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Identification and Characterization of a Novel Toxin–Antitoxin Module From FEBS Letters 581 (2007) 1727–1734 Identification and characterization of a novel toxin–antitoxin module from Bacillus anthracis Shivangi Agarwala, Shivani Agarwalb, Rakesh Bhatnagara,* a School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India b Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India Received 10 November 2006; revised 3 March 2007; accepted 20 March 2007 Available online 30 March 2007 Edited by Judit Ova´di (95–135 aa) [9]. When the bacterium looses the plasmid during Abstract Comparative genome analysis of Bacillus anthracis revealed a pair of linked genes encoding pemK (K, killer protein) a segregational event, the degradation of antitoxin by cellular and pemI (I, inhibitory protein) homologous to pem loci of other proteases renders the toxin free to execute its lethal effect. organisms. Expression of PemK in Escherichia coli and Bacillus Therefore, these modules have been implicated in maintaining anthracis was bacteriostatic whereas the concomitant expression the stability of extra-chromosomal elements in the host ensur- of PemI reversed the growth arrest. PemK expression effectively ing propagation of only plasmid-inherited population. The dif- inhibited protein synthesis with no significant effect on DNA rep- ferent decay rate of these toxins and antitoxins has been lication. Coexpression and interaction of these proteins con- envisioned to be the molecular basis of toxin activation in plas- firmed it to be a Type II addiction module. Thermal mid-free cells. Such a genetic unit has been termed as an denaturation analysis reflected poor conformational stability of ‘Addiction module’ because the cells become addicted to the PemI as compared to PemK. Circular dichroism analysis indi- short-lived antitoxin and its de novo synthesis is essential for cated that PemI contains twice the amount of b-sheets as PemK. Gel retardation assays demonstrated that PemI binds to its up- cell survival [10]. The operons encoding toxins and antitoxins stream DNA sequence. This study reports the first evidence of are autoregulated at the transcription level either by a complex an active chromosome encoded toxin–antitoxin locus in B. of toxins with the cognate antitoxins or by the antitoxins alone anthracis. [8,10,11]. The intracellular target and the mechanism of action Ó 2007 Federation of European Biochemical Societies. Pub- for majority of the toxins are unknown. However, functions lished by Elsevier B.V. All rights reserved. such as inhibition of DNA gyrase, DnaB dependent replicative helicase and cell membrane depolarization have been attrib- Keywords: Toxin–antitoxin locus; Addiction module; Post uted to few of the plasmid encoded toxins while the chromo- segregational killing; Plasmid inheritance; Bacillus anthracis somal TA modules are a part of the cellular machinery functioning in response to stress and environmental stimuli [12–18]. Antibiotics, which are general inhibitors of transcrip- tion or translation, are reported to inhibit continuous expres- sion of the labile antitoxin and activation of highly stable 1. Introduction toxin conferring cell death [8]. B. anthracis, a causative agent for anthrax [19] is a potential Bacterial system has an astounding capacity to develop resis- biological warfare agent [20]. Therefore, it is pertinent to deci- tance determinants in a very short span of time leading to their pher the role of TA operon present in B. anthracis. The genetic multidrug resistance. Therefore, it is of paramount importance organization of the chromosomal TA locus and the products to develop newer antibiotics with greater potential and fewer of the genes identified from B. anthracis are similar to the side effects against bacterial aggression. The extensive charac- emerging paradigm of addiction modules for programmed cell terization of toxin–antitoxin addiction modules in many bacte- death prevalent in other prokaryotes [21–23]. Based on all ria suggests that programmed cell death is a general these observations, the present study describes a novel group phenomenon in prokaryotes also [1–6]. Most of these modules of Type II [13] TA system in B. anthracis. were initially identified only on plasmids, where they are asso- ciated with post segregational killing [7] and stable plasmid inheritance during cell division [8]. 2. Materials and methods Toxin–antitoxin (TA) loci are usually organized as an oper- on, where the first cistron encodes a small (65–85 aa) labile 2.1. Materials antitoxin and the second one encodes a large stable toxin Plasmid pET-28a (+) from Novagen (Madison, WI, USA) and pGEX-4Tl from Amersham Biosciences (Uppsala, Sweden) were used for heterogeneous gene expression. Restriction enzymes were from *Corresponding author. Fax: +91 11 26717040. Fermentas GmbH (Germany). Ni2+-NTA agarose resin was from Qia- E-mail address: [email protected] (R. Bhatnagar). gen (Hilden, Germany), Glutathione Sepharose 4FF resin, Nitrocellu- lose membrane, isopropyl b-D-thiogalactopyranoside (IPTG), Abbreviations: IPTG, isopropyl b-D-thiogalactopyranoside; rToxin, antibiotics and bovine serum albumin were from Amersham Biosci- 6·-histidine tagged recombinant toxin; rAntitoxin, 6·-histidine tagged ences. Anti histidine murine monoclonal (HIS-probe), alkaline phos- recombinant antitoxin; GST, glutathione S-transferase; TA, toxin–a- phatase conjugated antibodies and substrate (BCIP/NBT) were from ntitoxin; SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel/elec- Sigma (St. Louis, MO, USA). Bradford reagent used for protein trophoresis; SSD, salmon sperm DNA estimation was from Bio-Rad (CA, USA). Radioisotopes, [35S] 0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.03.051 1728 S. Agarwal et al. / FEBS Letters 581 (2007) 1727–1734 methionine, [methyl-3H] thymidine and [c-32P] ATP were from BRIT 2.6. Effect of expression of PemK on protein synthesis and DNA (CCMB, Hyderabad, India). Oligonucleotides were synthesized by synthesis in vivo Microsynth, Switzerland. E. coli BL21 (DE3) cells containing either pETSAT alone or pET- E. coli strains DH5a and BL21 (DE3) (Novagen) were used as hosts SAT and pGEXSAAT together were grown in minimal M9 media for cloning and expression, respectively. Avirulent strain of Bacillus [26] without methionine. Cell cultures were taken at different time anthracis (Sterne 34F2, pXO2 negative) and Bacillus subtilis 168 were intervals and mixed with 5 lCi of [35S] methionine (for protein synthe- used as hosts for homologous gene expression. sis) or 2 lCi of [methyl-3H] thymidine (for DNA synthesis). Also, 1 ml culture was aliquoted to measure OD at 600 nm. After 1 min of incor- 2.2. Expression and purification of PemK and PemI of B. anthracis poration at 37 °C, the samples were chased with 0.5 mg of unlabelled The open reading frame corresponding to pemK of B. anthracis was methionine or thymidine for 10 min. Pellets were harvested and resus- PCR amplified using forward primer (50-GACCGGATCCTTGATT- pended in 200 ll of cold 20% trichloroacetic acid solution and centri- GTAAAACGCGGCGAC-30) and a reverse primer (50-GCCC- fuged at 20,000 · g for 30 min at 4 °C. The samples were washed AAGCTTAAAATC TATTAGTCCTAAACT-30), containing a Bam- twice with 200 ll of chilled 96% ethanol and precipitates were trans- HI and HindIII restriction site, respectively (underlined). The ampli- ferred to the vials. Counts were measured in a liquid scintillation coun- fied DNA fragment was ligated to BamHI/HindIII sites of pET-28 a ter (Tricarb, DPackard, 1600TR). The amount of incorporated expression vector to obtain pETSAT plasmid. To construct pETSAAT radioactivity per OD600 (specific rate of synthesis) was plotted against plasmid, pemI was PCR amplified employing the oligonucleotides (50- time. GACCCATATGGGATCCGTGTCCGAATCAGAATCAAGTGTA- 0 0 ACT-3 ) and (5 -GCCCGTCGACGAATTCCCCCCCGCTAACTA- 2.7. Effect of expression of PemK and PemI in B. anthracis 0 AGCGTTC-3 ), containing BamHI and SalI restriction sites, respec- Gram-positive-E. coli shuttle vector featuring high structural stabil- tively (underlined). This fragment was cloned in BamHI/SalI digested ity (pHCMC05) donated to Bacillus Genetic Stock Center (BGSC) by pET-28a vector. pemI gene was also cloned in pGEX-4T1 (pGEX- Dr. Wolfgang Schumann [27] was procured. The forward and reverse SAAT) vector to facilitate interaction studies. The integrity of the primers used for pemK amplification were 50-GACCGGATCCTT- cloned genes was verified by automated dideoxy DNA sequencing. GATTGTAAAACGCGGCGAC-30 and 50-CCTCTAGAACGCGTA The expression of recombinant proteins from these constructs was in- AAATCTATTAGTCCTAAACT-30. pemI was PCR amplified using duced at an optical density of 0.6 at 600 nm by the addition of IPTG forward primer, 50-GCCCGGATCCGTGTCCGAATCAAGTGTA- (1 mM) for 3 h at 37 °C. PemK and PemI with His6-tag at N-terminus 0 0 2+ ACTACT-3 and reverse primer 5 -GACCTCTAGAACGCGTCCC- were purified from the soluble fraction using Ni -NTA affinity chro- CCCGCTAACTAAGCGTTC-30. BamHI and XbaI sites (underlined) matography as described by Qiagen. Purification of PemI with gluta- were added to the forward and reverse primers, respectively. PCR thione S-transferase (GST) tag at the N-terminus was accomplished amplified pemK and pemI were cloned in pHCMC05 vector to generate using glutathione sepharose affinity chromatography as per the manu- pSpacpemk and pSpacpemI, respectively. Both the genes were there- facturer’s instructions.
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