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TRANSCRIPT
1
Relaxed cleavage specificity within the RelE toxin family 1
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Nathalie Goeders1, PierreLuc Drèze1 and Laurence Van Melderen1# 3
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1Laboratoire de Génétique et Physiologie Bactérienne, IBMM, Faculté des Sciences, 5
Université Libre de Bruxelles (ULB), 12 rue des Professeurs Jeener et Brachet, B: 6041 6
Gosselies, Belgium. 7
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Running title: Heterogeneous cleavage patterns of RelE toxins 9
10
#Corresponding author: 11
Laurence Van Melderen 12
E‐mail: [email protected] 13
14
Keywords: toxin‐antitoxin system, mRNA interferase, RNA degradation 15
Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.02266-12 JB Accepts, published online ahead of print on 29 March 2013
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Abstract 16
Bacterial type II toxin‐antitoxin systems are widespread in bacteria. Among them, the 17
RelE toxin family is one of the most abundant. The RelEK‐12 toxin of E. coli K‐12 18
represents the paradigm for this family and has been extensively studied, both in vivo 19
and in vitro. RelEK‐12 is an endoribonuclease that cleaves mRNAs that are translated by 20
the ribosome machinery as these transcripts enter the A site. Earlier in vivo reports 21
showed that RelE cleaves preferentially in the 5’‐end coding region of the transcripts, in 22
a codon‐independent manner. To investigate whether the molecular activity as well as 23
the cleavage pattern are conserved within the members of this toxin family, RelE‐like 24
sequences were selected in Proteobacteria, Cyanobacteria, Actinobacteria and 25
Spirochaetes and tested in E. coli. Our results show that these RelE‐like sequences are 26
part of toxin‐antitoxin gene pairs and that they inhibit translation in E. coli by cleaving 27
transcripts that are being translated. Primer extension analyses show that these toxins 28
exhibit specific cleavage patterns in vivo, both in terms of frequency and location of 29
cleavage sites. We did not observe codon‐dependent cleavage, but rather a trend to 30
cleave upstream purines, and between the second and third position of codons, except 31
for the actinobacterial toxin. Our results suggest that RelE‐like toxins might have 32
evolved to rapidly and efficiently shut down translation in a large spectrum of bacterial 33
species, which correlates with the observation that TA systems are spreading by 34
horizontal gene transfer. 35
36
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Introduction 38
Bacteria thrive in ever changing environments and therefore have evolved regulatory 39
mechanisms allowing rapid modulation of gene expression and thereby adaptation. 40
Among these mechanisms, ribonucleases (RNAses) play an important regulatory role by 41
adjusting RNA levels (for review, [1]). Escherichia coli encodes more than 20 RNases 42
involved in different processes such as RNA quality control, RNA maturation and stress 43
response modulation. In addition, E. coli encodes numerous RNases that act as 44
bacteriocins (for review, [2]) or belong to CRISPR systems (for review, [3]) and toxin‐45
antitoxin (TA) systems (for review, [4]). 46
TA systems are classified in different types depending on the nature and mode of action 47
of the antitoxin, the toxin always being a protein. Type II systems are generally 48
composed of two genes organized in an operon, the first gene encoding an antitoxin 49
protein and the second a toxin. These systems are abundant in bacterial genomes. In 50
some bacterial species such as Nitrosomonas europaea (Proteobacteria) and Chlorobium 51
chlorochromatii (Chlorobi), predicted type II TA systems represent around 2.5% of the 52
total predicted ORFs of these genomes [5]. Type II systems are associated with mobile 53
genetic elements such as plasmids and phages as well as with chromosomes in which 54
they may be part of or constitute by themselves genomic islands/islets (for review, [4]) . 55
It has been proposed that they move between genomes through horizontal gene 56
transfer. The question of their biological roles remains debated although several 57
interesting hypotheses have emerged notably their implications in programmed cell 58
death, stress response, persistence, stabilization of large genomic regions or mobile 59
genetic elements (for review, [4, 6]). 60
Interestingly, the vast majority of type II toxins that have been identified and 61
characterized so far are endoribonucleases (also denoted as mRNA interferases) (see 62
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notably, [7‐14]. One of the best characterized is the RelEK‐12 toxin of the relBEK12 system 63
of E. coli K‐12 [7]. RelEK‐12 is an endoribonuclease cleaving mRNAs in a translation‐64
dependent manner. Free RelEK‐12 enters the ribosomal A site and binds to the ribosome 65
30S subunit [15, 16]. Resolution of the three‐dimensional structure of the RelEK‐12‐66
ribosome complex showed that subsequent to complex formation, the target mRNA in 67
the A site is re‐orientated so that RelEK‐12 catalyzes cleavage of the transcript [16]. Three 68
RelEK‐12 residues are essential for this activity: Y87 which re‐orients and stabilizes the 69
mRNA to allow the nucleophilic attack, R61 which stabilizes the cleavage transition state 70
and R81 which acts as a general acid [16]. RelEK‐12 cleaves preferentially in the 5’‐region 71
of the target mRNA, usually between the second and the third nucleotide of codons or 72
between two codons [17]. The RelBK‐12 antitoxin wraps around the toxin, thereby 73
inhibiting its entry in the A site and leading to structural rearrangements that disrupt 74
the RelEK‐12 catalytic site [18, 19]. 75
RelE‐like toxins belong to the widespread type II ParE/RelE super‐family [5, 20]. Type II 76
toxin super‐families are based on similarities at the level of amino acid sequence and 77
three‐dimensional structure prediction/determination [5, 21]. Interestingly, this super‐78
family is composed of two functionally distinct families (although not structurally) that 79
are either endoribonucleases as mentioned above (RelE family) or proteins inhibiting 80
DNA‐gyrase and DNA replication (ParE family) [6, 22]. This suggests that these two 81
families share a common ancestor and have functionally diverged during evolution. 82
Several RelE‐like proteins (such as YoeB [14, 23], MqsR [24], YafQ [25] and YgjN [10]) of 83
E. coli K‐12 have been characterized both at the activity and structural levels. While they 84
all share a common fold with RelEK‐12 and most of them cleave mRNAs in a translation‐85
dependent manner, differences are observed at the cleavage specificity level. The YoeB 86
toxin cleaves predominantly between the start and the second codon [23] and minor 87
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cleavage sites are observed downstream in the target mRNA, preferentially upstream of 88
purines [26]. YafQ cleaves preferentially AAA codons [25] while YgjN does not show any 89
specificity [10]. MqsR has been shown to act in a translation‐independent manner both 90
in vivo and in vitro with a preference for GC(U/A) codons [10, 24]. Three RelE‐like toxins 91
from different Proteobacteria (Brucella abortus, Helicobacter pylori and Proteus vulgaris) 92
were characterized. Toxins from B. abortus and H. pylori were shown to cleave RNAs in a 93
translation‐independent manner in vitro [27, 28]. For H. pylori RelE‐like, cleavage occurs 94
preferentially upstream of purines. Finally, a RelE‐like from Proteus vulgaris was shown 95
to cleave preferentially at AAA sequences in a translation‐dependent manner [29]. 96
To gain further insights into cleavage specificity within the RelE family, we investigated 97
the mechanism of action of RelE homologues found in distantly related bacterial species. 98
The activity of 6 RelE‐like sequences from different phyla was tested in E. coli. These 99
RelE‐like sequences are part of type II TA systems. They cleave the E. coli lpp and ompA 100
transcripts in a translation‐dependent manner without showing strong codon specificity 101
although these toxins tend to cleave upstream of purines and between the second and 102
third position of codons. 103
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Materials and methods 105
Strains, Plasmids and Media 106
See Table S1. 107
Strains 108
An E. coli strain deleted for the 10 type II TA systems identified at the time we started 109
this work was constructed to avoid any interference with the activity of the RelE‐like 110
toxins tested [30, 31]. Note that the toxins of these 10 systems are all mRNA 111
interferases. MG1655Δ10 strain was constructed starting from MG1655Δ5 (ΔmazEF, 112
ΔrelBE, ΔchpB, ΔdinJyafQ, ΔyefMyoeB) [32] by successive deletions of the yafNO, prlF113
yhaV, hicAB, ygjMN and mqsRA loci. Deletions were constructed using the mini‐ lambda 114
RED system as described in [33]. Loci replacement by antibiotic resistance genes was 115
checked by PCR amplification and antibiotic resistance genes were subsequently 116
removed using the pCP20 thermo‐sensitive plasmid as described in [34]. The ygjMN and 117
mqsRA deletions were P1 transduced into MG1655Δ8 and MG1655Δ9, respectively as in 118
MG1655Δ7 recombination did occur preferentially at the FRT sites rather than at the 119
loci of interest. 120
The MG1655Δ10 strain was used for all the experiments described in this work except 121
for the lpp mRNA northern blots. These experiments were performed in a MG1655Δ10 122
Δlpp::kan strain (constructed by P1 transduction of lpp::kan in MG1655Δ10) 123
transformed with the pSC710 or pSC711 plasmids [35]. 124
Toxin and antitoxinexpressing plasmids 125
Toxin and antitoxin genes were amplified by PCR on genomic DNA using the appropriate 126
primers (see Table S2). Toxin PCR fragments, digested by the XbaI and PstI restriction 127
enzymes, were cloned in the pBAD33 vector digested by the same enzymes. Antitoxin 128
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PCR fragments, digested by the EcoRI and PstI restriction enzymes, were cloned into the 129
pKK223‐3 vector digested by the same enzymes. 130
Media 131
Luria‐Bertani medium (LB) and M9 minimal medium (KH2PO4 (22 mM), Na2HPO4 (42 132
mM), NH4Cl (19 mM), MgSO4 (1 mM), CaCl2 (0.1 mM), NaCl (9 mM), vitamin B1 (1mg ml‐133
1)) supplemented with casamino acids (0.2%) and carbon sources (glucose 1%, glycerol 134
1%, arabinose 1%) were used to grow bacteria. Ampicillin and chloramphenicol were 135
added at respective final concentration of 100 mg ml‐1 and 20 mg ml‐1. Isopropyl β‐D‐1‐136
thiogalacto‐pyranoside (IPTG) was used at final concentrations of 0.01 mM or 1mM. 137
Toxicity and antitoxicity assays 138
Colonies of MG165510 containing the pBAD33 vector or its derivatives carrying the 139
toxin genes as well as the pKK223‐3 vector or its derivatives carrying the cognate 140
antitoxin genes were diluted in 10‐2M MgSO4. Ten l of the serial dilutions were plated 141
on M9 minimum solid media containing the appropriate antibiotics and inducers. 142
Colony forming units were observed after overnight incubation of the plates at 37°C. 143
Northern blots 144
Overnight cultures were diluted and grown to an OD600nm ~ 0.6 in LB media and 145
expression of the toxins was induced by addition of 1% arabinose. Total RNAs were 146
extracted at times indicated in the figures with the RNeasy® MinElute® Cleanup Qiagen 147
kit following manufacturer specifications or using the hot phenol method as described 148
in [36]. Five g of total RNA extracts were separated on 1% agarose gels and transferred 149
to a nylon membrane in 20 X SSC buffer. The membrane was hybridized overnight with 150
the labeled primers (Ambion® NorthernMax® Kit). Primers were labeled with 3 l of 151
[�‐32P]‐ATP (specific activity: 3,000 Ci/mmol). The Promega T4 polynucleotide kinase 152
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was used for primer phosphorylation. Labeled primers were purified by PAGE (12% 153
acrylamide). The experiments were performed at least twice. 154
Primer extensions 155
Ten g of total RNA were hybridized with 0.2 pmol of labeled primers for 10 min. Primer 156
sequences are described in [17]. The mixture was cooled on ice. Reverse transcription 157
was performed with the Superscript® III Reverse Transcriptase (Invitrogen). 158
Sequencing reactions were preformed using the USB Thermo Sequenase Cycle 159
Sequencing Kit. Cleavage patterns of the lpp, ompA and the rpsA mRNAs were tested 160
before induction and 30 min after toxin expression. The lpp transcript (237 bp) was 161
analyzed using one primer covering 195 nucleotides. In order to cover the full‐length 162
ompA mRNA (1,041 nucleotides), five primers were used, allowing the read of a total of 163
957 nucleotides with 126 nucleotides for ompA1, 180 for ompA2, 213 for ompA3 and 164
ompA4 and 225 for ompA5 [17]. The rpsA primer covers the 5’‐end 188 nucleotides 165
over the 1,674 nucleotides of the full‐length rpsA. The experiments were performed at 166
least twice. 167
Potential cleavage rate in the original host genomes 168
For each toxin, the prevalence of the three codons cleaved most frequently in the lpp 169
and ompA transcripts were estimated in bacterial genomes based on the codon usage in 170
each species 171
http://exon.gatech.edu/GeneMark/metagenome/CodonUsageDatabase/. Considering 172
that these codons are cleaved with an efficiency of 100%, the inverse of the sum of 173
frequencies estimates the toxin cleavage rate in the different genomes. 174 175
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Results 176
Detection of RelElike toxins in bacterial genomes 177
The ParE/RelE super‐family is one of the most abundant type II toxin super‐families in 178
bacterial genomes [5]. Previous studies revealed that these toxins are quite divergent, 179
although they share or are predicted to adopt a common RelE‐fold [21]. To test whether 180
toxins belonging to this super‐family share common molecular mechanisms and 181
cleavage specificity despite protein sequence divergence, six toxins detected on the 182
chromosomes of distantly related bacterial species were selected for further analyses 183
(RelEO157 from Escherichia coli O157:H7 (�‐Proteobacteria), RelERpa from 184
Rhodopseudomonas palustris BisB18 (α‐Proteobacteria), RelESme from Sinorhizobium 185
meliloti (α‐Proteobacteria), RelETde from Treponema denticola (Spirochaetes), RelEMav 186
from Mycobacterium avium (Actinobacteria) and RelENsp from Nostoc sp. 187
(Cyanobacteria)) (Table S3). These putative toxins appear to be part of genomic islands 188
as indicated by their GC content and their different distribution among isolates from the 189
same species (data not shown). 190
Amino acid sequence comparisons confirmed that RelE‐like sequences are quite 191
divergent, even those originating from the same or closely related bacterial species 192
(Figure 1). Table 1 shows that the RelE‐like sequences present 24% to 47% of similarity 193
and 15% to 28% of identity with the canonical RelEK‐12. Note that RelETde presents 52% 194
identity and 66% similarity with the YoeBK‐12 toxin. Similarity between the different 195
RelE‐like sequences is even lower with only a few percent of similarity between RelESme 196
and RelETde or RelEO157. Despite this very low conservation at the amino acid sequence 197
level, predicted secondary and tertiary structures indicated that these RelE‐like 198
sequences share a similar fold with RelEK‐12 toxin (data not shown). 199
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Functional and structural data regarding RelEK‐12 indicate that the active‐site residues 200
R61, R81 and Y87 are essential for mRNAs cleavage as single mutations lead to a 201
decreased activity [16]. Amino acid sequence alignments show that these residues are 202
conserved in the eight RelE‐like sequences, although to different extents (Figure 1). 203
While R61 is conserved in the 8 sequences, Y87 and R81 are less conserved (5 and 3 out 204
of 8, respectively). The M. avium RelEMav possesses a phenylalanine at the position 205
corresponding to Y87 like the H. pylori HP0894 RelE‐like toxin (F88) and the E. coli YafQ 206
(F91) [28, 37]. In addition, RelEMav contains part of a HP0894 motif involved in substrate 207
recognition (E107, L108 and F109) [28]. 208
To test the toxic activity of the RelE‐like sequences, the corresponding genes were 209
cloned in the pBAD33 vector under the control of the pBAD promoter. These constructs 210
as well as the pBAD33‐relEK12 plasmid were transformed in E. coli MG1655 deleted of 211
10 type II TA systems (MG165510, see materials and methods) to avoid any 212
interference with the 10 endogenous mRNA interferases encoded by these loci [30, 31]. 213
Transformation of MG165510 with the pBAD33‐yoeBK12 plasmid was not successful 214
even in the presence of glucose to repress expression from the pBAD promoter most 215
likely due to the absence of the chromosomal copy of the system ([38]. Figure 2A shows 216
that ectopic overexpression of the 6 relE‐like genes inhibits E. coli growth. 217
218
RelE toxins are associated with antitoxins belonging to different superfamilies 219
The 6 RelE‐like toxins are associated with predicted antitoxins belonging to three 220
different super‐families i.e. Phd, HigA and RelB (Fig. S1, Table S3). The toxins of T. 221
denticola and Nostoc sp. are associated with predicted relBTde and phdNsp genes, 222
respectively. These loci exhibit a canonical organization in which the antitoxin genes 223
precede that of the toxins. In contrast, the four other systems present a reverse 224
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organization in which the toxin gene is located upstream of the predicted antitoxin gene. 225
Toxins from R. palustris, S. meliloti, M. avium and E. coli O157:H7 are associated with 226
predicted HigA antitoxins (Fig. S1, Table S3). 227
The predicted antitoxin genes were cloned in the pKK223‐3 vector under the control of 228
the pTac promoter. These antitoxin‐containing plasmids were transformed in the 229
MG1655Δ10 strain containing the toxin‐expressing plasmids (Fig. 2A). Except for the 230
relENspphdNsp gene pair, co‐expression of the predicted antitoxins with their respective 231
toxins restores normal growth, showing that these gene pairs constitute functional TA 232
systems (Fig. 2B). Co‐expression of PhdNsp with RelENsp only partially restores E. coli cell 233
growth (data not shown). To visualize the expression of this putative antitoxin by 234
western blot, a N‐terminal FLAG tag was added to PhdNsp and surprisingly, this version 235
of the antitoxin completely restored cell growth upon co‐expression with relENsp (Fig. 236
2B) while addition of the FLAG tag to the C‐terminal did not restore cell growth. 237
Altogether these data indicate that the PhdNsp is indeed an antitoxin and that blocking its 238
N‐terminus might increase either its translation or its stability. 239
Translationdependent mRNA cleavage 240
It was shown previously that over‐expression of RelESme, RelEMav and RelEO157 leads to a 241
decrease of the global translation rate in E. coli [5]. As expected, ectopic over‐expression 242
of RelERpa, RelENsp and RelETde toxins also inhibits translation (Fig. S2). A series of 243
northern blot experiments was then carried out to test whether these toxins affect 244
mRNA stability in a translation‐dependent manner as observed for the canonical RelEK‐245
12 toxin. Ectopic over‐expression of the 6 RelE‐like toxins leads to ompA degradation 246
(Fig. 3). Translation‐dependence was tested using a well‐established mRNA assay 247
consisting of a version of the lpp mRNA that is not translated due to the mutation of the 248
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start codon in a lysine AAG codon [35]. Degradation of this mRNA was tested in the 249
MG1655Δ10 Δlpp::kan strain. While degradation was observed in the case of wild‐type 250
lpp mRNA, the mutant transcript remained stable in all cases indicating that the RelE 251
homologues cleave mRNAs in a translation‐dependent manner (Fig. S3). 252
Cleavage pattern specificity 253
Primer extension experiments on the lpp and ompA mRNAs were performed under over‐254
expression of the different RelE toxins. Interestingly, expression of these toxins leads to 255
different cleavage patterns, in terms of cleavage specificity as well as frequency (Fig. 4, 256
5, S4 and Table 2). No cleavage is detected in the 5’‐UTR region of the test mRNAs, 257
confirming that the 6 RelE homologues cleave mRNAs in a translation‐dependent 258
manner. The toxins did not show codon specificity although 5 of them preferentially cut 259
between the second and third position and upstream of a purine. 260
RelEK‐12 generates 33 cleavages in the lpp mRNA (28 major and 5 minor cleavages) and 261
35 in ompA (18 major and 17 minor cleavages) (Fig. 4, 5, S4 and Table 2). RelEK‐12 262
cleaves the lpp mRNA throughout the full‐length sequence while the ompA mRNA is 263
preferentially cleaved in the 5' region. Cleavages occur preferentially between the 264
second and third nucleotide (61%) and mostly upstream of a purine (70%). Among 265
these, 74% occur upstream of a G. The 3 codons most frequently cleaved (CAG, 266
glutamine; CUG, leucine and GCG, alanine) represent 37% of the sites cleaved by RelEK‐12 267
(Table 2). 268
While the cleavage pattern of lpp by RelEO157 is similar to that of RelEK‐12 (i.e. throughout 269
the mRNA), cleavage pattern of ompA is different (Fig. 4, 5, S4 and Table 2). Only minor 270
cleavages were detected in the 5’‐end of ompA, most of them occurring after the first 271
200 first bp (Fig. 4). Fewer cleavage sites were detected as compared to RelEK‐12 (46 vs 272
68). Nineteen cleavages (15 major) were detected in the lpp mRNA and 27 (24 major) in 273
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ompA. As for RelEK‐12, RelEO157 most frequently cleaves between the second and third 274
nucleotide (90%) and mostly upstream of a G (70%). Among these, 70% occur between 275
a U and a G. The 3 codons most frequently cleaved (CUG, leucine; CAG, glutamine and 276
AUG codon) represent 65% of the total number of sites cleaved by RelEO157 (Table 2). 277
The RelERpa toxin cleaves the lpp and ompA mRNAs throughout the full‐length sequence 278
with respectively 14 (11 major) and 41 cuts (30 major) (Fig. 4, 5, S4 and Table 2). 279
Cleavages occur preferentially between the second and third nucleotide (69%) and 280
frequently upstream purines (93%). The 3 codons most frequently cleaved (CAG, 281
glutamine; AAA lysine; CCG, proline) represent 58% of the sites cleaved by RelERpa. Note 282
that RelERpa cleaves 100% (15/15) of CAG codons covered by the primer extension 283
experiments (Table 2). 284
For RelESme, a smaller number of cleavage sites were detected both in lpp (11 cuts and 285
10 major) and ompA (11 minor cuts) (Fig. 4, 5, S4 and Table 2). The cleavage pattern of 286
lpp and ompA by RelESme is quite different than that observed with the other RelE‐like 287
toxins as no cleavage is detected in the 5’‐end of the transcripts (first cleavage in lpp at 288
position 77 and 68 in ompA) (Fig. 5). Cleavages occur preferentially between the second 289
and third nucleotide (82%) and before purines (82%). The 3 codons most frequently 290
cleaved (CAG, glutamine; AAA, lysine and AUG codon) represent 63% of the total 291
number of cleavage sites (Table 2). 292
The RelENsp toxin generates 33 (21 major) and 8 major cleavage sites in ompA and lpp 293
mRNAs, respectively (Fig. 4, 5, S4 and Table 2). The cleavage patterns of lpp and ompA 294
are quite similar, the mRNAs being cleaved throughout the full‐length sequences (Fig. 5). 295
Cleavages occur preferentially between the second and third nucleotide (56%) and the 296
first and second nucleotide (33%). RelENsp cleaves preferentially upstream of an A 297
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(71%). The 3 codons most frequently cleaved (GAA, glutamic acid; AAA lysine; GCA, 298
alanine) represent 59% of the total number of sites cleaved by RelENsp (Table 2). 299
The number of cleavage sites for the RelETde toxin was low as compared to the other 300
RelE‐like toxins (1 cleavage site in the lpp mRNA and 4 in ompA). In both mRNAs, the 301
second AAA codon is cleaved by RelETde. To investigate whether the RelETde activity is 302
sequence‐ or position‐dependent, we used the rpsA mRNA which encodes an ACU codon 303
in second position. In this experiment, the YoeBK‐12 toxin was included as it shows a high 304
degree of similarity with RelETde (52% identity, 66% similarity) (Table 1). In addition, 305
YoeBK‐12 was previously reported to cleave the second codon of mRNAs [26]. Fig. 6 306
shows that overall similar cleavage patterns and specificities are observed in the rpsA 307
mRNA for RelEK‐12, RelETde and YoeBK‐12. As observed for YoeBK‐12, RelETde cleaves the 308
rpsA mRNA at the second codon, between the second and the third nucleotide 309
(upstream of a U). However, in contrast to the other test mRNAs, additional cleavage 310
sites were observed further in the rpsA transcript. Cleavages occurred mainly between 311
the second and third nucleotide (82%) and preferentially upstream of purines (75 %). 312
Sequence analysis revealed that the regions upstream (~ 12 nucleotides) the cleavage 313
sites have in common a G‐A rich region presenting similarities with the GAAG Shine‐314
Dalgarno sequence (Fig. 7A). These regions might play a role in cleavage specificity, for 315
instance by causing ribosome pausing and/or inducing secondary structures [39]. 316
Cleavage by RelEMav presents some specific features (Fig. 4, 5 and S4). While the lpp 317
mRNA is cleaved regularly (17 cleavages, 11 major), ompA is preferentially cleaved in 318
the 3’‐end region (22 cleavages, 16 major). Only very minor cleavages were observed in 319
the mRNA regions covered by ompA1 and ompA2 primers (covering the 5’‐end of the 320
ompA mRNA). In addition, although preferentially cleaving upstream of a G, RelEMav 321
cleaves preferentially between codons (90%) (Fig. 7B). 322
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Discussion 323
This work highlights the general conservation of molecular mechanisms used by the 324
members of the RelE family of type II toxin. Although originating from distantly related 325
bacterial species and sharing low amino sequence similarities, the toxins characterized 326
in this work are mRNA interferases cleaving RNA in a translation‐dependent manner. To 327
determine cleavage specificity, primer extension experiments upon RelE‐like toxins 328
overexpression were performed in a MG1655 strain deleted of the 10 well‐characterized 329
type II systems. We thought to avoid secondary cleavage sites from endogeneous type II 330
toxins, although we cannot rule out the contribution of other or unknown 331
endoribonucleases. Our data show that RelE‐like toxins do not cleave at specific codons, 332
but rather exhibit a trend to cleave upstream of purines, and between the second and 333
third position of codons, which is similar to the activity of the canonical RelEK‐12 toxin. 334
Using the highly expressed ompA and lpp as mRNA‐test, we found that these toxins 335
appear to preferentially cleave in vivo at codons that are abundant in bacterial genomes. 336
Note that in vitro analyses using purified toxins and translational complexes will be 337
needed to correlate translation rate and cleavage specificity using artificial mRNAs 338
containing rare codons. Nevertheless, our data show that RelEK‐12 preferentially cleaves 339
the glutamine CAG, the leucine CUG and the alanine GCG codons, as described before 340
(Table 3) [17]. Considering these 3 preferential cleavage sites (representing only 37% 341
of the RelEK‐12 mediated cuts) and their prevalence in the E. coli genome (117.3/1000 342
codons, Table 3), and assuming that these codons are efficiently cleaved, the initial 343
cleavage rate for RelEK‐12 is one cleavage every 9 nucleotides. This estimation is similar 344
for the other RelE‐like toxins (RelEO157 and RelENsp every 10 nucleotides and RelERpa 345
every 14). Regarding RelEMav, target codons are also well represented since it 346
preferentially cleaves before codons starting with G (Fig. 7). Thus, the relaxed 347
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specificity of these RelE‐like toxins allow them to be quite efficient and quite broad in 348
terms of substrates. Interestingly, we observed that some codons are cleaved by 349
different RelE‐like toxins (Table 3). For instance, the CAG glutamine codon is cleaved by 350
RelEK‐12, RelEO157, RelERpa and RelESme. The AAA lysine codon is also cleaved by RelERpa, 351
RelENsp, RelESme and RelETde (Table 3). This codon is generally found at the second 352
position of highly expressed mRNAs [40]. 353
Altogether, our in vivo data suggest that these RelE‐like toxins are active and able to 354
exert their function (s) in a large variety of bacterial species. Considering that TA 355
systems are located on mobile genetic elements and that they invade bacterial 356
chromosomes through horizontal gene transfer and subsequent integration as genomic 357
islands, it is likely that a relaxed specificity has been selected by evolution. 358
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Acknowledgements 360
The authors are grateful to Kenn Gerdes and the members of his laboratory for 361
providing the pSC710 and pSC711 plasmids and for their help with the primer extension 362
experiments. N.G. is supported by the National Research Fund, Luxembourg (908853). 363
This work was supported by the Fonds de la Recherche Scientifique (FRSM‐3.4530.04), 364
the Fondation Van Buuren and the Fonds Brachet. We thank the scientific community 365
for kindly providing us with genomic DNA and bacterial strains. 366
367
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Figure legends 369
FIGURE 1. Alignment of the RelE‐like sequences with RelEK‐12 and YoeBK‐12 370
Amino acid sequences were aligned with MAFFT 371
(http://www.ebi.ac.uk/Tools/msa/mafft/). Residues highlighted in dark green are 372
conserved in 80% of the sequences, in medium green in 70% and 50% conservation is 373
shown in light green. Arrows indicate residues important for the catalytic activity of 374
RelK‐12. * indicates identical amino acids, : and . indicate similar residues. Boxes show 375
conserved motifs. The ELF motif in RelEMav is conserved in HP0894 [28]. 376
377
FIGURE 2. The RelE‐like toxins inhibit E. coli growth and constitute TA systems 378
(1A) Serial dilutions of the MG1655∆10 containing the pBAD33 control vector or its 379
derivatives with the relE‐like genes were plated on M9 minimal media containing either 380
glucose (1%) or arabinose (1%) and the appropriate antibiotics. (1B) Serial dilutions of 381
the MG1655∆10 containing the pBAD33 and pKK223‐3 control vectors or their 382
derivatives with the relE‐like genes and their cognate antitoxin genes were plated on M9 383
minimal media containing either glucose (1%) or arabinose (1%) and IPTG (1mM), and 384
the appropriate antibiotics. Note that the pSC101‐lacIq plasmid was co‐transformed 385
with the pKK223‐3‐relBK12 to repress relBK12 expression since it appears to be toxic 386
(data not shown). IPTG was used at a final concentration of 0.01mM to induce antitoxin 387
expression. Plates were incubated overnight at 37°C and survival was estimated. 388
389
FIGURE 3. The RelE‐like toxins induce mRNA cleavage 390
The MG1655∆10 containing the pBAD33 control vector or its derivatives with the relE‐391
like genes were grown in LB to an OD600nm ~ 0.6. Toxin expression was then induced by 392
addition of arabinose (1%). Total RNAs were extracted at 0, 15, 30, 45 and 60 min after 393
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toxin induction and were used for northern blot analysis of the ompA mRNA using 394
specific probe as described in Materials and Methods. 395
396
FIGURE 4. RelE‐like toxins expression leads to distinct cleavage patterns on the lpp and 397
ompA transcripts 398
Primer extension analysis of the lpp (A) and the ompA transcripts using ompA1 (B) and 399
ompA5 (C) primers. The MG1655∆10 strain containing the pBAD33 control vector or its 400
derivatives with the relE‐like genes were grown in LB with the appropriate antibiotic at 401
37°C to an OD600nm ~ 0.6. Toxin expression was then induced by addition of arabinose 402
(1%) for 30 min. Total RNAs were extracted as described in Materials and Methods. 403
Major and minor cleavage sites are indicated by filled and open circles, respectively. FL: 404
full length. 405
406
FIGURE 5. Location and frequency of RelE‐like toxins cleavage sites 407
A schematic representation to scale of the lpp (A) and ompA (B) transcripts, with each 408
grey box representing 100 nucleotides. Small and large bars represent major and minor 409
cleavage sites, respectively. 410
411
FIGURE 6. RelEK‐12, YoeBK‐12 and RelETde toxins expression leads to similar cleavage 412
patterns on the rpsA transcripts 413
Primer extension analysis of the rpsA transcript. The MG1655∆10 strain containing the 414
pBAD33 control vector or its derivatives expressing the RelEK‐12, YoeBK‐12 and RelETde 415
toxin were grown in LB with the appropriate antibiotic at 37°C to an OD600nm ~ 0.6. 416
Toxin expression was then induced by addition of arabinose (1%) for 30 min. Total 417
RNAs were extracted as described in Materials and Methods. Major and minor cleavage 418
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sites are indicated by filled and open circles, respectively. 419
*: full length transcript from rpsA3 promoter [41]. 420
421
FIGURE 7. Consensus sequences around the cleavage sites of the RelETde and RelEMav 422
toxins. (A) Conserved motif upstream the RelETde cleavage site. The A/GXXGAAXC/A 423
motif is conserved around 12 nucleotides upstream the RelETde cleavage site (arrow). 424
Logo sequence were constructed using (weblogo.berkeley.edu/logo.cgi). (B) The 425
RelEMav toxin cleaves preferentially between codons ending and starting with guanines. 426
The cleavage site is represented by an arrow. The upstream codon (codon 1) and the 427
downstream codon (codon 2), as well as the nucleotide position (1, 2, 3 in codon 1 and 428
4, 5, 6 in codon 2) are indicated. 429
430
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References 431
1. Arraiano CM, Andrade JM, Domingues S, Guinote IB, Malecki M, Matos RG, 432 Moreira RN, Pobre V, Reis FP, Saramago M et al: The critical role of RNA 433 processing and degradation in the control of gene expression. FEMS 434 Microbiol Rev 2010, 34(5):883‐923. 435
2. Cascales E, Buchanan SK, Duche D, Kleanthous C, Lloubes R, Postle K, Riley M, 436 Slatin S, Cavard D: Colicin biology. Microbiol Mol Biol Rev 2007, 71(1):158‐229. 437
3. Horvath P, Barrangou R: CRISPR/Cas, the immune system of bacteria and 438 archaea. Science 2010, 327(5962):167‐170. 439
4. Hayes F, Van Melderen L: Toxinsantitoxins: diversity, evolution and function. 440 Crit Rev Biochem Mol Biol 2011, 46(5):386‐408. 441
5. Leplae R, Geeraerts D, Hallez R, Guglielmini J, Dreze P, Van Melderen L: Diversity 442 of bacterial type II toxinantitoxin systems: a comprehensive search and 443 functional analysis of novel families. Nucleic Acids Res 2011, 39(13):5513‐444 5525. 445
6. Hallez R, Geeraerts D, Sterckx Y, Mine N, Loris R, Van Melderen L: New toxins 446 homologous to ParE belonging to threecomponent toxinantitoxin systems 447 in Escherichia coli O157:H7. Mol Microbiol 2010, 76(3):719‐732. 448
7. Christensen SK, Mikkelsen M, Pedersen K, Gerdes K: RelE, a global inhibitor of 449 translation, is activated during nutritional stress. Proc Natl Acad Sci U S A 450 2001, 98(25):14328‐14333. 451
8. Jorgensen MG, Pandey DP, Jaskolska M, Gerdes K: HicA of Escherichia coli 452 defines a novel family of translationindependent mRNA interferases in 453 bacteria and archaea. J Bacteriol 2009, 191(4):1191‐1199. 454
9. Christensen‐Dalsgaard M, Gerdes K: Two higBA loci in the Vibrio cholerae 455 superintegron encode mRNA cleaving enzymes and can stabilize plasmids. 456 Mol Microbiol 2006, 62(2):397‐411. 457
10. Christensen‐Dalsgaard M, Jorgensen MG, Gerdes K: Three new RelE458 homologous mRNA interferases of Escherichia coli differentially induced by 459 environmental stresses. Mol Microbiol 2010, 75(2):333‐348. 460
11. Zhang Y, Yamaguchi Y, Inouye M: Characterization of YafO, an Escherichia coli 461 toxin. J Biol Chem 2009, 284(38):25522‐25531. 462
12. Zhang Y, Zhang J, Hara H, Kato I, Inouye M: Insights into the mRNA cleavage 463 mechanism by MazF, an mRNA interferase. J Biol Chem 2005, 280(5):3143‐464 3150. 465
13. Zhang Y, Zhang J, Hoeflich KP, Ikura M, Qing G, Inouye M: MazF cleaves cellular 466 mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol 467 Cell 2003, 12(4):913‐923. 468
14. Grady R, Hayes F: AxeTxe, a broadspectrum proteic toxinantitoxin system 469 specified by a multidrugresistant, clinical isolate of Enterococcus faecium. 470 Mol Microbiol 2003, 47(5):1419‐1432. 471
15. Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, Ehrenberg M: The bacterial 472 toxin RelE displays codonspecific cleavage of mRNAs in the ribosomal A 473 site. Cell 2003, 112(1):131‐140. 474
16. Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, 475 Ramakrishnan V, Brodersen DE: The structural basis for mRNA recognition 476 and cleavage by the ribosomedependent endonuclease RelE. Cell 2009, 477 139(6):1084‐1095. 478
on May 9, 2019 by guest
http://jb.asm.org/
Dow
nloaded from
22
17. Hurley JM, Cruz JW, Ouyang M, Woychik NA: Bacterial toxin RelE mediates 479 frequent codonindependent mRNA cleavage from the 5' end of coding 480 regions in vivo. J Biol Chem 2011, 286(17):14770‐14778. 481
18. Li GY, Zhang Y, Inouye M, Ikura M: Inhibitory mechanism of Escherichia coli 482 RelERelB toxinantitoxin module involves a helix displacement near an 483 mRNA interferase active site. J Biol Chem 2009, 284(21):14628‐14636. 484
19. Boggild A, Sofos N, Andersen KR, Feddersen A, Easter AD, Passmore LA, 485 Brodersen DE: The Crystal Structure of the Intact E. coli RelBE Toxin486 Antitoxin Complex Provides the Structural Basis for Conditional 487 Cooperativity. Structure 2012. 488
20. Anantharaman V, Aravind L: New connections in the prokaryotic toxin489 antitoxin network: relationship with the eukaryotic nonsensemediated 490 RNA decay system. Genome Biol 2003, 4(12):R81. 491
21. Guglielmini J, Van Melderen L: Bacterial toxinantitoxin systems: Translation 492 inhibitors everywhere. Mob Genet Elements 2011, 1(4):283‐290. 493
22. Jiang Y, Pogliano J, Helinski DR, Konieczny I: ParE toxin encoded by the broad494 hostrange plasmid RK2 is an inhibitor of Escherichia coli gyrase. Mol 495 Microbiol 2002, 44(4):971‐979. 496
23. Zhang Y, Inouye M: The inhibitory mechanism of protein synthesis by YoeB, 497 an Escherichia coli toxin. J Biol Chem 2009, 284(11):6627‐6638. 498
24. Yamaguchi Y, Park JH, Inouye M: MqsR, a crucial regulator for quorum sensing 499 and biofilm formation, is a GCUspecific mRNA interferase in Escherichia 500 coli. J Biol Chem 2009, 284(42):28746‐28753. 501
25. Prysak MH, Mozdzierz CJ, Cook AM, Zhu L, Zhang Y, Inouye M, Woychik NA: 502 Bacterial toxin YafQ is an endoribonuclease that associates with the 503 ribosome and blocks translation elongation through sequencespecific and 504 framedependent mRNA cleavage. Mol Microbiol 2009, 71(5):1071‐1087. 505
26. Kamada K, Hanaoka F: Conformational change in the catalytic site of the 506 ribonuclease YoeB toxin by YefM antitoxin. Mol Cell 2005, 19(4):497‐509. 507
27. Heaton BE, Herrou J, Blackwell AE, Wysocki VH, Crosson S: Molecular structure 508 and function of the novel BrnT/BrnA toxinantitoxin system of Brucella 509 abortus. J Biol Chem 2012, 287(15):12098‐12110. 510
28. Han KD, Matsuura A, Ahn HC, Kwon AR, Min YH, Park HJ, Won HS, Park SJ, Kim DY, 511 Lee BJ: Functional identification of toxinantitoxin molecules from 512 Helicobacter pylori 26695 and structural elucidation of the molecular 513 interactions. J Biol Chem 2011, 286(6):4842‐4853. 514
29. Hurley JM, Woychik NA: Bacterial toxin HigB associates with ribosomes and 515 mediates translationdependent mRNA cleavage at Arich sites. J Biol Chem 516 2009, 284(28):18605‐18613. 517
30. Garcia‐Pino A, Christensen‐Dalsgaard M, Wyns L, Yarmolinsky M, Magnuson RD, 518 Gerdes K, Loris R: Doc of prophage P1 is inhibited by its antitoxin partner 519 Phd through fold complementation. J Biol Chem 2008, 283(45):30821‐30827. 520
31. Winther KS, Gerdes K: Ectopic production of VapCs from Enterobacteria 521 inhibits translation and transactivates YoeB mRNA interferase. Mol 522 Microbiol 2009, 72(4):918‐930. 523
32. Tsilibaris V, Maenhaut‐Michel G, Mine N, Van Melderen L: What is the benefit to 524 Escherichia coli of having multiple toxinantitoxin systems in its genome? J 525 Bacteriol 2007, 189(17):6101‐6108. 526
on May 9, 2019 by guest
http://jb.asm.org/
Dow
nloaded from
23
33. Datsenko KA, Wanner BL: Onestep inactivation of chromosomal genes in 527 Escherichia coli K12 using PCR products. Proc Natl Acad Sci U S A 2000, 528 97(12):6640‐6645. 529
34. Cherepanov PP, Wackernagel W: Gene disruption in Escherichia coli: TcR and 530 KmR cassettes with the option of Flpcatalyzed excision of the antibiotic531 resistance determinant. Gene 1995, 158(1):9‐14. 532
35. Christensen SK, Gerdes K: RelE toxins from bacteria and Archaea cleave 533 mRNAs on translating ribosomes, which are rescued by tmRNA. Mol 534 Microbiol 2003, 48(5):1389‐1400. 535
36. Masse E, Escorcia FE, Gottesman S: Coupled degradation of a small regulatory 536 RNA and its mRNA targets in Escherichia coli. Genes Dev 2003, 17(19):2374‐537 2383. 538
37. Armalyte J, Jurenaite M, Beinoraviciute G, Teiserskas J, Suziedeliene E: 539 Characterization of Escherichia coli dinJyafQ toxinantitoxin system using 540 insights from mutagenesis data. J Bacteriol 2012, 194(6):1523‐1532. 541
38. Christensen SK, Maenhaut‐Michel G, Mine N, Gottesman S, Gerdes K, Van 542 Melderen L: Overproduction of the Lon protease triggers inhibition of 543 translation in Escherichia coli: involvement of the yefMyoeB toxin544 antitoxin system. Mol Microbiol 2004, 51(6):1705‐1717. 545
39. Wen JD, Lancaster L, Hodges C, Zeri AC, Yoshimura SH, Noller HF, Bustamante C, 546 Tinoco I: Following translation by single ribosomes one codon at a time. 547 Nature 2008, 452(7187):598‐603. 548
40. Brock JE, Paz RL, Cottle P, Janssen GR: Naturally occurring adenines within 549 mRNA coding sequences affect ribosome binding and expression in 550 Escherichia coli. J Bacteriol 2007, 189(2):501‐510. 551
41. Lemke JJ, Sanchez‐Vazquez P, Burgos HL, Hedberg G, Ross W, Gourse RL: Direct 552 regulation of Escherichia coli ribosomal protein promoters by the 553 transcription factors ppGpp and DksA. Proc Natl Acad Sci U S A 2011, 554 108(14):5712‐5717. 555
556 557
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TABLE 1 Similarity and identity between the RelE‐like toxins and the RelEK‐12 and 559
YoeBK‐12 canonical toxins 560
561 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 66
562 563 Amino acid sequence identity (%) and similarity (%) are indicated in grey and white, 564
respectively, as determined using the NEEDLE alignment software 565
(http://www.ebi.ac.uk/Tools/psa/emboss_needle/index.html 566
567
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TABLE 2 Cleavage sites the RelE‐like toxins in the lpp and ompA transcripts 568
569 570
571
572
573
574
575
576
577
578
579
580
Number of sites that are cleaved by the toxins in the E. coli lpp and/or ompA transcripts 581
are indicated. The sequence of the 3 codons that are the most frequently cleaved in the 582
lpp and ompA transcripts is indicated as well as the number of cleaved codons over the 583
total number of these codons in the 2 transcripts, in relative numbers (between 584
brackets) and in %. The last column (%) represents the proportion of these 3 cleaved 585
codons relative to the total number of cuts mediated by the toxins. In bold, the codons 586
that are common to different toxins. 587
588
589
590
591
592
Toxins
Bacterial species
Number of cleavage in lpp and ompA
The 3 most frequently cleaved codons in lpp and ompA
lpp
ompA
lpp and ompA
codon (relative number)
%
codon (relative number)
%
codon (relative number)
%
%
RelEK‐12 E. coli K‐12
33 35 68 CAG
(11/15) 73
CUG (10/27) 37
GCG (4/4) 100
37
RelEO157 E. coli
O157:H7 19 27 46
CUG (18/27) 67
CAG (7/15) 47
AUG (5/9) 27
65
RelERpa R.
palustris 14 41 55
CAG (15/15) 100
AAA (11/18)
61
CCG (6/12) 50
58
RelENsp Nostoc sp
8 33 41 AAA
(11/18) 61
GCA (7/13) 54
GAA (6/8) 75
59
RelESme S.
meliloti 11 11 22
CAG (7/15) 47
AAA (4/18) 22
AUG (3/9) 33
63
RelETde T.
denticola 1 4 5 AAA (2/18) 11
CCA(1/3) 33
AAG (1/5) 20
80
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TABLE 3 Most frequently cleaved codons and their occurrence in bacterial genomes 593
594
The 3 most frequently cleaved codons were determined in the lpp and ompA transcripts 595
(see table 2). The occurrence of these codons is indicated in frequency per 1000 codons 596
(0/00). The last column indicates the sum of the prevalence of the 3 most cleaved 597
codons. 598
Toxins Bacterial species
Most frequently cleaved codons and their occurrence (0/00)1st 2nd 3rd Total
RelEK‐12 E. coli K‐12
CUG 53.8
GCG 34.3
CAG 29.2 117.3
RelEO157 E. coli
O157:H7 CUG 51.4
CAG 29.5
AUG 24.7
105.6
RelERpa R. palustris CCG 37.6
CAG 26.5
AAA 7.3
71.4
RelENsp Nostoc sp GAA 46.5
AAA 34.5
GCA 22.6
103.6
RelESme S. meliloti AUG 22.2
CAG 24.9
AAA 6.7
53.8
RelETde T. denticola AAA 60.6
AAG 25.6
CCA 2.7
89.2
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