relaxed cleavage specificity within the rele toxin familytable 1 similarity and identity between...

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Relaxed Cleavage Specificity within the RelE Toxin Family Nathalie Goeders, Pierre-Luc Drèze, Laurence Van Melderen Laboratoire de Génétique et Physiologie Bactérienne, IBMM, Faculté des Sciences, Université Libre de Bruxelles (ULB), Gosselies, Belgium Bacterial type II toxin-antitoxin systems are widespread in bacteria. Among them, the RelE toxin family is one of the most abun- dant. The RelE K-12 toxin of Escherichia coli K-12 represents the paradigm for this family and has been extensively studied, both in vivo and in vitro. RelE K-12 is an endoribonuclease that cleaves mRNAs that are translated by the ribosome machinery as these transcripts enter the A site. Earlier in vivo reports showed that RelE K-12 cleaves preferentially in the 5=-end coding region of the transcripts in a codon-independent manner. To investigate whether the molecular activity as well as the cleavage pattern are conserved within the members of this toxin family, RelE-like sequences were selected in Proteobacteria, Cyanobacteria, Actino- bacteria, and Spirochaetes and tested in E. coli. Our results show that these RelE-like sequences are part of toxin-antitoxin gene pairs, and that they inhibit translation in E. coli by cleaving transcripts that are being translated. Primer extension analyses show that these toxins exhibit specific cleavage patterns in vivo, both in terms of frequency and location of cleavage sites. We did not observe codon-dependent cleavage but rather a trend to cleave upstream purines and between the second and third positions of codons, except for the actinobacterial toxin. Our results suggest that RelE-like toxins have evolved to rapidly and efficiently shut down translation in a large spectrum of bacterial species, which correlates with the observation that toxin-antitoxin systems are spreading by horizontal gene transfer. B acteria thrive in ever-changing environments; therefore, they have evolved regulatory mechanisms allowing rapid modula- tion of gene expression and adaptation. Among these mecha- nisms, ribonucleases (RNAses) play an important regulatory role by adjusting RNA levels (for a review, see reference 1). Escherichia coli has more than 20 RNases involved in different processes, such as RNA quality control, RNA maturation, and stress response modulation. In addition, E. coli carries numerous RNases that act as bacteriocins (for a review, see reference 2) or belong to CRISPR systems (for a review, see reference 3) and toxin-antitoxin (TA) systems (for a review, see reference 4). TA systems are classified into different types depending on the nature and mode of action of the antitoxin, with the toxin always being a protein. Type II systems are generally composed of two genes organized in an operon, the first gene encoding an antitoxin protein and the second a toxin. These systems are abundant in bacterial genomes. In some bacterial species, such as Nitrosomonas europaea (Proteobacteria) and Chlorobium chlorochromatii (Chlo- robi), predicted type II TA systems represent around 2.5% of the total predicted open reading frames (ORFs) of these genomes (5). Type II systems are associated with mobile genetic elements, such as plasmids and phages, as well as with chromosomes, in which they may be part of or constitute by themselves genomic islands/ islets (for a review, see reference 4). It has been proposed that they move between genomes through horizontal gene transfer. The question of their biological roles remains debated, although sev- eral interesting hypotheses have emerged, notably their implica- tions in programmed cell death, stress response, persistence, sta- bilization of large genomic regions, or mobile genetic elements (for a review, see reference 4). Interestingly, the vast majority of type II toxins that have been identified and characterized so far are endoribonucleases (also denoted as mRNA interferases) (714). One of the best character- ized is the RelE K-12 toxin of the relBE K-12 system of E. coli K-12 (7). RelE K-12 is an endoribonuclease that cleaves mRNAs in a transla- tion-dependent manner. Free RelE K-12 enters the ribosomal A site and binds to the ribosome 30S subunit (15, 16). Resolution of the three-dimensional structure of the RelE K-12 -ribosome complex showed that subsequent to complex formation, the target mRNA in the A site is reoriented so that RelE K-12 catalyzes cleavage of the transcript (16). Three RelE K-12 residues are essential for this activ- ity: Y87, which reorients and stabilizes the mRNA to allow the nucleophilic attack; R61, which stabilizes the cleavage transition state; and R81, which acts as a general acid (16). RelE K-12 cleaves preferentially in the 5= region of the target mRNA, usually between the second and third nucleotide of codons or between two codons (17). The RelB K-12 antitoxin wraps around the toxin, thereby in- hibiting its entry in the A site and leading to structural rearrange- ments that disrupt the RelE K-12 catalytic site (18, 19). RelE-like toxins belong to the widespread type II ParE/RelE superfamily (5, 20). Type II toxin superfamilies are based on sim- ilarities at the level of amino acid sequence and three-dimensional structure prediction/determination (5, 21). Interestingly, this su- perfamily is composed of two functionally (although not structur- ally) distinct families that are either endoribonucleases, as men- tioned above (RelE family), or proteins inhibiting DNA-gyrase and DNA replication (ParE family) (6, 22). This suggests that these two families share a common ancestor and have functionally diverged during evolution. Several RelE-like proteins, such as YoeB (14, 23), MqsR (24), YafQ (25), and YgjN (10), of Esche- richia coli K-12 have been characterized both at the activity and structural levels. While they all share a fold with RelE K-12 and most Received 27 December 2012 Accepted 20 March 2013 Published ahead of print 29 March 2013 Address correspondence to Laurence Van Melderen, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JB.02266-12. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.02266-12 June 2013 Volume 195 Number 11 Journal of Bacteriology p. 2541–2549 jb.asm.org 2541 on March 7, 2020 by guest http://jb.asm.org/ Downloaded from

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Page 1: Relaxed Cleavage Specificity within the RelE Toxin FamilyTABLE 1 Similarity and identity between the RelE-like toxins and the RelE K-12 and YoeB K-12 canonical toxins a Toxin RelE

Relaxed Cleavage Specificity within the RelE Toxin Family

Nathalie Goeders, Pierre-Luc Drèze, Laurence Van Melderen

Laboratoire de Génétique et Physiologie Bactérienne, IBMM, Faculté des Sciences, Université Libre de Bruxelles (ULB), Gosselies, Belgium

Bacterial type II toxin-antitoxin systems are widespread in bacteria. Among them, the RelE toxin family is one of the most abun-dant. The RelEK-12 toxin of Escherichia coli K-12 represents the paradigm for this family and has been extensively studied, bothin vivo and in vitro. RelEK-12 is an endoribonuclease that cleaves mRNAs that are translated by the ribosome machinery as thesetranscripts enter the A site. Earlier in vivo reports showed that RelEK-12 cleaves preferentially in the 5=-end coding region of thetranscripts in a codon-independent manner. To investigate whether the molecular activity as well as the cleavage pattern areconserved within the members of this toxin family, RelE-like sequences were selected in Proteobacteria, Cyanobacteria, Actino-bacteria, and Spirochaetes and tested in E. coli. Our results show that these RelE-like sequences are part of toxin-antitoxin genepairs, and that they inhibit translation in E. coli by cleaving transcripts that are being translated. Primer extension analyses showthat these toxins exhibit specific cleavage patterns in vivo, both in terms of frequency and location of cleavage sites. We did notobserve codon-dependent cleavage but rather a trend to cleave upstream purines and between the second and third positions ofcodons, except for the actinobacterial toxin. Our results suggest that RelE-like toxins have evolved to rapidly and efficiently shutdown translation in a large spectrum of bacterial species, which correlates with the observation that toxin-antitoxin systems arespreading by horizontal gene transfer.

Bacteria thrive in ever-changing environments; therefore, theyhave evolved regulatory mechanisms allowing rapid modula-

tion of gene expression and adaptation. Among these mecha-nisms, ribonucleases (RNAses) play an important regulatory roleby adjusting RNA levels (for a review, see reference 1). Escherichiacoli has more than 20 RNases involved in different processes, suchas RNA quality control, RNA maturation, and stress responsemodulation. In addition, E. coli carries numerous RNases that actas bacteriocins (for a review, see reference 2) or belong to CRISPRsystems (for a review, see reference 3) and toxin-antitoxin (TA)systems (for a review, see reference 4).

TA systems are classified into different types depending on thenature and mode of action of the antitoxin, with the toxin alwaysbeing a protein. Type II systems are generally composed of twogenes organized in an operon, the first gene encoding an antitoxinprotein and the second a toxin. These systems are abundant inbacterial genomes. In some bacterial species, such as Nitrosomonaseuropaea (Proteobacteria) and Chlorobium chlorochromatii (Chlo-robi), predicted type II TA systems represent around 2.5% of thetotal predicted open reading frames (ORFs) of these genomes (5).Type II systems are associated with mobile genetic elements, suchas plasmids and phages, as well as with chromosomes, in whichthey may be part of or constitute by themselves genomic islands/islets (for a review, see reference 4). It has been proposed that theymove between genomes through horizontal gene transfer. Thequestion of their biological roles remains debated, although sev-eral interesting hypotheses have emerged, notably their implica-tions in programmed cell death, stress response, persistence, sta-bilization of large genomic regions, or mobile genetic elements(for a review, see reference 4).

Interestingly, the vast majority of type II toxins that have beenidentified and characterized so far are endoribonucleases (alsodenoted as mRNA interferases) (7–14). One of the best character-ized is the RelEK-12 toxin of the relBEK-12 system of E. coli K-12 (7).RelEK-12 is an endoribonuclease that cleaves mRNAs in a transla-tion-dependent manner. Free RelEK-12 enters the ribosomal A site

and binds to the ribosome 30S subunit (15, 16). Resolution of thethree-dimensional structure of the RelEK-12-ribosome complexshowed that subsequent to complex formation, the target mRNAin the A site is reoriented so that RelEK-12 catalyzes cleavage of thetranscript (16). Three RelEK-12 residues are essential for this activ-ity: Y87, which reorients and stabilizes the mRNA to allow thenucleophilic attack; R61, which stabilizes the cleavage transitionstate; and R81, which acts as a general acid (16). RelEK-12 cleavespreferentially in the 5= region of the target mRNA, usually betweenthe second and third nucleotide of codons or between two codons(17). The RelBK-12 antitoxin wraps around the toxin, thereby in-hibiting its entry in the A site and leading to structural rearrange-ments that disrupt the RelEK-12 catalytic site (18, 19).

RelE-like toxins belong to the widespread type II ParE/RelEsuperfamily (5, 20). Type II toxin superfamilies are based on sim-ilarities at the level of amino acid sequence and three-dimensionalstructure prediction/determination (5, 21). Interestingly, this su-perfamily is composed of two functionally (although not structur-ally) distinct families that are either endoribonucleases, as men-tioned above (RelE family), or proteins inhibiting DNA-gyraseand DNA replication (ParE family) (6, 22). This suggests thatthese two families share a common ancestor and have functionallydiverged during evolution. Several RelE-like proteins, such asYoeB (14, 23), MqsR (24), YafQ (25), and YgjN (10), of Esche-richia coli K-12 have been characterized both at the activity andstructural levels. While they all share a fold with RelEK-12 and most

Received 27 December 2012 Accepted 20 March 2013

Published ahead of print 29 March 2013

Address correspondence to Laurence Van Melderen, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.02266-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.02266-12

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of them cleave mRNAs in a translation-dependent manner, dif-ferences are observed at the cleavage specificity level. The YoeBtoxin cleaves predominantly between the start and the secondcodon (23), and minor cleavage sites are observed downstream inthe target mRNA, preferentially upstream of purines (26). YafQpreferentially cleaves AAA codons (25), while YgjN does not showany specificity (10). MqsR has been shown to act in a translation-independent manner both in vivo and in vitro with a preference forGC(U/A) codons (10, 24). Three RelE-like toxins from differentproteobacteria (Brucella abortus, Helicobacter pylori, and Proteusvulgaris) were characterized. Toxins from B. abortus and H. pyloriwere shown to cleave RNAs in a translation-independent mannerin vitro (27, 28). For H. pylori RelE-like toxin, cleavage occurspreferentially upstream of purines. Finally, a RelE-like toxin fromProteus vulgaris was shown to cleave preferentially at AAA se-quences in a translation-dependent manner (29).

To gain further insights into cleavage specificity within theRelE family, we investigated the mechanism of action of RelE ho-mologues found in distantly related bacterial species. The activityof 6 RelE-like sequences from different phyla was tested in E. coli.These RelE-like sequences are part of type II TA systems. Theycleave the E. coli lpp and ompA transcripts in a translation-depen-dent manner without showing strong codon specificity, althoughthese toxins tend to cleave upstream of purines and between thesecond and third positions of codons.

MATERIALS AND METHODSStrains, plasmids, and media. For additional details on strains and plas-mids used in this study, see Table S1 in the supplemental material.

Strains. An E. coli strain, deleted for the 10 type II TA systems identi-fied at the time we started this work, was constructed to avoid any inter-ference with the activity of the RelE-like toxins tested (30, 31). Note thatthe toxins of these 10 systems are all mRNA interferases. The MG1655�10strain was constructed starting from MG1655�5 (�mazEF �relBE �chpB�dinJ-yafQ �yefM-yoeB) (32) by successive deletions of the yafNO, prlF-yhaV, hicAB, ygjMN, and mqsRA loci. Deletions were constructed usingthe mini-lambda RED system as described in reference 33. Locus replace-ment by antibiotic resistance genes was checked by PCR amplification,and antibiotic resistance genes were subsequently removed using thepCP20 thermosensitive plasmid as described in reference 34. The ygjMNand mqsRA deletions were P1 transduced into MG1655�8 andMG1655�9, respectively, because in MG1655�7, recombination oc-curred preferentially at the FLP recombination target (FRT) sites ratherthan at the loci of interest.

The MG1655�10 strain was used for all of the experiments describedin this work, except for the lpp mRNA Northern blotting. Those experi-ments were performed with an MG1655�10 �lpp::kan strain (constructedby P1 transduction of lpp::kan in MG1655�10) transformed with thepSC710 or pSC711 plasmid (35).

Toxin- and antitoxin-expressing plasmids. Toxin and antitoxingenes were amplified by PCR on genomic DNA using the appropriateprimers (see Table S2 in the supplemental material). Toxin PCR frag-ments, digested by the XbaI and PstI restriction enzymes, were cloned inthe pBAD33 vector digested by the same enzymes. Antitoxin PCR frag-ments, digested by the EcoRI and PstI restriction enzymes, were clonedinto the pKK223-3 vector digested by the same enzymes.

Media. Luria-Bertani (LB) medium and M9 minimal medium(KH2PO4 [22 mM], Na2HPO4 [42 mM], NH4Cl [19 mM], MgSO4 [1mM], CaCl2 [0.1 mM], NaCl [9 mM], vitamin B1 [1 mg ml�1]) supple-mented with Casamino Acids (0.2%) and carbon sources (1% glucose, 1%glycerol, 1% arabinose) were used to grow bacteria. Ampicillin and chlor-amphenicol were added at respective final concentrations of 100 and 20

mg ml�1. Isopropyl �-D-1-thiogalactopyranoside (IPTG) was used at fi-nal concentrations of 0.01 or 1 mM.

Toxicity and antitoxicity assays. Colonies of MG1655�10 containingthe pBAD33 vector or its derivatives carrying the toxin genes, as well as thepKK223-3 vector or its derivatives carrying the cognate antitoxin genes,were diluted in 10�2M MgSO4. Ten-�l aliquots of the serial dilutions wereplated on M9 minimum solid media containing the appropriate antibiot-ics and inducers. CFU were observed after overnight incubation of theplates at 37°C.

Northern blotting. Overnight cultures were diluted and grown to anoptical density at 600 nm (OD600) of �0.6 in LB medium, and expressionof the toxins was induced by addition of 1% arabinose. Total RNAs wereextracted at the times indicated in the figures with the RNeasy MinEluteCleanup Qiagen kit by following the manufacturer’s specifications or us-ing the hot phenol method as described in reference 36. Five-�g aliquotsof total RNA extracts were separated on 1% agarose gels and transferred toa nylon membrane in 20� SSC (1� SSC is 0.15 M NaCl plus 0.015 Msodium citrate) buffer. The membrane was hybridized overnight with thelabeled primers (Ambion NorthernMax kit). Primers were labeled with 3�l of [�-32P]ATP (specific activity, 3,000 Ci/mmol). The Promega T4polynucleotide kinase was used for primer phosphorylation. Labeledprimers were purified by PAGE (12% acrylamide). The experiments wereperformed at least twice.

Primer extensions. Ten �g of total RNA was hybridized with 0.2 pmolof labeled primers for 10 min. Primer sequences are described in reference17. The mixture was cooled on ice. Reverse transcription was performedwith Superscript III reverse transcriptase (Invitrogen). Sequencing reac-tions were performed using the USB Thermo Sequenase cycle sequencingkit. Cleavage patterns of the lpp, ompA, and rpsA mRNAs were testedbefore induction and 30 min after toxin expression. The lpp transcript(237 bp) was analyzed using one primer covering 195 nucleotides. Inorder to cover the full-length ompA mRNA (1,041 nucleotides), five prim-ers were used, allowing the reading of a total of 957 nucleotides with 126nucleotides for ompA1, 180 for ompA2, 213 for ompA3 and ompA4, and225 for ompA5 (17). The rpsA primer covers the 5=-end 188 nucleotidesover the 1,674 nucleotides of the full-length rpsA. The experiments wereperformed at least twice.

Potential cleavage rate in the original host genomes. For each toxin,the prevalence of the three codons cleaved most frequently in the lpp andompA transcripts were estimated in bacterial genomes based on the codonusage in each species (http://exon.gatech.edu/GeneMark/metagenome/CodonUsageDatabase/). Considering that these codons are cleaved withan efficiency of 100%, the inverse of the sum of frequencies estimates thetoxin cleavage rate in the different genomes.

RESULTSDetection of RelE-like toxins in bacterial genomes. The ParE/RelE superfamily is one of the most abundant type II toxin super-families in bacterial genomes (5). Previous studies revealed thatthese toxins are quite divergent, although they share or are pre-dicted to adopt a common RelE fold (21). To test whether toxinsbelonging to this superfamily share common molecular mecha-nisms and cleavage specificity despite protein sequence diver-gence, six toxins detected on the chromosomes of distantly relatedbacterial species were selected for further analyses (RelEO157 fromEscherichia coli O157:H7 [Gammaproteobacteria], RelERpa fromRhodopseudomonas palustris BisB18 [Alphaproteobacteria], Re-lESme from Sinorhizobium meliloti [Alphaproteobacteria], RelETde

from Treponema denticola [Spirochaetes], RelEMav from Mycobac-terium avium [Actinobacteria], and RelENsp from Nostoc sp. [Cya-nobacteria]) (see Table S3 in the supplemental material). Theseputative toxins appear to be part of genomic islands, as indicatedby their GC content and their different distribution among iso-lates from the same species (data not shown).

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Amino acid sequence comparisons confirmed that RelE-likesequences are quite divergent, even those originating from thesame or closely related bacterial species (Fig. 1). Table 1 shows thatthe RelE-like sequences present 24 to 47% similarity and 15 to28% identity with the canonical RelEK-12. Note that RelETde pres-ents 52% identity and 66% similarity to the YoeBK-12 toxin. Sim-ilarity between the different RelE-like sequences is even lower,with only a few percentage points of similarity between RelESme

and RelETde or RelEO157. Despite this very low conservation at theamino acid sequence level, predicted secondary and tertiary struc-tures indicated that these RelE-like sequences share a similar foldwith RelEK-12 toxin (data not shown).

Functional and structural data regarding RelEK-12 indicate thatthe active-site residues R61, R81, and Y87 are essential for mRNAcleavage, as single mutations lead to decreased activity (16).Amino acid sequence alignments show that these residues are con-served in the eight RelE-like sequences, although to different ex-

tents (Fig. 1). While R61 is conserved in the 8 sequences, Y87 andR81 are less conserved (5 and 3, respectively, out of 8). The M.avium RelE, RelEMav, possesses a phenylalanine at the positioncorresponding to Y87, similar to the H. pylori HP0894 RelE-liketoxin (F88) and E. coli YafQ (F91) (28, 37). In addition, RelEMav

contains part of an HP0894 motif involved in substrate recogni-tion (E107, L108, and F109) (28).

To test the toxic activity of the RelE-like sequences, the corre-sponding genes were cloned in the pBAD33 vector under the con-trol of the pBAD promoter. These constructs, as well as thepBAD33-relEK-12 plasmid, were transformed in E. coli MG1655deleted of 10 type II TA systems (MG1655�10; see Materials andMethods) to avoid any interference with the 10 endogenousmRNA interferases encoded by these loci (30, 31). Transforma-tion of MG1655�10 with the pBAD33-yoeBK-12 plasmid was notsuccessful, even in the presence of glucose to repress expressionfrom the pBAD promoter, most likely due to the absence of the

FIG 1 Alignment of the RelE-like sequences with RelEK-12 and YoeBK-12. Amino acid sequences were aligned with MAFFT (http://www.ebi.ac.uk/Tools/msa/mafft/). Residues highlighted in dark gray are conserved in 80% of the sequences, in medium gray for 70% conservation, and in light gray for 50% conservation.Arrows indicate residues important for the catalytic activity of RelEK-12. An asterisk indicates identical amino acids; colons and dots indicate similar residues.Boxes show conserved motifs. The ELF motif in RelEMav is conserved in HP0894 (28).

TABLE 1 Similarity and identity between the RelE-like toxins and the RelEK-12 and YoeBK-12 canonical toxinsa

Toxin RelEK-12 RelERpa RelENsp RelEMav RelEO157 RelESme RelETde YoeBK-12

RelEK-12 24 28 15 19 16 17 16RelERpa 47 28 14 13 20 20 17RelENsp 43 46 4 18 13 17 21RelEMav 27 24 7 15 13 15 19RelEO157 35 24 32 30 3 4 14RelESme 24 26 23 24 5 5 9RelETde 25 37 28 27 11 4 52YoeBK-12 30 31 36 29 30 23 66a Amino acid sequence identity (%) and similarity (%) are indicated in gray and white, respectively, as determined using the NEEDLE alignment software(http://www.ebi.ac.uk/Tools/psa/emboss_needle/index.html).

Heterogeneous Cleavage Patterns of RelE Toxins

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chromosomal copy of the system (38). Figure 2A shows that ecto-pic overexpression of the 6 relE-like genes inhibits E. coli growth.

RelE toxins are associated with antitoxins belonging to dif-ferent superfamilies. The 6 RelE-like toxins are associated withpredicted antitoxins belonging to three different superfamilies,i.e., Phd, HigA, and RelB (see Fig. S1 and Table S3 in the supple-mental material). The toxins of T. denticola and Nostoc sp. areassociated with predicted relBTde and phdNsp genes, respectively.

These loci exhibit a canonical organization in which the antitoxingenes precede those of the toxins. In contrast, the four other sys-tems present a reverse organization in which the toxin gene islocated upstream of the predicted antitoxin gene. Toxins from R.palustris, S. meliloti, M. avium, and E. coli O157:H7 are associatedwith predicted HigA antitoxins (see Fig. S1 and Table S3).

The predicted antitoxin genes were cloned in the pKK223-3vector under the control of the pTac promoter. These antitoxin-containing plasmids were transformed in the MG1655�10 straincontaining the toxin-expressing plasmids (Fig. 2A). Except for therelENsp-phdNsp gene pair, coexpression of the predicted antitoxinswith their respective toxins restores normal growth, showing thatthese gene pairs constitute functional TA systems (Fig. 2B). Coex-pression of PhdNsp with RelENsp only partially restores E. coli cellgrowth (Fig. 2B). To visualize the expression of this putative an-titoxin by Western blotting, an N-terminal Flag tag was added toPhdNsp (PhdNflag), and surprisingly, this version of the antitoxincompletely restored cell growth upon coexpression with relENsp

(Fig. 2B), while addition of the Flag tag to the C terminus did notrestore cell growth (data not shown). Altogether, these data indi-cate that PhdNsp is indeed an antitoxin, and that blocking its Nterminus might increase either its translation or its stability.

Translation-dependent mRNA cleavage. It was shown previ-ously that overexpression of RelESme, RelEMav, and RelEO157 leadsto a decrease of the global translation rate in E. coli (5). As ex-pected, ectopic overexpression of RelERpa, RelENsp, and RelETde

toxins also inhibits translation (see Fig. S2 in the supplementalmaterial). A series of Northern blot experiments was then carriedout to test whether these toxins affect mRNA stability in a trans-lation-dependent manner, as observed for the canonical RelEK-12

toxin. Ectopic overexpression of the 6 RelE-like toxins leads toompA degradation (Fig. 3). Translation dependence was testedusing a well-established mRNA assay consisting of a version of thelpp mRNA that is not translated due to the mutation of the startcodon in a lysine AAG codon (35). Degradation of this mRNA wastested in the MG1655�10 �lpp::kan strain. While degradation wasobserved in the case of wild-type lpp mRNA, the mutant transcriptremained stable in all cases, indicating that the RelE homologuescleave mRNAs in a translation-dependent manner (see Fig. S3).

Cleavage pattern specificity. Primer extension experimentson the lpp and ompA mRNAs were performed under overexpres-sion of the different RelE toxins. Interestingly, expression of thesetoxins leads to different cleavage patterns in terms of cleavagespecificity as well as frequency (Fig. 4 and 5 and Table 2; also seeFig. S4 in the supplemental material). No cleavage is detected inthe 5=-untranslated region (UTR) of the test mRNAs, confirmingthat the 6 RelE homologues cleave mRNAs in a translation-depen-dent manner. The toxins did not show codon specificity, although

FIG 2 RelE-like toxins inhibit E. coli growth and constitute TA systems. (A)Serial dilutions of the MG1655�10 strain, containing the pBAD33 controlvector or its derivatives with the relE-like genes, were plated on M9 minimalmedia containing either glucose (1%) or arabinose (1%) and the appropriateantibiotics. (B) Serial dilutions of the MG1655�10 strain, containing thepBAD33 and pKK223-3 control vectors or their derivatives with the relE-likegenes and their cognate antitoxin genes, were plated on M9 minimal mediacontaining either glucose (1%) or arabinose (1%), along with IPTG (1 mM)and the appropriate antibiotics. Note that the pSC101-lacIq plasmid wascotransformed with pKK223-3-relBK-12 to repress relBK-12 expression, since itappears to be toxic (data not shown). IPTG was used at a final concentration of0.01 mM to induce antitoxin expression. Plates were incubated overnight at37°C, and survival was estimated.

FIG 3 RelE-like toxins induce mRNA cleavage. The MG1655�10 strain, containing the pBAD33 control vector or its derivatives with the relE-like genes, wasgrown in LB to an OD600 of �0.6. Toxin expression was then induced by addition of arabinose (1%). Total RNAs were extracted at 0, 15, 30, 45, and 60 min aftertoxin induction and were used for Northern blot analysis of the ompA mRNA using specific probes as described in Materials and Methods.

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5 of them preferentially cut between the second and third positionand upstream of a purine.

RelEK-12 generates 33 cleavages in the lpp mRNA (28 major and5 minor cleavages) and 35 in ompA (18 major and 17 minor cleav-ages) (Fig. 4, 5, and Table 2; also see Fig. S4 in the supplementalmaterial). RelEK-12 cleaves the lpp mRNA throughout the full-length sequence, while the ompA mRNA is preferentially cleavedin the 5= region. Cleavages occur preferentially between the secondand third nucleotide (61%) and mostly upstream of a purine (70%).Among these, 74% occur upstream of a G. The 3 codons most fre-quently cleaved (CAG, glutamine; CUG, leucine; and GCG, alanine)represent 37% of the sites cleaved by RelEK-12 (Table 2).

While the cleavage pattern of lpp by RelEO157 is similar to thatof RelEK-12 (i.e., throughout the mRNA), the cleavage pattern ofompA is different (Fig. 4, 5, and Table 2; also see Fig. S4 in thesupplemental material). Only minor cleavages were detected inthe 5= end of ompA, with most of them occurring after the first 200bp (Fig. 4). Fewer cleavage sites were detected for RelEO157 thanfor RelEK-12 (46 versus 68). Nineteen cleavages (15 major) weredetected in the lpp mRNA and 27 (24 major) in ompA. As forRelEK-12, RelEO157 most frequently cleaves between the secondand third nucleotide (90%) and mostly upstream of a G (70%).Among these, 70% occur between a U and a G. The 3 codons mostfrequently cleaved (CUG, leucine; CAG, glutamine; and AUG

FIG 4 RelE-like toxin expression leads to distinct cleavage patterns on the lpp and ompA transcripts. Primer extension analysis of the lpp (A) and ompAtranscripts using ompA1 (B) and ompA5 (C) primers. The MG1655�10 strains, containing the pBAD33 control vector or its derivatives with the relE-like genes,were grown in LB with the appropriate antibiotic at 37°C to an OD600 of �0.6. Toxin expression was then induced by addition of arabinose (1%) for 30 min. TotalRNAs were extracted as described in Materials and Methods. Major and minor cleavage sites are indicated by filled and open circles, respectively. FL, full length.

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codon) represent 65% of the total number of sites cleaved byRelEO157 (Table 2).

The RelERpa toxin cleaves the lpp and ompA mRNAs through-out the full-length sequence, with 14 (11 major) and 41 (30 major)cuts, respectively (Fig. 4, 5, and Table 2; also see Fig. S4 in thesupplemental material). Cleavages occur preferentially betweenthe second and third nucleotide (69%) and frequently upstream ofpurines (93%). The 3 codons most frequently cleaved (CAG,glutamine; AAA lysine; and CCG, proline) represent 58% of thesites cleaved by RelERpa. Note that RelERpa cleaves 100% (15/15) of CAG codons covered by the primer extension experi-ments (Table 2).

For RelESme, a smaller number of cleavage sites was detectedboth in lpp (11 cuts, 10 major) and ompA (11 minor cuts) (Fig. 4,5, and Table 2; also see Fig. S4 in the supplemental material). Thecleavage pattern of lpp and ompA by RelESme is quite differentfrom that observed with the other RelE-like toxins, as no cleavageis detected in the 5= end of the transcripts (first cleavage in lpp atposition 77 and 68 in ompA) (Fig. 5). Cleavages occur preferen-tially between the second and third nucleotide (82%) and before

purines (82%). The 3 codons most frequently cleaved (CAG, glu-tamine; AAA, lysine; and AUG, codon) represent 63% of the totalnumber of cleavage sites (Table 2).

The RelENsp toxin generates 33 (21 major) cleavage sites and 8major cleavage sites in ompA and lpp mRNAs, respectively (Fig. 4,5, and Table 2; also see Fig. S4 in the supplemental material). Thecleavage patterns of lpp and ompA are quite similar, with themRNAs being cleaved throughout the full-length sequences(Fig. 5). Cleavages occur preferentially between the second andthird nucleotide (56%) and the first and second nucleotide (33%).RelENsp cleaves preferentially upstream of an A (71%). The 3codons most frequently cleaved (GAA, glutamic acid; AAA lysine;and GCA, alanine) represent 59% of the total number of sitescleaved by RelENsp (Table 2).

The number of cleavage sites for the RelETde toxin was lowcompared to those of the other RelE-like toxins (1 cleavage site inthe lpp mRNA and 4 in ompA). In both mRNAs, the second AAAcodon is cleaved by RelETde. To investigate whether the RelETde

activity is sequence or position dependent, we used the rpsAmRNA which encodes an ACU codon in the second position. In

FIG 5 Location and frequency of RelE-like toxin cleavage sites. A schematic representation of the lpp (A) and ompA (B) transcripts is shown to scale, with eachgray box representing 100 nucleotides. Small and large bars represent major and minor cleavage sites, respectively.

TABLE 2 Cleavage sites of the RelE-like toxins in the lpp and ompA transcriptsa

Toxin (bacterial species)

No. of cleavages in:Three most frequently cleaved codons in lpp and ompA(relative no.), %

%lpp ompA lpp and ompA Codon 1 Codon 2 Codon 3

RelEK-12 (E. coli K-12) 33 35 68 CAG (11/15), 73 CUG (10/27), 37 GCG (4/4), 100 37RelEO157 (E. coli O157:H7) 19 27 46 CUG (18/27), 67 CAG (7/15), 47 AUG (5/9), 27 65RelERpa (R. palustris) 14 41 55 CAG (15/15), 100 AAA (11/18), 61 CCG (6/12), 50 58RelENsp (Nostoc sp.) 8 33 41 AAA (11/18), 61 GCA (7/13), 54 GAA (6/8), 75 59RelESme (S. meliloti) 11 11 22 CAG (7/15), 47 AAA (4/18), 22 AUG (3/9), 33 63RelETde (T. denticola) 1 4 5 AAA (2/18), 11 CCA (1/3), 33 AAG (1/5), 20 80a Numbers of sites that are cleaved by the toxins in the E. coli lpp and/or ompA transcripts are indicated. The sequence of the 3 codons that are the most frequently cleaved in the lppand ompA transcripts is indicated, as well as the number of cleaved codons over the total number of these codons in the 2 transcripts, in relative numbers (in parentheses) and inpercentages. The last column (%) represents the proportion of these 3 cleaved codons relative to the total number of cuts mediated by the toxins. In boldface are the codons that arecommon to different toxins.

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this experiment, the YoeBK-12 toxin was included, as it shows ahigh degree of similarity to RelETde (52% identity, 66% similarity)(Table 1). In addition, YoeBK-12 was previously reported to cleavethe second codon of mRNAs (26). Figure 6 shows that overallsimilar cleavage patterns and specificities are observed in the rpsAmRNA for RelEK-12, RelETde, and YoeBK-12. As observed forYoeBK-12, RelETde cleaves the rpsA mRNA at the second codon,between the second and third nucleotides (upstream of a U).However, in contrast to the other test mRNAs, additional cleavagesites were observed further in the rpsA transcript. Cleavages oc-curred mainly between the second and third nucleotide (82%)and preferentially upstream of purines (75%). Sequence anal-ysis revealed that the regions upstream (�12 nucleotides) ofthe cleavage sites have in common a G-A-rich region present-ing similarities to the GAAG Shine-Dalgarno sequence

(Fig. 7A). These regions might play a role in cleavage specific-ity; for instance, by causing ribosome pausing and/or inducingsecondary structures (39).

Cleavage by RelEMav presents some specific features (Fig. 4 and5; also see Fig. S4 in the supplemental material). While the lppmRNA is cleaved regularly (17 cleavages, 11 major), ompA is pref-erentially cleaved in the 3=-end region (22 cleavages, 16 major).Only very minor cleavages were observed in the mRNA regionscovered by ompA1 and ompA2 primers (covering the 5= end of theompA mRNA). In addition, although preferentially cleaving up-stream of a G, RelEMav cleaves preferentially between codons(90%) (Fig. 7B).

DISCUSSION

This work highlights the general conservation of molecular mech-anisms used by the members of the RelE family of type II toxin.Although originating from distantly related bacterial species andsharing low amino acid sequence similarities, the toxins charac-terized in this work are mRNA interferases cleaving RNA in atranslation-dependent manner. To determine cleavage specificity,primer extension experiments upon RelE-like toxin overexpres-sion were performed in an MG1655 strain deleted of the 10 well-characterized type II systems. We sought to avoid secondary cleav-age sites from endogenous type II toxins, although we cannot ruleout the contribution of other or unknown endoribonucleases.Our data show that RelE-like toxins do not cleave at specificcodons but rather exhibit a trend to cleave upstream of purinesand between the second and third positions of codons, which issimilar to the activity of the canonical RelEK-12 toxin.

Using the highly expressed ompA and lpp as an mRNA test, wefound that these toxins appear to preferentially cleave in vivo atcodons that are abundant in bacterial genomes. Note that in vitroanalyses using purified toxins and translational complexes will beneeded to correlate translation rate and cleavage specificity using

FIG 6 RelEK-12, YoeBK-12, and RelETde toxin expression leads to similar cleav-age patterns on the rpsA transcripts. Primer extension analysis of the rpsAtranscript is shown. The MG1655�10 strains, containing the pBAD33 controlvector or its derivatives expressing the RelEK-12, YoeBK-12, and RelETde toxins,were grown in LB with the appropriate antibiotic at 37°C to an OD600 of �0.6.Toxin expression was then induced by addition of arabinose (1%) for 30 min.Total RNAs were extracted as described in Materials and Methods. Major andminor cleavage sites are indicated by filled and open circles, respectively. Theasterisk indicates the full-length transcript from the rpsA3 promoter (41).

FIG 7 Consensus sequences around the cleavage sites of the RelETde andRelEMav toxins. (A) Conserved motif upstream of the RelETde cleavage site. TheA/GXXGAAXC/A motif is conserved around 12 nucleotides upstream of theRelETde cleavage site (arrow). Logo sequences were constructed using a web-based application (weblogo.berkeley.edu/logo.cgi). (B) The RelEMav toxincleaves preferentially between codons ending and starting with guanines. Thecleavage site is represented by an arrow. The upstream codon (codon 1) andthe downstream codon (codon 2), as well as the nucleotide position (1, 2, and3 in codon 1 and 4, 5, and 6 in codon 2), are indicated.

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artificial mRNAs containing rare codons. Nevertheless, our datashow that RelEK-12 preferentially cleaves the glutamine CAG, theleucine CUG, and the alanine GCG codons, as described before(Table 3) (17). Considering these 3 preferential cleavage sites (rep-resenting only 37% of the RelEK-12-mediated cuts) and their prev-alence in the E. coli genome (117.3/1,000 codons; Table 3), andassuming that these codons are efficiently cleaved, the initialcleavage rate for RelEK-12 is one cleavage every 9 nucleotides. Thisestimation is similar for the other RelE-like toxins (RelEO157 andRelENsp every 10 nucleotides, and RelERpa every 14). RegardingRelEMav, target codons are also well represented, since it preferen-tially cleaves before codons starting with G (Fig. 7). Thus, therelaxed specificity of these RelE-like toxins allow them to be quiteefficient and quite broad in terms of substrates. Interestingly, weobserved that some codons are cleaved by different RelE-like tox-ins (Table 3). For instance, the CAG glutamine codon is cleaved byRelEK-12, RelEO157, RelERpa, and RelESme. The AAA lysine codon isalso cleaved by RelERpa, RelENsp, RelESme, and RelETde (Table 3).This codon is generally found at the second position of highlyexpressed mRNAs (40).

Altogether, our in vivo data suggest that these RelE-like toxinsare active and able to exert their function(s) in a large variety ofbacterial species. Considering that TA systems are located on mo-bile genetic elements and that they invade bacterial chromosomesthrough horizontal gene transfer and subsequent integration asgenomic islands, it is likely that a relaxed specificity has been se-lected by evolution.

ACKNOWLEDGMENTS

We are grateful to Kenn Gerdes and the members of his laboratory forproviding the pSC710 and pSC711 plasmids and for their help with theprimer extension experiments.

N.G. is supported by the National Research Fund, Luxembourg(908853). This work was supported by the Fonds de la Recherche Scien-tifique (FRSM-3.4530.04), the Fondation Van Buuren, and the FondsBrachet.

We thank the scientific community for kindly providing us withgenomic DNA and bacterial strains.

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1st 2nd 3rd Total

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