapping flanks. Colonies that did not form the yellow product after exposure to catechol were picked onto LBT plates to confirm loss of the plasmid backbone. Candidate clones were screened by Southern hybridization analysis for correct deletion of mazEF. Replacement of mazEF with the overlapping flanks created strain RF100 (ΔmazEF). To create a construct for the deletion of MSMEG_1277 and MSMEG_1278 (phd/doc), the kanamycin resistance (Kmr) cassette, encoded by aphA-3, was amplified from pUC18K (33.Ménard R. Sansonetti P.J. Parsot C. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells.J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (615) Google Scholar) by using primers 5′mcspUC F and 3′mcspUC R. The resulting 850-bp product was digested with EcoRI and PstI. PCR products of ∼1000 bp flanking the phd/doc genes of M. smegmatis were amplified by using the primer phd/docKOLF F with primer phd/docKOLF R (left flank) and primer phd/docKORF F with primer phd/docKORF R (right flank). The left flank PCR product was digested with SpeI and EcoRI, and the right flank PCR product was digested with PstI and SpeI. Both flanking products and the kanamycin cassette were ligated into the SpeI site of the pBluescript II KS plasmid. The resulting assembly, left flank/Kmr/right flank, was subcloned as a SpeI fragment into pX33 (30.Gebhard S. Tran S.L. Cook G.M. The Phn system of Mycobacterium smegmatis. A second high-affinity ABC transporter for phosphate.Microbiology. 2006; 152: 3453-3465Crossref PubMed Scopus (56) Google Scholar) generating the pRF221 plasmid. The expected double crossover event would result in a nonpolar deletion insertion at the phd/doc locus, eliminating 73% of the phd/doc coding sequence in exchange for the kanamycin resistance marker. Allelic replacement of phd/doc was carried out essentially as described previously (32.Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10955-10960Crossref PubMed Scopus (386) Google Scholar) and was achieved by growing a culture of M. smegmatis carrying pRF221 in Middlebrook 7H9-ADC medium with kanamycin at 28 °C with agitation (200 rpm) to an A600 of ∼0.5, followed by plating onto low salt LBT plates (2 g NaCl liter−1) containing kanamycin and 10% sucrose at 40 °C, selecting for double crossover events. Replacement of phd/doc with the kanamycin marker created strain RF101 (Δphd/doc::aphA-3). To create a ΔmazEF ΔvapBC Δphd/doc triple mutant, a ΔmazEF ΔvapBC double mutant was made. Strain RF100 was transformed with pJR113 (22.Robson J. McKenzie J.L. Cursons R. Cook G.M. Arcus V.L. The vapBC operon from Mycobacterium smegmatis is an autoregulated toxin-antitoxin module that controls growth via inhibition of translation.J. Mol. Biol. 2009; 390: 353-367Crossref PubMed Scopus (82) Google Scholar), and the mutant was created as described previously, and this is strain RF102. This strain was then transformed with pRF221, and the triple mutant (ΔTAtriple; RF105) was created using the two-step crossover protocol. All crossover events were analyzed by Southern hybridization. For Southern hybridization analysis, EcoRI-digested genomic DNA (SmaI-digested genomic DNA was used for the creation of the ΔmazEF ΔvapBC double mutant) of the putative mutants was separated on a 0.8% agarose/Tris acetate/EDTA gel and transferred onto a nylon membrane (Hybond-N+; Amersham Biosciences) by vacuum blotting. Probes were labeled using Gene Images AlkPhos Direct Labeling and Detection System (Amersham Biosciences). To create a transcriptional fusion of the mazEF operon, a PCR product encompassing 556 bp of DNA upstream of mazE and 107 bp of its coding region was amplified using the primers mazElac F and mazElac R. The product was cloned into the BamHI and Asp-718 sites of pJEM15 (34.Timm J. Lim E.M. Gicquel B. Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ. The pJEM series.J. Bacteriol. 1994; 176: 6749-6753Crossref PubMed Google Scholar), creating plasmid pRF210. To create a transcriptional fusion of the phd/doc operon, a PCR product encompa" @default.
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• W2024398218 date "2012-02-01" @default.
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• W2024398218 title "Toxin-Antitoxin Systems of Mycobacterium smegmatis Are Essential for Cell Survival" @default.
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