Structure and activity of AbiQ, a lactococcal endoribonuclease belonging to the type III toxin-antitoxin system

Structure and activity of AbiQ, a lactococcalendoribonuclease belonging to the type IIItoxin–antitoxin system

Julie E. Samson,1 Silvia Spinelli,2

Christian Cambillau2 and Sylvain Moineau1*1Département de biochimie, de microbiologie et debio-informatique, Faculté des sciences et génie,Université Laval, Québec, Canada, G1V 0A6.2Architecture et Fonction des MacromoléculesBiologiques, UMR 6098, CNRS – Aix-MarseilleUniversity, Case 932, 163 Avenue de Luminy, 13288MARSEILLE Cedex 09, France.

Summary

AbiQ is a phage resistance mechanism found on anative plasmid of Lactococcus lactis that abort viru-lent phage infections. In this study, we experimentallydemonstrate that AbiQ belongs to the recentlydescribed type III toxin–antitoxin systems. When over-expressed, the AbiQ protein (ABIQ) is toxic and causesbacterial death in a bacteriostatic manner. Northernand Western blot experiments revealed that the abiQgene is transcribed and translated constitutively, andits expression is not activated by a phage product.ABIQ is an endoribonuclease that specifically cleavesits cognate antitoxin RNA molecule in vivo. The crystalstructure of ABIQ was solved and site-directed muta-genesis identified key amino acids for its anti-phageand/or its RNase function. The AbiQ system is the firstlactococcal abortive infection system characterized todate at a structural level.

Introduction

In most environments, bacteria and phages are engaged ina seemingly endless arms race. Phages evolve and adaptto improve their fitness when facing new hosts. This phe-nomenon is highlighted by the diversity in phage genomes.Conversely, bacteria have developed a plethora of strate-gies to cope with phage infections (Labrie et al., 2010). Thenature of this predator–prey interaction shapes microbialpopulations by maintaining their equilibrium and coexist-

ence. Studying phage–host relation is of primary impor-tance to, among others, industrial applications suchas food fermentations and possibly controls our ownmicrobiome.

Lactococcus lactis is a Gram-positive lactic acid bacte-rium used to produce an array of fermented dairy products.This microorganism, which is generally recognized as safe,has also been leveraged to produce proteins of medical orindustrial interest (Mierau and Kleerebezem, 2005). Aswith any microbes, this bacterium can be infected byvirulent phages present in their environment (Moineau andLévesque, 2005). Thus, economically relevant bacterialcultures can be lost due to phage lysis. To circumvent thisproblem, it is possible to use or to construct bacterialstrains resistant to phage infection (Garneau and Moineau,2011). Natural anti-phage systems are divided into severalgroups based on the phage multiplication step they affect:inhibition of phage adsorption, inhibition of phage DNAejection, cleavage of nucleic acids (e.g. CRISPR-Cas andRestriction-Modification), and abortion of phage infection(Abi) (Labrie et al., 2010). Abi is a general term that groupsall defence systems that acts after phage DNAejection andbefore cell lysis as well as leading to bacterial cell death,thereby limiting phage spreading.

Lactococcus lactis has been a productive model to dis-cover abortive mechanisms as 23 different lactococcal Abisystems have been reported (Chopin et al., 2005; Durmazand Klaenhammer, 2007; Holubova and Josephsen, 2007;Haaber et al., 2008). Usually, the resistance phenotype ismediated by a single host gene, except for AbiE, AbiG,AbiL, AbiT and AbiU, which are encoded by two genes,AbiR, encoded by possibly four genes and AbiS, com-posed of a specific DNAstructure (Dai et al., 2001; Joseph-sen and Neve, 2004; Chopin et al., 2005; Yang et al.,2006). Most of these genes have been found on plasmids(Chopin et al., 2005). Their mode of action has been poorlycharacterized due to their diversity and to the lack ofin-depth knowledge of lactococcal phage biology, althoughsome progress has been made lately. Nonetheless, themode of action of lactococcal Abi systems appears to behighly diverse and system-dependent.

For example, AbiD1 was shown to be activated by aphage protein and to interfere with a RuvC-like endonucle-

Accepted 11 December, 2012. *For correspondence. [email protected]; Tel. (+1) 418 656 3712; Fax (+1)418 656 2861.

Molecular Microbiology (2013) 87(4), 756–768 � doi:10.1111/mmi.12129First published online 7 January 2013

© 2012 Blackwell Publishing Ltd

ase to inhibit phage proliferation (Bidnenko et al., 1998;2009). On the other hand, the reverse transcriptase-related protein AbiK mediates phage resistance by polym-erizing an untemplated DNA (Fortier et al., 2005; Wanget al., 2011). Lactococcal AbiP, a protein anchored to themembrane by its N-terminal domain, has been shown toarrest phage DNA replication and to inhibit the switch off ofphage early transcripts (Domingues et al., 2004; 2008).AbiV blocks the activation of late gene transcriptionprobably by inhibition of general translation (Haaberet al., 2010). AbiZ may interact with the phage holin tocause premature cell lysis (Durmaz and Klaenhammer,2007).

The gene coding for AbiQ system was isolated from theL. lactis native plasmid pSRQ900 (Emond et al., 1998).This protein of 172 amino acids (20.3 kDa) is positivelycharged with a predicted pI of 9.6. Phage infection abortedby the AbiQ system leads to the accumulation of non-mature forms of the viral DNAwithin the host (Emond et al.,1998). Recent bioinformatic analyses predicted that theAbiQ mechanism is related to another Abi mechanismnamed ToxIN also found on a plasmid but of Pectobacte-rium atrosepticum (Fineran et al., 2009). ToxIN is also atype III toxin–antitoxin (TA) system. TA systems have alsoother roles including among others plasmid maintenance,response to environmental stresses, persister cellsand programmed bacterial death (Magnuson, 2007;Yamaguchi et al., 2011). They are composed of a toxicprotein that kills bacteria if it is not neutralized by anantitoxin. The nature of the neutralizing relationshipdefines the type of TA system (Yamaguchi et al., 2011).Type I systems are composed of an RNA antitoxin thatinactivates the mRNA of the toxin; type II systems involveinteraction of the protein antitoxin with the toxic protein;and type III systems involve an antitoxic RNA moleculeinteracting with its toxic protein. ToxIN is the only type III TAcharacterized to date and its role as an abortive phageinfection mechanism has been demonstrated (Fineranet al., 2009).

A type III locus contains an antitoxin composed of atandem array of nucleotide direct repeats followed by atoxin-encoding gene. Palindromic repeat sequences thatform a hairpin structure are found between the TA compo-nents and act as a transcriptional terminator that regulatesthe levels of antitoxin RNAversus toxin transcripts (Fineranet al., 2009). This genetic organization is also found in theAbiQ system. Specifically, a promoter region is followed by2.8 repeats of 35 nucleotides, a rho-independent termina-tor, a consensus ribosome binding site (RBS) sequenceand the abiQ gene. Comparative analyses showed that theABIQ protein shares 31% sequence identity with ToxN.However, the sequences of the antitoxins antiQ and toxIare quite different as is the number of repeats since toxIcontains 5.5 repeats of 36 nucleotides. The ToxN protein

has been recently crystallized with its cognate antitoxinRNA ToxI, which revealed a heterohexameric triangularassembly of three ToxN proteins interspersed by three36 nt ToxI RNA pseudoknots. It was also shown that ToxNis a ribonuclease (Blower et al., 2011). A recent surveybased on structure similarity searches combined withprotein comparison showed that type III TA systems arelikely diverse and abundant in the microbial world (Bloweret al., 2012).

The aim of this study was to characterize the AbiQsystem through biochemical and structural analyses todemonstrate the TA nature of this phage resistancemechanism. To this end, we have solved its 3D structureand identify amino acids important for catalysis and RNAbinding. Biochemical experiments demonstrated theendoribonuclease activity of ABIQ and its ability to cleaveits cognate antitoxin RNA.

Results

AbiQ is a toxin–antitoxin system

In order to demonstrate that AbiQ is a toxin–antitoxinsystem, we first tried to separately clone the two compo-nents of the AbiQ system (antiQ repeats and abiQ gene)into two different vectors under control of the AbiQ nativepromoter in both L. lactis and Escherichia coli. No clonecontaining the abiQ gene alone was obtained, even whenwe tried to introduce the abiQ gene on a plasmid into astrain harbouring the putative antitoxin sequence. Second,the lactococcal nisin-inducible expression system NICEwas used in an attempt to better control ABIQ proteinexpression (de Ruyter et al., 1996). The abiQ gene wascloned and placed under the control of the inducible nisApromoter in the expression vector pNZ8010 and success-fully transformed into the laboratory strain L. lactisMG1363. A plasmid containing the intact abiQ gene wasthus obtained and an attempt was made to transfer thisplasmid into the host strain for nisin regulated gene expres-sion L. lactis NZ9000. Again, plasmids obtained fromchloramphenicol-resistant transformants contained majordeletions or mutations in the abiQ sequence, suggestingthat this gene is highly toxic even when expressed at a lowlevel.

To address this difficulty, the vector pNZ8010-abiQ wasco-transformed with the vector pTRKH2 (O’Sullivan andKlaenhammer, 1993) containing the antitoxin sequence(pTRKH2-antiQ) into L. lactis NZ9000. Chloramphenicol-resistant transformants without modification in abiQ werefinally obtained and grown. Growth was then followed,post-nisin induction, by measuring the optical density at600 nm for 4 h. The bacterial growth of the ABIQ-inducedcells essentially stopped when nisin was added comparedwith the controls without abiQ that continue to grow

AbiQ, a type III toxin–antitoxin system 757

© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 87, 756–768

(Fig. 1A). This confirmed thatABIQ was toxic to L. lactis. Todetermine if this effect was bacteriostatic or bactericidal,the induced cells were collected and transferred into freshmedia without nisin and bacterial growth was measured byfollowing the OD600 for 7 h. The recovery period of theabiQ-containing bacteria was longer than the controlswithout abiQ, but the bacteria began to grow again, indi-cating that the AbiQ system acts in a bacteriostatic manner(Fig. 1B).

The abiQ toxin gene is transcribed and translatedconstitutively during phage infection

The laboratory phage-sensitive strain L. lactis IL1403with or without the AbiQ system cloned in the pNZ123vector (de Vos, 1987) was infected with the virulentphage P008 to study AbiQ expression. Samples weretaken from non-infected cells and at various time inter-vals following phage addition. RNA or proteins wereextracted as indicated in Experimental procedures. Afterelectrophoresis in an agarose gel, a 32 nt oligonucle-otide DNA probe complementary to a sequence in themiddle of the abiQ gene was used to detect the specificRNA transcript in Northern blot experiments. In parallel,the protein extracts were migrated in a polyacrylamidegel and the ABIQ-6 His-tagged protein was detectedusing anti-His antibodies in Western blot experiments.Of note the presence of the His-tag did not interfere withAbiQ anti-phage activity as the EOP (10-5) of phageP008 was similar on strains carrying tagged and non-tagged ABIQ protein.

As shown in Fig. 2A, abiQ was constitutively tran-scribed in the non-infected cells and decreased over timeduring phage infection, starting 20 min post infection. Asimilar RNA profile was observed when a probe targetingthe bacterial recA gene was used, suggesting that thedecrease was a general phenomenon associated with theabortive system and cell death (Fig. 2A). The size of theunique transcript (~ 700 nt) corresponded to transcriptionof the complete operon and was in accordance with thegenetic analyses (702 nt). On the other hand, the ABIQprotein was present in similar quantity during the entiresampling period (Fig. 2A). This indicated that ABIQ wasstable in the cell, which is a characteristic of toxin TAproteins (Yamaguchi et al., 2011). The expression of theantitoxin is presented below.

ABIQ is an RNase that cleaves its antitoxin in vivo

The fate of the antitoxin RNA molecule was also ana-lysed in a similar manner, except that an additionalstrain was used, namely a strain containing only antiQ.Total RNA from non-infected or P008-infected cells con-taining only the antitoxin or the complete AbiQ systemwere migrated in a polyacrylamide gel and transferred tonylon membranes. Northern blots were performed usingone repeat of the antiQ sequence as a probe. As shownin Fig. 2B, the antiQ was constitutively produced in non-infected cells and in phage-infected cells. Moreover, theexpression of the antiQ was constant over time inphage-infected cells, in sharp contrast with abiQ tran-scripts (Fig. 2), suggesting that the antiQ RNA moleculesare either more produced or more stable than the abiQmRNA. Of note, phage products did not cleave the long

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Fig. 1. Effect of ABIQ overexpression on bacterial growth.A. L. lactis NZ9000 with different combinations ofvectors/recombinant plasmids was grown to an OD600 of 0.2 prior toinduction with 5 ng ml-1 nisin. The graph represents thepost-induction growth of the different cultures over 4 h.B. Second growth curve of the induced culture. The 4 h inducedbacteria were transferred into fresh medium without nisin. Thegrowth was followed for 7 h.Symbols: diamonds, NZ9000+pTRKH2+pNZ8010; circles,NZ9000+pTRKH2-antiQ+pNZ8010; triangles,NZ9000+pTRKH2-antiQ+pNZ8010-abiQ.

758 J. E. Samson, S. Spinelli, C. Cambillau and S. Moineau �


antitoxin molecule since one unique band (approxi-mately 120 nt) corresponding to the entire repeat regionis detected during the phage infection of cells containingonly the antitoxin (Fig. 2B).

Interestingly, in cells containing the complete AbiQsystem (antiQ and abiQ), the antitoxin was cleaved in vivoresulting in multiple bands on the gel (Fig. 2B). This indi-cates that ABIQ specifically cuts the antitoxin or activatean RNase activity in the bacteria, possibly within therepeats as it was also shown for ToxIN (Blower et al.,2011). The antitoxin was also cleaved at similar levelsthroughout the infection without significant differences inphage-infected cells compared with the non-infectedcontrol, confirming the stability of the antitoxin moleculesand/or the ABIQ protein. Altogether, these data stronglysuggest that ABIQ is an endoribonuclease or activates abacterial RNase.

Crystal structure of ABIQ

The structure of ABIQ was solved by molecular replace-ment using the structure of ToxN as starting model (2XDD)(Blower et al., 2011) with which it shares 31% sequenceidentity (Fig. 3A). The polypeptide chain of ABIQ could betraced between residues Met1 and Lys168, with only a fewresidues not visible in the electron density maps, residuesLys54–Gly56 and Ser169–Gln172, which are probablydisordered (Fig. 3B). The protein is composed of a centralsix-stranded anti-parallel b-sheet with connectivity 6-1-2-3-5-4. Six a-helices surround the b-sheet, with three ofthem covering one face of the sheet (a1, a5, a6) and twocovering the opposite face (a3, a4). The last one, a2, sitson the side of the sheet (Fig. 3B). As mentioned above, theelectron map density at position 51, at the end of strand 3(Fig. 3B) clearly indicates the presence of a Leu residue,instead of a Ser for the native enzyme (Fig. S1).

Contrary to ABIQ, ToxN was solved in complex with itsantitoxin RNA ToxI (Blower et al., 2011). They form a 3 + 3hexamer with a ToxN at each edge of the triangle made byone repeat pseudoknot structure of the antitoxin (Fig. 4A).When superposing ABIQ to one of the ToxN molecule(Fig. 4B), we notice an overall excellent fit, since ther.m.s.d. deviation between Ca atoms is only 1.5 Å. Devia-tions occur mainly in loops in which residues of ABIQ areinserted or deleted compared with ToxN (Fig. 3A), namelyloops around ABIQ residues Tyr27–Asn29, Glu59–Lys66,Asp106–Lys108 and Asp145–Tyr148 (Fig. 4B). Notewor-thy, the loop Glu59–Lys66 is just after the active-sitenucleophile (Ser51) and is in part disordered. It is possiblethat this loop needs its cognate antitoxin RNA to becomeordered, as in the ToxIN complex.

Because of the similarity between ABIQ and ToxN, wecould use the ToxI–ToxN structure as a template to modelthe putative ABIQ/antiQ complex. The resulting in silicochimera ABIQ–ToxI displays very interesting features(Fig. 5). The Ser51 is close to the RNAcleavage site; its OHmoiety is at 3.0 Å from the O2′ atom of the 2′–3′ cyclicphosphate. This group is formed between the backbonephosphate group from the RNAmolecule and the 2′O of theribose of adenine 32. In the ToxIN structure, it probablyresults from the nucleophilic serine on the 2′O of the riboseof adenine 32, which in turn performs the nucleophilicattack on the P=O group and leads to cyclization (Neu-bauer et al., 2009; Blower et al., 2011). A similar mecha-nism can be suggested for ABIQ. Also most of the RNAbases fit well inside the active-site crevice, i.e. bases uracil22 to adenine 32 at the 5′ end and -3 to -1 at the 3′ end. Incontrast, bases cytosine 0 to adenine 1 at the 3′ end clashwith ABIQ, indicating that AbiQ antitoxin RNA should followa different path of 4 residues after the cleavage site 3′ end.More remote favourable interactions are also establishedwith bases adenine 6 – uracil 7 and uracils 16–17.

Fig. 2. Northern and Western blots to detect abiQ and antiQ RNAor ABIQ protein in L. lactis during a time-course infection withphage P008. Samples were taken from a non-infected (NI) cultureand at 0, 10, 20 and 30 min. The genetic organization of the AbiQconstruction used during the infection is represented under thecorresponding gels. Black line below the AbiQ organizationcorresponds to the relative position of the probes used to detectabiQ or antiQ RNA. The diamonds represent the repeats (antiQ),the white arrows the abiQ gene, the black arrows the putativepromoter and the hairpins the putative rho-independent terminators.A. Northern blots using a probe targeting abiQ (abiQ transcription)or recA (recA transcription). Western blots using anti-His antibodies(Rockland) that recognized the ABIQ-tagged protein (translation).B. Northern blots using a probe targeting antiQ. In the antiQgenetic organization at the right of the gels, the diamondscorrespond to the repeat transcribed according to the transcriptsize.



Mutagenesis of key amino acids to inactivateABIQ activities

Based on the structure, different amino acids probablyimportant for the activities of ABIQ were identified(Fig. 6A). Site-directed mutagenesis of these amino acidswas accomplished and the activity of the mutated proteinswas measured by evaluating the phage P008 EOP. RNAcleavage of the antitoxin by ABIQ was also determined invivo using Northern blot analysis (Fig. 6B). For all the

mutants, except Lys55Ala and Arg69Glu, the anti-phageactivity of mutated ABIQ protein was completely abolished(EOP of 1.0) (Fig. 6A). Thus, the amino acids Tyr27, Pro49,Ser51, Ser52, Lys54, Lys60 and Arg67 are important forthe phage resistance phenotype. Conversely, only themutation Ser51Leu completely abolished the RNA cleav-age of the antitoxin by ABIQ (Fig. 6B). This was expectedsince this serine is likely the nucleophilic residue. Interest-ingly, the amino acid substitution Ser51Thr still cleaved theantitoxin molecule (Fig. 6B). This threonine residue is the

Fig. 3. The ABIQ sequence and crystal structure.A. ABIQ sequence aligned with the sequence of ToxN with Multalin (Corpet, 1988; Gouet et al., 2003). The conserved residues are with a redbackground. The ABIQ secondary structure is represented above its sequence. Important residues for ABIQ are identified by red marks andfor ToxN by blue marks. Triangles identify the nucleophiles positions. Blue circles pinpoint the ToxN residues important for catalysis, and plussigns, those important for RNA binding. The mutated sites of ABIQ are also indicated: ‘=’ means no effect on the RNA cleavage, and ‘-’means RNA cleavage decreased based on results presented in Fig. 6B.B. Stereo view of the overall fold of ABIQ in ribbon representation and rainbow colouring (Nt→Ct: blue to red). The beta strands are numberedfrom b1 to b6 and the helices from a1 to a6. The segment not visible in the electron density map (Lys54–Gly56) is identified by a grey line.



amino acid found in the ToxN protein active site (Bloweret al., 2011). Partial cleavage of the antitoxin was observedfor the mutations Pro49Ala, Ser52Ala and Arg67Glu whencompared with the ABIQ wild-type profile since there wasan accumulation of the larger transcripts and a decrease in

the amount of smaller transcripts. For the other mutations,even if the anti-phage activity was abolished, the proteinsretain the capacity to cleave their antitoxins.

To verify the effect of the antitoxin cleavage when anABIQ-mutant strain was infected with phage P008, a time-

Fig. 4. Comparison of ABIQ and ToxN.A. Superimposition of ABIQ (ribbon with rainbow colouring) on ToxN monomer 1 (beige) belonging to the ToxN3–ToxI3 hexameric complex. TheRNA is in light green sticks representation.B. Close-up view of ABIQ–ToxN superposition, with the diverging loops identified (same colours as in A).

Fig. 5. The in silico generated chimeric ABIQ–ToxI complex.A. ABIQ in ribbon representation and rainbow colouring and ToxI in light green sticks. Note the position of the cleaved bases (adenine -3,adenine 32), and the closeness of Ser51 (modelled on the Xray determined position of Leu51) to the cyclic phosphate of base adenine 32.B. View of the surface of the above mentioned ABIQ–ToxI complex. ABIQ surface is coloured beige. Note that the RNA position fits well theABIQ surface, except for bases cytosine 0 to adenine 1 (black arrow).



course infection with a strain containing ABIQ wild typeand a strain containing the Ser52Ala mutation was per-formed (Fig. 6C). Similar profiles were obtained with anaccumulation of the larger transcripts in the mutant ABIQcompared with the wild-type control.

Discussion

Toxin–antitoxin systems are widespread in bacteria andhave been demonstrated to play key roles in regulatinggrowth during stresses such as phage infection. In thelatter, this regulation is achieved by the death of the phage-infected TA-containing bacteria for the survival of the wholepopulation. Here, we have shown through a series ofexperiments that the lactococcalAbiQ mechanism belongsto type III TAsystem and thatABIQ is an endoribonuclease.

The operon is composed of 2.8 direct repeats followed bya gene coding for a toxic protein similar to ToxN, the soletype III TA characterized before this study. First, It was notpossible to clone abiQ alone under its native or induciblepromoter, indirectly demonstrating the toxic nature of theprotein. Then, when overexpressed in bacteria containingits cognate antitoxin (antiQ), the ABIQ protein inhibited thebacterial growth in a bacteriostatic manner, a phenotypeassociated with TA systems.

The AbiQ system is encoded on a low-copy nativeplasmid pSRQ900 (10.8 kb) found in a raw milk L. lactisisolate (Emond et al., 1998). It is well documented thatthis bacterium contains several plasmids that provideadditional activities such as lactose utilization, proteolysis,and of interest here, phage resistance (Duckworth et al.,1981; Klaenhammer, 1987; Boucher et al., 2001). In addi-

Fig. 6. RNase assay to determine the AbiQ activity against its cognate antitoxin.A. Site-directed mutations in the ABIQ protein and phage P008 EOP evaluation.B. Northern blot to visualize the antitoxin (antiQ) cleavage profile achieved by the RNase activity of wild-type and mutant ABIQ. The probeconsists of a single repeat of the antitoxin.C. Effect of the phage P008 on the antitoxin cleavage by the ABIQ mutant Ser52Ala during a time-course infection.



tion, it is tempting to speculate that AbiQ plays a role inplasmid maintenance to assure transfer of the plasmids tothe daughter cells and maintain key functions for growth inthe phage-containing milk environment. A similar phe-nomenon was observed in E. coli. The hok/sok system(type I TA) serves in plasmid R1 maintenance and indefence against phage T4 (Pecota and Wood, 1996).These functions were also suggested for the ToxINsystem (Fineran et al., 2009).

To shed further light on its activities, we started byevaluating the transcription and translation of the abiQgene. The transcription of abiQ is constitutive and the sizeof the transcript corresponds to the size predicted bybioinformatics analysis for the complete operon, includingthe repeats (antiQ). This result means that the terminatorlocated between the antitoxin repeats and abiQ gene isleaky, and that no additional promoter is present sinceonly one RNA band is detected. It also confirms that thetoxin and antitoxin were co-transcribed as observed forthe ToxIN system (Fineran et al., 2009).

During a time-course infection with virulent phage P008,the amount of abiQ transcripts decreased rapidly 20 minpost infection. This finding could be attributed to the deathof the bacteria, the RNase activity of ABIQ or the lowstability of the mRNA molecule. The first hypothesis is themost plausible, since a similar pattern of transcription wasalso observed for the host recA gene. Interestingly, theantiQ RNA was produced at a similar level throughout thephage infection and was not cut by any phage product. Thissmall RNA was probably very stable or constantly pro-duced. This is rather surprising since antitoxin moleculesare usually more labile than their cognate toxin in TAsystems. The ABIQ protein was translated constitutivelyand its concentration was constant during the phage infec-tion. This protein either was always produced or was stablein L. lactis. In any case, this is consistent with the constantpresence of the antitoxin in the cell, as it is needed toprotect it from the toxic effect of ABIQ. The ToxN-FLAG-tagged protein level during a time-course experiments withP. atrosepticum phages jA2 and jM1 was also constant,similarly to the ABIQ protein level reported here (Bloweret al., 2009). Thus, stable protein expression even during aphage infection is probably a general phenomenon for typeIII TA systems. However, a similar time-course phageinfection combined with Northern blot experiments todetect toxI- and toxN-specific transcripts as yet to be runwith the ToxIN system but would be of interest to determinewhether the above is a general observation for type III TAsystems.

Because it only kills the phage-infected bacteria, ABIQendoribonuclease must be somehow activated or madeavailable by a phage component. In vivo experimentspresented here suggest that by cleaving its antitoxin, ABIQis sequestered and not available to attack its bacterial

targets. However, when the system is stressed through aphage infection, the ratio of toxin–antitoxin may becomeunbalanced or the antitoxin may be somehow titrated out(Fineran et al., 2009). We can eliminate a few hypotheseswith the data presented here. First, the antitoxin antiQ wasnot cleaved by a phage protein nor was abiQ. In addition,there is no activation of the abiQ transcription and ABIQtranslation, indicating that the phage does not play a role inincreasing the amount of ABIQ in the cells. However, if aphage protein or product binds to antiQ to sequester theantitoxin, free ABIQ could act on its cellular targets leadingto cell death. On the other hand,ABIQ could bind directly toa phage product, which will change its activity and direct ittowards its bacterial targets. The phage AbiQ’s activatorcomponent is likely expressed late during the phage infec-tion, which would explain why the phage DNA is replicatedbut not maturated in the capsid as reported previously(Emond et al., 1998).

The ABIQ 3D structure was determined without its anti-toxin and was found to be very similar to the ToxN structurerecently solved. Moreover, the ToxI–ToxN binding seems tobe in a large part compatible to a putative antiQ–ABIQbinding. Despite the fact that the sequence of antiQ andtoxI are non-homologous and the number of repetition inthe antitoxin operon sequences are different (2.8 repeatsfor antiQ and 5.5 repeats for toxI) (Fineran et al., 2009), thetoxI structure has revealed a pseudoknot correspondingapproximately to one repetition of the complete toxI-codingsequence that could be also predicted for antiQ by bioin-formatics analyses (supplementary Fig. S2, Blower et al.,2011; 2012). However, contrary to toxI, the antiQ pseudo-knot is formed in the middle of a repetition and not at theboundaries of the repeats (supplementary Fig. S2, Reederand Giegerich, 2004; Blower et al., 2012).

Based on the structure and on the complex model, weconstructed site-directed mutations likely to impact RNaseand anti-phage activities of AbiQ (Fig. 6). Surprisingly, onlyone mutation, Ser51Leu, completely abolished both func-tions. This mutated form of ABIQ could be overexpressedin E. coli without significant impact on the bacterial growth,demonstrating the importance of the RNase activity on thecell toxicity activity of AbiQ. This amino acid Ser51 is thebest candidate to perform the nucleophilic attack on thecleavable RNA phosphate (for a review on nucleasesmechanism see Yang, 2011). This is in contrast with ToxN,for which the residue Ser53, similar to ABIQ Ser52, hasbeen proposed to perform the nucleophilic attack. Note-worthy, ToxN mutation Ser53Ala did not completely abolishthe endonuclease activity, in sharp contrast with our resultsfor ABIQ mutation Ser51Leu (Blower et al., 2011). Interest-ingly, when ABIQ Ser51 was replaced by a threonine, theresidue found in a similar position in ToxN, the RNaseactivity of ABIQ was still functional but its anti-phage activ-ity was abolished.



Of note, the Tyr27 residue appears to be important onlyfor the anti-phage mechanism since, when mutated, theAbiQ anti-phage activity is abolished while its RNaseactivity is still functional, suggesting that both activitiesare not entirely related. Residues Pro49 and Ser52 muta-tions diminished both ABIQ activities since there was anaccumulation of the large transcript and cells werephage-sensitive. Pro49 is two positions before the nucle-ophilic Ser51 residue and probably imposing the properconformation of the catalytic machinery. It is also con-served in ToxN. Ser52 was also shown to be important forcatalysis. Its role however is difficult to assign, since theOH moiety is turned opposite to the RNA. A probableexplanation is that it might move upon RNA binding, asprobably does the loop Lys54–Asn57, which is disorderedin the absence of RNA antitoxin in our structure. In thiscontext, Ser52 may very well play the role as one of theoxyanion stabilizer.

Partial RNA cleavage and no anti-phage activity havealso been obtained for the Arg67Glu which are likely to beinvolved in RNA binding and, thus, have an important rolein stabilization of the Michaelis complex. Lysines close tothe active site could bring positive charges important foroxyanion stabilization. On the other hand, ABIQ mutationsLys54Ala, Lys55Ala and Lys60Ala showed no appreciabledifference in antiQ RNA cleavage, thereby they probablydo not play a role as members of oxyanion stabilizing

residues, while this role was fulfilled by the homologousToxN Lys55. Conversely, Lys54Ala and Lys60Ala elimi-nate the anti-phage activity and these residues play a keyrole in phage resistance. Therefore, while the Xray datamade it possible to significantly rationalize the nucle-ophilic and antiQ RNA binding features of ABIQ, such aprogress could not be reached concerning its anti-phageactivity. Studies are underway to address this.

In conclusion, we have determined that the lactococcalAbiQ anti-phage mechanism is a type III TA system. This isthe first TA described in L. lactis and the second type III TAcharacterized to date. Similarities as well as differenceswith the other studied type III TA system (ToxIN) werehighlighted indicating, among others, that additionalstudies are needed on other type III TA systems to proposea general model. Elucidating the anti-phage activity shouldprovide new insights into methods to control phage popu-lations. From an application perspective, besides theadvantage of phage resistance for industrial bacterialstrains, AbiQ could serve as natural selection markers forthe development of food-grade plasmids.

Experimental procedures

Bacterial strains and phages

The bacterial strains and phages used in this study are listedin Table 1. L. lactis strains were grown at 30°C in M17 broth

Table 1. Bacterial strains, phage and plasmids used in this study.

Bacterial strains,phage or plasmids Relevant characteristics References and sources

Escherichia coliMG1655 F- l- ilvG- rfb-50 rph-1 Blattner et al. (1997)NEB 10 Beta araD139 D(ara-leu)7697 fhuA lacX74 galK (f80 D(lacZ)M15) mcrA galU recA1

endA1 nupG rpsL (StrR) D(mrr-hsdRMS-mcrBC)New England Biolabs

Rosetta F- ompT hsdSB(rB- mB

-) gal dcm (DE3) pLysSRARE2 (CamR) NovagenXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZDM15

Tn10 (Tetr)]Stratagene

Lactococcus lactisIL1403 Laboratory strain, plasmid free, host of phage P008 Chopin et al. (1984)MG1363 Laboratory strain, plasmid free, cloning host Wegmann et al. (2007)NZ9000 MG1363 derivative, inducible strain for the NICE system Kuipers et al. (1998)

PhageP008 Phage infecting L. lactis IL1403, 936 group, sensitive to AbiQ Mahony et al. (2006)

PlasmidspDEST17 Gateway IPTG-inducible expression vector of E. coli, 6.4 kb, AmpR InvitrogenpDONR201 Gateway intermediate vector, 4.5 kb, KmR InvitrogenpNZ123 Shuttle vector L. lactis–E. coli, 2.5 kb, CmR de Vos (1987)pNZ123-AbiQ AbiQ complete system cloned in pNZ123 at EcoRI site, CmR This studypNZ123-AbiQ6H AbiQ complete system cloned in pNZ123 at EcoRI site, C-terminal

6His-tagged protein, CmRThis study

pNZ123-antiQ Antitoxin region cloned in pNZ123 at EcoRI site, CmR This studypNZ8010 NICE-inducible expression vector in L. lactis, CmR de Ruyter et al. (1996)pSRQ928 pNZ123 + 2.2 kb fragment containing AbiQ, CmR Emond et al. (1998)pTRKH2 High-copy-number vector, EmR, 6.9 kb O’Sullivan and Klaenhammer (1993)pTRKH2-antiQ Antitoxin region cloned in pTRKH2 at EcoRV–BamHI site, EmR This study



(Oxoid) supplemented with 0.5% glucose (GM17). Whennecessary, chloramphenicol or erythromycin (5 mg ml-1) wasadded to the media for plasmid maintenance. For phageP008 propagation, L. lactis IL1403 was grown to an opticaldensity at 600 nm (OD600) of 0.2 before the addition of 104

phages and 10 mM CaCl2. The culture was incubated untilcomplete bacterial cell lysis had occurred and the resultinglysate was filtered using a 0.45 mm syringe filter. The effi-ciency of plaquing (EOP) was measured by dividing the titreof the phage on the AbiQ+ strain by the titre of the phage onthe AbiQ- strain. To obtain a high phage titre, one litre ofphage lysate was separated on a discontinuous caesiumchloride gradient (Sambrook and Russell, 2001). E. colistrains were grown at 37°C in Luria–Bertani (LB) or BrainHeart Infusion (BHI) broths. Chloramphenicol (34 mg ml-1),ampicillin (100 mg ml-1), erythromycin (150 mg ml-1) or kan-amycin (25 mg ml-1) was added to the medium as needed.

Plasmids, primers and DNA manipulations

The plasmids used in this study are described in Table 1 andthe primers in supplementary material (Table S1). E. coliplasmids were prepared using Qiagen plasmid purificationkits as indicated by the manufacturer. L. lactis plasmid DNAwas obtained with Qiagen plasmid purification kits with thefollowing modifications. To increase bacterial lysis, cells weretreated by adding 30 mg ml-1 lysozyme directly to the P1buffer. The samples were incubated at 37°C for 20 min beforecontinuing with the normal protocol. Restriction endonucle-ase (Roche), Taq DNA polymerase (Invitrogen), Pwo DNApolymerase (Roche), Antarctic Phosphatase (New EnglandBiolabs) and T4 DNA ligase (Invitrogen) enzymes were usedaccording the manufacturer’s instructions. Cloning proce-dures were carried out as described elsewhere (Sambrookand Russell, 2001) and with primers listed in Table S1(supplementary material). L. lactis strains were electro-transformed using a Gene Pulser II apparatus (Holo and Nes,1989). E. coli transformation was performed by thermal treat-ment. Clones were confirmed by sequencing of the inserts atthe Plateforme de séquençage et de génotypage desgénomes of the CHUL centre. The antibiotic selective pres-sure was maintained throughout the study to ensure mainte-nance of the plasmids in the various cells.

Bacterial cell toxicity assay

First, a PCR product of the region containing the antitoxinantiQ was cloned into the vector pTRKH2 at the EcoRV–BamHI restriction sites using the vector pSRQ928 (Emondet al., 1998) as a template. This construct was obtained inE. coli XL1-Blue and then introduced into L. lactis NZ9000.Second, a PCR product of the abiQ gene, using pRSQ928 asa template, was cloned into the nisin-inducible vectorpNZ8010 at the BamHI–PstI restriction sites (de Ruyter et al.,1996). The resulting plasmid was transformed into L. lactisMG1363 to avoid toxic production of the protein and then intoL. lactis NZ9000 or NZ9000+pTRKH2-antiQ for proteinexpression. Plasmids pTRKH2 and pNZ8010 have compatiblereplicons and different antibiotic selection markers to assuretheir maintenance. For the toxicity assay, 10 ml of inoculated

GM17 media containing 1% of the different strains wereincubated at 30°C to reach an OD600 of 0.2 prior to inductionwith 5 ng ml-1 nisin. The OD600 was followed for 4 h afterinduction. To verify whether AbiQ was bacteriostatic, theinduced cultures were centrifuged and resuspended in 10 mlof fresh GM17 media with or without 5 ng ml-1 nisin. Bacterialgrowth was followed for 7 h at 30°C. This bacterial toxicityexperiment was repeated twice.

Time-course phage infection

A PCR product containing the AbiQ system was cloned intothe pNZ123 vector at the EcoRI restriction site usingpSRQ928 as template. The vector pNZ123 is a small high-copy-number plasmid in L. lactis and can be selected usingchloramphenicol. The construction pNZ123-AbiQ relied onthe native promoter for the expression of the AbiQ-operon(Emond et al., 1998; Boucher et al., 2001). To enable proteindetection by Western blot, primers were designed to includesix histidines and thereby create a fusion protein. Theseplasmids were transformed into L. lactis IL1403 and we per-formed a time-course infection with the virulent lactococcalphage P008. Fifty millilitres of GM17 was inoculated (1%)with IL1403 + AbiQ and grown at 30°C to an OD600 of 0.5. Thecells were centrifuged at 8000 r.p.m. for 5 min at room tem-perature in a Beckman GSA rotor and resuspended in 6 ml offresh media. One ml of culture was removed (non-infected),while 10 mM CaCl2 and phages at a multiplicity of infection(moi) of 5 were added to the remaining 5 ml of culture. Onemillilitre of samples were withdrawn at different times after thebeginning of the infection (0, 10, 20, 30 and 40 min), centri-fuged 1 min at high speed and frozen at -80°C.

Northern experiments

Total RNA from the time-course phage infection (1 ml) wasextracted from phage-free and phage-infected L. lactis cellsusing Trizol reagent (Invitrogen) with the addition of a lys-ozyme pre-treatment (60 mg ml-1 for 10 min at 37°C) toincrease lysis of these Gram-positive cells. RNAsamples weretreated with DNase I (Roche) at 37°C for 20 min to eliminateresidual DNA and protected with RNase inhibitor (Roche).RNA concentration was determined using a NanoDrop 2000.Five micrograms of RNA was loaded on a 1% formaldehyde-agarose denaturating gel or on a 10% polyacrylamide/8 Murea gel, subjected to electrophoresis and transferred to anylon membrane (Sambrook and Russell, 2001). Northern blotexperiments were performed using a 32P radiolabelled (Perkin-Elmer) DNA probe complementary to the abiQ gene (5′-GGGGTATTAATTCGCTGTCAGGAACTGGAATC-3′) or theantiQ antitoxin (5′-GCTCCAATTTTATCAATTCCAACTATGGCTTGGATA-3′) (Fortier et al., 2006).

Western experiments

To extract total proteins, 1 ml of the phage time-courseinfected bacteria were centrifuged and the pellets resus-pended in 200 ml of lysis buffer [10 mM Tris-HCl pH 8.0, 0.3%SDS, 1 mM EDTA, 60 mM DTT and protease inhibitor(Roche)]. Cells were lysed by sonication using a Sonifier



W-350 apparatus (6 ¥ 45 s, output control 3, duty cycle 80%,hold). The suspension was centrifuged at 17 000 g for 30 minat 4°C, the supernatants were recovered and protein concen-trations were determined using the Bradford standardmethod (Bio-Rad) with bovine serum albumin as standard.Samples (1 mg) were subjected to electrophoresis in 4–15%tris-glycine polyacrylamide pre-cast gels (Bio-Rad) and elec-trotransferred to polyvinyldene fluoride membranes (PALLCorporation). The membranes were blocked overnight at 4°Cwith 5% (w/v) non-fat dry milk in phosphate-buffered salinesupplemented with 0.1% Tween 20 (PBS-T). Western blotwas performed for 1 h using the antibody IgG anti-6His(Rockland) resuspended in the blocking buffer at a concen-tration of 1:1000 followed by four 10 min washes in PBS-Tbuffer. Then, membranes were treated for 1 h with the secondantibody, anti-IgG-HRP, diluted in the blocking buffer at aconcentration of 1:10 000. The membranes were washedfour times for 10 min each in PBS-T with a final 10 minequilibration with PBS buffer. Detection was done by expos-ing the membranes to Kodak chemiluminescent films usingthe Amersham ECL Plus reagent as indicated by the manu-facturer (GE Healthcare).

ABIQ protein purification

The abiQ gene was amplified by PCR using pSRQ928 astemplate and primers, which added sequences necessaryfor the Gateway technology (Invitrogen). This amplicon wascloned into the pDONR201 vector and transferred into theexpression vector pDEST17 using the lambda recombinaseGateway enzymes. This construction added 6 histidines tothe N-terminus to allow purification of the protein by affinitychromatography in an E. coli strong expression induciblesystem. Plasmids were subcloned into E. coli NEB 10-betaand transformed into the expression strain Rosetta 2 (DE3)pLysS. Potential clones were screened by sequencing theinsert and one clone containing the mutation Ser51Leu wasused for protein purification since a wild-type clone wasnever isolated despite numerous attempts. Two litres ofculture were grown in Turbo broth (AthenaES) at 37°C to anOD600 of 0.6. Protein expression was induced by adding0.5 mM IPTG, and the culture incubated overnight at 25°C.The culture was harvested by centrifugation at 4000 g for10 min, resuspended in 50 ml of lysis buffer (50 mM Tris,300 mM NaCl, 10 mM imidazole, pH 8.0) supplementedwith 0.25 mg ml-1 lysozyme, 10 mg ml-1 DNase, 20 mMMgSO4 and EDTA-free antiproteases (Roche), and frozen at-80°C. After thawing on ice, the protein lysate was soni-cated (output control 4, duty cycle 80%, 3 ¥ 45 s), clearedat 12 000 r.p.m. for 30 min at 4°C, and filtered using a0.45 mm syringe filter. The protein was purified by nickelaffinity chromatography using a 5 ml His-Trap column on afast protein liquid chromatography apparatus (AKTA, GEHealthcare) with a stepwise gradient of imidazole. Theprotein was then subjected to gel filtration using a Superdex200 column in the following buffer: 10 mM Tris, 300 mMNaCl, pH 8.0. The concentration of the protein was deter-mined using a NanoDrop 1000 with the absorbance at 280corrected for the difference in absorption coefficient due toamino acid composition of the protein monomer with Prot-Param tool (web.expasy.org/protparam/).

Crystallography

Optimal crystallography conditions were screened using thecommercial kits MDL, Wizard 2, Stura and JSCG+.Avolume of100 nl of the kits was mixed to 100–300 nl of ABIQ at 4.6 mgml-1 using a Cartesian nano-dispensing robot (Sulzenbacheret al., 2002). The protein crystallized by mixing 200 nl ofprotein at 4.6 mg ml-1 with 100 nl of 1 M Na/K tartrate asprecipitant, and in 100 mM Mes, pH 6.0 buffer, at 20°C. Crys-tals suitable for X-ray diffraction were obtained after 2 months.A crystal was cryo-cooled with glycerol and a data set wascollected at 2.16 Å resolution at the SOLEIL Proxima1 beam-line. After processing the data sets using the XDS program(Kabsch, 2010), the scaling was performed with XSCALE(Table 2). Crystals belong to the P212121 space group with celldimensions a = 50.1 Å, b = 54.5 Å and c = 64.2 Å. The Vmcalculated for one ABIQ molecule in the asymmetric unit was2.08 Å3 Da-1, corresponding to a solvent volume of 39%. Thestructure was solved by molecular replacement with MOLREP(Vagin and Teplyakov, 2010) in CCP4 (Collaborative Compu-tational Project, Number 4, 1994). Structure refinement wasperformed with AutoBUSTER (Blanc et al., 2004) alternatedwith model rebuilding using COOT (Emsley et al., 2010),leading to R/Rfree values of 19.6% and 22.3%, respectively, andall residues in the preferred or allowed regions of the Ramach-andran plot (Table 2). Figures were made with Pymol(DeLano). The co-ordinates have been deposited at the PDBwith the entry number 4GLK. The in silico chimera model ofABIQ–ToxI was obtained by performing a rigid-body fitting ofthe ABIQ structure onto the model of ToxN in the ToxNIstructure (2XDD) (Blower et al., 2011). Since the structure ofthe antiQ RNA is still unknown, the ToxI RNA structure waskept in the in silico chimera model.

Site-directed mutagenesis and RNase assay

The plasmid pNZ123-AbiQ isolated from E. coli MG1655 wasused as a template for the PCR reactions. Amplification of the

Table 2. Data collection and refinement statistics.

Data collection/refinement Soleil Proxima 1a

Data collectionWavelength (Å) 0.9791Resolution limits (Å) 45.0–2.16 (2.22–2.16)Rmerge (%) 9.0 (61.0)No. of observations 86620 (6333)No. unique reflections 9856 (753)Mean((I)/sd(I)) 17.1 (3.7)Completeness (%) 99.7 (99.6)Multiplicity 13.7/13.1

RefinementResolution (Å) 27.7–2.16 (2.42–2.16)No. of reflections 9880 (2734)No. protein/water/glycerol atoms 1351/70/18No. test set reflections 694 (173)Rwork/Rfree (%) 19.6/22.3 (23.3/31.8)r.m.s.d. bonds (Å)/angles (°) 0.09/1.18B-wilson/B-average 35.5/37.4Ramachandran plot 97.5/2.5Preferred/allowed %

a. Parentheses refer to the highest resolution bin.



plasmid was achieved using primers designed to create spe-cific substitutions in AbiQ protein. The residual templateplasmid was removed using DpnI (NEB) that cleaves methyl-ated DNA. An aliquot of the digested PCR product was trans-formed into E. coli XL1-Blue and sequenced-confirmedplasmids were transferred into L. lactis IL1403. The plasmidconstructs in L. lactis were sequenced-confirmed. To verify ifthe antitoxin was cleaved by ABIQ, 10 ml of bacterial culturewas grown in GM17 media to an OD600 of 0.5. After centrifu-gation, bacterial pellets were frozen at -80°C. Extraction of theRNA, migration on polyacrylamide gel, and Northern assayswere performed as described above using antiQ probe.

Acknowledgements

We would like to thank Barbara-Ann Conway for editorialassistance and Maxime Bélanger for technical assistance.This work was supported by the Natural Sciences and Engi-neering Research Council of Canada (Strategic programme)as well as the Ministère du Développement économique, del’Innovation et de l’Exportation, Programme de soutien à larecherche: Programme de soutien à des initiatives interna-tionales de recherche et d’innovation. J.E.S. is the recipientof a scholarship from the Fonds Québécois de Recherche surla Nature et les Technologies. S.M. holds a Tier 1 CanadaResearch Chair in Bacteriophages.

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Structure and activity of AbiQ, a lactococcal endoribonuclease belonging to the type III toxin-antitoxin system