HflXr, a homolog of a ribosome-splitting factor,mediates antibiotic resistanceMélodie Duvala,b,c, Daniel Dard, Filipe Carvalhoa,b,c, Eduardo P. C. Rochae,f, Rotem Sorekd, and Pascale Cossarta,b,c,1

aDépartement de Biologie Cellulaire et Infection, Unité des Interactions Bactéries-Cellules, Institut Pasteur, F-75015 Paris, France; bINSERM, U604, F-75015 Paris,France; cInstitut National de la Recherche Agronomique (INRA), Unité sous-contrat 2020, F-75015 Paris, France; dDepartment of Molecular Genetics, WeizmannInstitute of Science, 76100 Rehovot, Israel; eMicrobial Evolutionary Genomics, Institut Pasteur, 75015, France; and fCNRS, UMR3525, 75015 Paris, France

Contributed by Pascale Cossart, October 24, 2018 (sent for review June 20, 2018; reviewed by Julian Davies and Susan Gottesman)

To overcome the action of antibiotics, bacteria have evolved a varietyof different strategies, such as drugmodification, target mutation, andefflux pumps. Recently, we performed a genome-wide analysis ofListeria monocytogenes gene expression after growth in the presenceof antibiotics, identifying genes that are up-regulated upon antibiotictreatment. One of them, lmo0762, is a homolog of hflX, which encodesa heat shock protein that rescues stalled ribosomes by separating theirtwo subunits. To our knowledge, ribosome splitting has never beendescribed as an antibiotic resistance mechanism. We thus investigatedthe role of lmo0762 in antibiotic resistance. First, we demonstratedthat lmo0762 is an antibiotic resistance gene that confers protectionagainst lincomycin and erythromycin, and that we renamed hflXr (hflXresistance). We show that hflXr expression is regulated by a transcrip-tion attenuation mechanism relying on the presence of alternativeRNA structures and a small ORF encoding a 14 amino acid peptidecontaining the RLR motif, characteristic of macrolide resistance genes.We also provide evidence that HflXr is involved in ribosome recy-cling in presence of antibiotics. Interestingly, L. monocytogenespossesses another copy of hflX, lmo1296, that is not involved inantibiotic resistance. Phylogenetic analysis shows several eventsof hflXr duplication in prokaryotes and widespread presence ofhflXr in Firmicutes. Overall, this study reveals the Listeria hflXr asthe founding member of a family of antibiotic resistance genes.The resistance conferred by this gene is probably of importancein the environment and within microbial communities.

HflX | Listeria monocytogenes | ribosome splitting | molecular evolution |riboregulation

To treat bacterial infections, the use of bacteriostatic or bac-tericidal antibiotics remains the gold standard. These mole-

cules act at many levels of the bacterial metabolism to preventreplication or to promote death. Beside their use as therapeutics,antibiotics are also found in the environment, since microor-ganisms use them to outcompete and survive within microbialcommunities, and many antibiotic-producing organisms such asStreptomyces spp. live in the soil (1).To overcome the action of antibiotics, bacteria have evolved dif-

ferent resistance strategies. Resistance can be intrinsic, i.e., a naturalproperty of the bacterium, such as the presence of an outer mem-brane in Gram-negative bacteria, that protects peptidoglycan fromvancomycin (2); or acquired, e.g., gain of plasmid-mediated antibi-otic resistance genes. The main resistance mechanisms can be clas-sified in three major families: those that prevent the drugs fromentering the cell or that actively pump them out, those that inactivatethe antibiotic, and those that modify the target so that it cannot berecognized by the antibiotic (3). The genes involved in thesemechanisms are often induced in presence of antibiotics, using var-ious regulatory systems (4). One of these, called “attenuation,” relieson the presence of a 5′ regulatory region that folds into alternativeRNA structures controlling either transcription or translation of theresistance gene.Listeria monocytogenes is a food-borne pathogen responsible for

listeriosis, a rare but lethal disease, that affects immunocompromisedindividuals, as well as pregnant women and elderly people (5).

Although listeriosis can be efficiently treated with ampicillin andgentamicin, resistance to various antibiotics, including lincosamides,gentamicin, ampicillin, streptomycin, erythromycin, kanamycin,and rifampicin (6–12) has been reported in both food andclinical isolates.In a previous study (13), we used a method “term-seq” to map

the 3′ ends of all RNAs in bacteria grown under various condi-tions, including in the presence or absence of antibiotics. Wediscovered a previously unknown antibiotic resistance gene inL. monocytogenes EGDe laboratory strain, lmo0919, which isinduced in the presence of lincomycin, an antibiotic that blockstranslation by binding to the 70S ribosome in the peptidyl-transfer center, thereby preventing the transpeptidylation step(14). Another gene, lmo0762, is also induced in the presence oflincomycin. This gene is located downstream of rli80, a smallRNA that contains a putative ORF encoding a 14-aa peptide. Bysequence comparison, we found that the protein encoded bylmo0762 is homologous to Escherichia coli and Staphylococcusaureus HflX, GTPase proteins that bind to 70S ribosomes (15–18)and recycle blocked ribosomes during heat shock in a GTP-dependent manner (15, 19, 20). Moreover, HflX was recentlydescribed as a RNA helicase that modulates rRNA conforma-tion of heat-damaged 50S subunits in E. coli (21), and which isalso able to split 100S disomes in S. aureus (20). The specificactivation of lmo0762 by sublethal doses of antibiotics suggeststhat this gene is involved in antibiotic resistance. However, to our

Significance

Antibiotics have beenwidely used to treat bacterial infections andare also found in the environment. Bacteria have evolved variousresistance mechanisms, allowing them to overcome antibioticexposure and raising important health issues. Here, we report abacterial antibiotic resistance mechanism, based on ribosomesplitting and recycling, ensuring efficient translation even inpresence of lincomycin and erythromycin, two antibiotics thatblock protein synthesis. This mechanism is mediated by a HflX-likeprotein, encoded by lmo0762 in Listeria monocytogenes, whoseexpression is tightly regulated by a transcriptional attenuationmechanism. This gene increases bacterial fitness in the environ-ment. Our results raise the possibility that other antibiotic-induced resistance mechanisms remain to be discovered.

Author contributions: M.D., D.D., F.C., E.P.C.R., R.S., and P.C. designed research; M.D.,D.D., F.C., and E.P.C.R. performed research; M.D., D.D., F.C., E.P.C.R., R.S., and P.C. ana-lyzed data; and M.D., D.D., F.C., E.P.C.R., R.S., and P.C. wrote the paper.

Reviewers: J.D., University of British Columbia; and S.G., National Institutes of Health.

The authors declare no conflict of interest.

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

Data deposition: Data were deposited in the European Nucleotide Database (ENA), www.ebi.ac.uk/ena/ (accession no. PRJEB25942).1To whom correspondence should be addressed. Email: pascale.cossart@pasteur.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810555115/-/DCSupplemental.

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knowledge, such a mechanism of ribosome splitting has not beenreported in the context of antibiotic resistance.In this study, we assessed the role of lmo0762 in antibiotic

resistance. We showed that lmo0762 confers resistance to lin-comycin and erythromycin, two antibiotics that block translation.Thus, we renamed it hflXr, for hflX resistance. We analyzed itsexpression regulation, and showed that an attenuation mecha-nism leading to premature transcription termination controlshflXr expression. This attenuation mechanism relies on the 14-aaORF encoded by rli80, which strikingly contains the arginine–leucine–arginine (RLR) motif, a signature feature for macrolideresistance gene leader peptides (22). Moreover, by analyzing thepolysome profiles of bacteria grown in the presence of erythro-mycin, we provide data suggesting that HflXr promotes recyclingof blocked 70S ribosomes and thus translation. Finally, we iden-tified another L. monocytogenes homolog of hflX, lmo1296, whichis not involved in antibiotic resistance. Phylogenetic analysisrevealed that duplication occurred several times, independently, inseveral major clades of prokaryotes, and that many Firmicutespossess a hflX copy closely related to hlfXr. Overall, these datasuggest that this antibiotic resistance mechanism, as with otherresistance mechanisms, is probably of importance for survival inthe environment and within microbial communities.

Resultslmo0762 Is an Antibiotic Resistance Gene. In a previous study, weanalyzed the expression profile of all Listeria genes in the pres-ence of subinhibitory concentrations of lincomycin and discov-ered that lmo0762 is induced (ref. 13 and Fig. 1A; data arepublicly available in the European Nucleotide Database, acces-sion no. PRJEB25942). This gene is located downstream of asmall RNA, rli80, and while rli80 appears to be transcribed inboth the absence (black) or the presence (green) of lincomycin,lmo0762 is primarily transcribed in the presence of the antibi-otic. To verify lmo0762 induction by lincomycin at the proteinlevel, we created a strain in which a Flag tag was introduced atthe C terminus of the Lmo0762 protein. We extracted totalproteins from this strain after growth in the absence or presence

of subinhibitory concentration of the antibiotic, and performed aWestern blot using an anti-Flag antibody (Fig. 1B, Left). Theresults confirmed that Lmo0762 levels are significantly increasedin presence of lincomycin. This up-regulation prompted us to in-vestigate whether lmo0762 plays a role in antibiotic resistance.To test this hypothesis, we constructed different strains: a

mutant strain with a deleted rli80-lmo0762 region (Δlmo0762); acomplemented strain, where the rli80-lmo0762 region, under thecontrol of its native promoter, was reintroduced at a differentlocus of the Δlmo0762 strain (Δlmo0762-cpt); and a strain con-stitutively expressing Lmo0762, independently of the presence ofantibiotics, due to a mutation in the upstream regulatory regionthat we describe below (SI Appendix, Fig. S1, const). We con-firmed by qRT-PCR that lmo0762 is induced by lincomycin atsimilar levels in the wild type (WT) and Δlmo0762-cpt strains (SIAppendix, Fig. S2). We then performed a minimum inhibitoryconcentration (MIC) assay on these strains using various anti-biotics (Fig. 1C, Left and SI Appendix, Fig. S3), and we observedthat the Δlmo0762 strain is twofold more sensitive to erythro-mycin than the WT and Δlmo0762-cpt strains, a result consistentwith effects observed with several macrolide resistance deter-minants (23). In contrast, the constitutively overexpressing conststrain is fourfold more resistant to erythromycin than the WT.We confirmed by Western blot that Lmo0762 levels also increasein presence of erythromycin (Fig. 1B, Right), even though in-duction at the transcription level is lower than in presence oflincomycin (Fig. 1D). However, while lmo0762 expression is in-duced by lincomycin exposure, the Δlmo0762 strain did not showincreased sensitivity to lincomycin, and Lmo0762 overexpressionresulted in increased sensitivity to lincomycin, rather than theexpected resistance. Given that we previously identified anothergene, lmo0919, as a lincosamide resistance gene (13), we hy-pothesized that lmo0919 may be masking the effect of thelmo0762 deletion. Thus, we constructed a double mutant strain,Δlmo0919Δlmo0762, that we complemented by reintroducing thelocus rli80-lmo0762 elsewhere in the genome, as previously de-scribed (Δlmo0919Δlmo0762-cpt), and we also introduced themutation that led to lmo0762 constitutive overexpression

Fig. 1. lmo0762 is an antibiotic resistance gene. (A) RNA-seq profile of rli80-lmo0762 locus obtained from L. monocytogenes grown in absence (black) or inpresence of lincomycin (0.5 μg/mL, green). (B) Western blot analysis performed on WT (no Flag) and a strain expressing Lmo0762 with a Flag at the C terminus(Flag). EF-Tu was used as a loading control. (C) Schematic representation of MIC assay. The colors indicate that the tested strain is more sensitive (blue) ormore resistant (red) to the antibiotic compared with the reference (ref) strain. The MIC values are indicated in the boxes after 48-h incubation (in μg/mL). (D) In-duction of lmo0762 and lmo1296 upon antibiotic treatment calculated by qPCR in comparison with the endogenous level before addition of the antibiotic. Data arerepresented as mean ± SEM. We used a one-way ANOVA on ΔCt values for statistics, using biological replicates as pairing factors. *P < 0.05, **P < 0.01; ns, non-significant.

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(Δlmo0919-const). We used these four strains to perform MICassays (Fig. 1C, Right and SI Appendix, Fig. S3), and strikingly,we found that the double mutant is fourfold more sensitive tolincomycin compared with the Δlmo0919 strain, and that re-sistance is restored in the complemented strain. Moreover,overexpression of Lmo0762 (Δlmo0919-const) renders the strainmore resistant to lincomycin than the Δlmo0919 strain. Overall,we conclude that lmo0762 is an antibiotic resistance gene, con-ferring protection against lincomycin and erythromycin.

Lmo0762 Is a Homolog of the Bacterial Ribosome-Splitting ProteinHflX. To explore the mode of action of Lmo0762, we searchedfor homologs in other species using BLASTp and HMM proteinprofiles. The top hits were homologs of HflX in various bacteria,including S. aureus, Bacillus cereus, and E. coli (SI Appendix, TableS1). HflX has been described as a heat-shock stress-responseGTPase that can split and recycle ribosomes that have becomeimmobilized due to heat stress (15, 19, 20). We thus renamedlmo0762 hflXr, for hflX resistance. Surprisingly, we discoveredanother homolog of hflX in L. monocytogenes, lmo1296 (SI Ap-pendix, Table S1). The two proteins encoded by hflXr and lmo1296contain a predicted GTP-binding domain and a 50S ribosome-binding domain (SI Appendix, Table S2), like all other 8,527 ho-mologs of this family that were found within 8,113 genomes.To decipher whether lmo1296 also participates in antibiotic re-

sistance, we first analyzed its induction upon antibiotic exposure.qRT-PCR analysis of RNAs extracted from bacteria grown inpresence or absence of antibiotics showed weak or no induction oflmo1296 upon antibiotic exposure in comparison with lmo0762 (Fig.1D). We then performed MIC assay on a strain lacking lmo1296(Δlmo1296), which showed no increased susceptibility to erythro-mycin, or any other antibiotics tested, compared with WT strain (Fig.2A and SI Appendix, Fig. S3). We also created the hflXr-lmo1296

double deletion strain (Δlmo0762-Δlmo1296), which showed nofurther susceptibility compared with the Δlmo0762 strain. Fi-nally, we reintroduced the lmo1296 gene, under the control ofrli80, in the Δlmo0762 mutant (SI Appendix, Fig. S2A, Δlmo0762-cpt1296). We confirmed by qRT-PCR that in this strain, lmo1296is induced by the antibiotic at a level similar to that of hflXr in theWT or Δlmo0762-cpt strains due to rli80 regulation (SI Appendix,Fig. S2B), and we tested the strain in a MIC assay. We observedthat unlike Δlmo0762-cpt, the Δlmo0762-cpt1296 strain remainssensitive to erythromycin (Fig. 2A and SI Appendix, Fig. S3), thusindicating that lmo1296 is not involved in antibiotic resistance.To unravel the evolutionary history of hflXr and its homologs,

we reconstructed the phylogeny of hflX genes among prokaryotes(Fig. 2B and SI Appendix, Fig. S4). Strikingly, the phylogenetictree shows that lmo1296 and hflXr are well separated in two largeclades, both containing almost only Firmicutes. This duplicationevent is probably old, since it is shared by many bacteria from theclade, and the trees of the two subfamilies closely recapitulatethe tree of Firmicutes (SI Appendix, Fig. S5). Interestingly, otherphyla also harbor a duplication in the hflX genes, e.g., α-, β-, γ-,and δ-proteobacteria and Archaea. These events occurred in-dependently from the one of Firmicutes, highlighting the im-portance of hflX gene duplication. The analysis of 163 pangenomesfrom bacteria revealed that the genes of this family, when present,are in the core genome (98% in >90% of the strains, SI Appendix,Fig. S6). Finally, the analysis of genetic neighborhood of the twosubfamilies showed much higher conservation for lmo1296 than forhflXr (Datasets S1 and S2). Based on these elements, it is temptingto speculate that the ancient duplication of hflX led to two proteinswith specialized functions in Firmicutes, explaining why duplicatescooccur, one of which being involved in antibiotic resistance (hflXr).If correct, this means that other genes of this subfamily may provideantibiotic resistance. Such genes were found in important patho-gens, such as B. cereus, Bacillus anthracis, and Clostridium difficile.

HflXr Recycles Ribosomes upon Antibiotic Exposure.Ribosome stalling isa phenomenon induced by heat shock that results in translationarrest. Some antibiotics can also impair translation, and resistanceproteins that remove the antibiotic from the stalled ribosome havepreviously been described (tetO, tetM, and ABC-F transporters)(24–26). However, a mechanism by which the ribosome is split andrecycled has not been described in the context of antibiotic resistanceand could constitute another class of antibiotic resistance factors.To assess if HflXr promotes antibiotic resistance via ribosome

splitting and recycling, we analyzed the polysome profiles in WTand Δlmo0762 bacteria grown in the presence or absence ofantibiotic. Given that lmo0919 may mask the effect of hflXr inthe presence of lincomycin, we used erythromycin in this ex-periment. The results show a greater quantity of 70S in theΔlmo0762 compared with WT in presence of erythromycin, whilethis difference is not visible in absence of antibiotic (Fig. 3 and SIAppendix, Fig. S7). A comparable difference was previously ob-served in E. coli, in the context of heat shock (19). Such an ac-cumulation of 70S ribosomes in absence of hflXr suggests thatthis gene participates in ribosome recycling, probably by splittingribosomes halted upon antibiotic exposure.

hflXr Transcription Is Regulated by a Ribosome-Dependent AttenuationMechanism. As shown above, hflXr expression is induced by lin-comycin and erythromycin (Fig. 1 A and D). Moreover, hflXr islocated downstream of rli80, a constitutively transcribed smallRNA that we hypothesized to act as a regulatory switch (Fig.1A). Using the term-seq data for L. monocytogenes grown in theabsence of antibiotics (13), we found an accumulation of 3′ readsimmediately downstream of the rli80 riboregulator, suggesting aregulatory mechanism relying on premature termination (4) (Fig.1A, black arrow). A hallmark of such riboregulators is the abilityto display mutually exclusive RNA folding patterns that either

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stabilize or destabilize the intrinsic transcriptional terminator,turning the downstream gene transcription “off” or “on,” respectively(4, 27, 28), in a process named “attenuation.” We thus searchedfor such alternative RNA structures in the rli80 sequence, usingthe PASIFIC algorithm (29). We found that rli80 can indeed foldinto either a terminator structure, that would lead to prematuretermination and to an accumulation of short RNAs, or alter-natively into a structure acting as an anti-terminator, whichprevents the formation of the terminator, allowing the synthesisof full-length hflXr mRNA (Fig. 4A and SI Appendix, Fig. S8). Inaddition, we found that rli80 contains an ORF encoding a 14-aapeptide, which harbors an RLR motif (Fig. 4A), a hallmarksignature found in small ORFs of other macrolide sensingattenuators (SI Appendix, Table S3). The RLR motif controlstranslation arrest by disturbing the transpeptidation step due tothe amino acid geometry (22), thus promoting the expression ofthe downstream macrolide resistance gene (30, 31).Based on this model as well as on the induction by antibiotics,

we hypothesized that hlfXr transcription is controlled either bydirect binding of the antibiotic to the mRNA (riboswitch) or by aribosome-mediated attenuation mechanism due to ribosomalstalling on the rli80 ORF. To discriminate between these twopossibilities, we performed RNA-seq to measure the lincomycin-dependent induction of hflXr in a strain expressing the 23SrRNA methyltransferase ErmC which renders ribosomes in-sensitive to lincomycin (32). The results show that in ErmC-expressing bacteria, hflXr expression was no longer activated inresponse to the antibiotic (SI Appendix, Fig. S9A), suggestingthat the riboregulation depends on stalled ribosomes rather thanby direct binding of the antibiotic to the RNA. We further testedthis hypothesis by validating that the small ORF is translatedin vivo by generating a strain with a translational GFP fusion tothe C terminus of the small peptide (SI Appendix, Fig. S9B), thatshowed fluorescence under the microscope. To validate the at-tenuation mechanism, which involves different regulatory RNAstructures and the rli80 ORF, we mutated the start codon of theORF (ATG > ACG), as well as regions controlling the formationof the terminator, named anti-terminator and anti-anti-terminatorregions (Fig. 4A, brown dashed squares). We analyzed the RNA-seq profile of the rli80-lmo0762 locus in these different mutantstrains, in the absence and presence of lincomycin (Fig. 4B and SIAppendix, Fig. S9C, data available in the ENA database, accessionno. PRJEB25942). In agreement with the above hypothesis, mu-tating the anti-terminator region or the ORF start codon, both of

which are predicted to stabilize the off conformation of rli80,prevented activation of lmo0762 expression during lincomycinexposure and led to lower mRNA abundance in qRT-PCR assay(SI Appendix, Fig. S10). In contrast, mutating the anti-anti-terminator region (i.e., the strain that we previously namedconst), which is predicted to maintain the on conformation of theriboregulator, led to constitutive readthrough and expression ofhflXr, regardless of the presence of lincomycin, as well as en-hanced antibiotic resistance (Fig. 1C and SI Appendix, Figs. S3 andS10). Taken together, these results show that rli80 controls theexpression of hflXr via a ribosome-dependent transcription at-tenuation mechanism, such that HflXr protein expression is in-duced in response to ribosome stalling.

DiscussionIn this work, we describe an antibiotic mechanism of resistanceto lincomycin and erythromycin in L. monocytogenes which ismediated by Lmo0762, an HflX homolog, that we renamedHflXr, for HflX resistance. We showed that deletion of the generenders the bacteria more sensitive to erythromycin and linco-mycin, while its overexpression renders them more resistant. Theinduction of the gene in the presence of antibiotics is mediatedby an attenuation mechanism that involves a small ORF encodinga peptide containing a RLR motif. This tripeptide is a signature ofmacrolide resistance genes, since it is commonly found in smallORFs in leader regions that regulate their expression (SI Ap-pendix, Table S3) (30, 31). Moreover, by analyzing the polysomeprofiles in bacteria grown in the presence or absence of antibiotic,we provided evidence that the proportion of 70S ribosome in-creases upon erythromycin treatment in a strain lacking hflXr,which led us to propose that the mechanism of action of HflXr isto split ribosomes, as for E. coli HflX. Surprisingly, in vitro ex-periments showed that erythromycin and lincomycin can inhibitthe GTPase activity of the E. coli HflX (15). Given that ribosomesplitting was described as a GTP-dependent mechanism (15, 19),

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Fig. 3. Lmo0762 is involved in recycling of antibiotic-stalled 70S ribosomes.Wild-type (blue) and Δlmo0762 (purple) bacteria were grown in BHI mediumuntil exponential phase and erythromycin (0.18 μg/mL) was added or not for1 h. Chloramphenicol was then added to the culture (2-min exposure at 5 mM)to stabilize the polysomes, and bacteria were pelleted and flash frozen. Thecellular content was extracted, and equal amount of lysate (A260) was loadedon a 5–50% sucrose gradient. After ultracentrifugation, the samples werecollected from top (0 mm) to bottom (80 mm) of the tubes and the absor-bance at 260 nm was monitored using a UV lamp. The baseline was correctedand the results were normalized based on the area under the curve.

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Fig. 4. Lmo0762 expression is regulated by a transcription attenuationmechanism. (A) The predicted rli80 RNA structures were analyzed using thePASIFIC algorithm, and two alternative conformations were predicted, onewith an intrinsic terminator (Left) that leads to a short transcript, and onewith an anti-terminator (Right) that leads to a long transcript that encodeslmo0762. Key regulatory regions were identified (anti-anti-terminator inred, anti-terminator in orange, and terminator in green/blue) and a short ORFof 14 aa (purple) is encoded in a region that encompasses the anti-anti-ter-minator region. Different mutants were created where regulatory regionswere removed (dashed brown squares) to decipher the regulatory mecha-nism. (B) RNA-seq profiles of WT and mutant bacteria, obtained as in Fig. 1A.

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further investigations will be required to reconcile these data withour results.Interestingly, L. monocytogenes encodes another hflX homo-

log, lmo1296, which is not involved in antibiotic resistance. ManyFirmicutes possess two copies of hflX, and similar independentduplications are observed in other clades of prokaryotes (Fig.2B). Given that many Firmicutes possess a hflX gene that belongsto the subfamily of hflXr, it seems reasonable to think that theantibiotic resistance mechanism that we described in Listeria forhflXr could be conserved in many other bacteria. This duplica-tion represents an example of how bacteria can employ commonstress response factors as antibiotic resistance genes. In addition,our work is now strengthened by two recently published studieswhere functional metagenomic databases constructed fromantibiotic-rich environments pointed to hflX from Simkanianegevensis and Emergencia timonensis as a putative resistancegene (23, 33). Based on our phylogenetic analysis, hflX fromEubacterium spp., which is closely related to E. timonensis, be-longs to the hflXr family. S. negevensis is not a Firmicute and itshflX belongs to subfamilies which have not been studied forantibiotic resistance. These observations reinforce the conclu-sions of our study and further suggest that this antibiotic re-sistance mechanism is likely spread in the environment. It isinteresting to note that other ribosome rescue mechanismscontribute to basal antibiotic resistance, e.g., tmRNA, whoseinactivation renders E. coli more susceptible to antibiotics, andleads to impaired protein synthesis in presence of antibiotics (34,35). However, a distinction should be made with hflXr, given thatthe tmRNA is a general stress factor, while hflXr duplication,specialization, and transcription regulation favor the idea thatthis gene is dedicated to response to antibiotics. In addition,functional metagenomic proves that hflX from other species thatbelong to the same subfamily as hflXr confer resistance to mac-rolides when transferred to other bacteria, which, to our knowl-edge, is not the case for tmRNA (23, 33). However, it has beenshown that the sigma factor sigR induced by antibiotics in Strep-tomyces coelicolor regulates the transcription of various ribosome-associated genes, and notably tmRNA and HflX (36). Hence,further analysis will be required to determine whether thetranstranslation and HflXr may act in a synergistic manner inribosome recycling, or whether tmRNA may compensate thelack of hflXr in some bacterial species. Finally, the level of re-sistance conferred by hflXr may appear weak in clinical settingsaccording to EUCAST breakpoints, but given that homologs ofhflXr are spread in Firmicutes, this gene is probably of impor-tance in an environmental context and within microbial com-munities, conferring resistance to antibiotics that may be foundin the soil, such as lincomycin and erythromycin.The benefit provided by hflXr in bacteria exposed to linco-

mycin and to erythromycin seems different, since it was necessaryto delete lmo0919, another lincomycin resistance gene, to ob-serve the effect of the hflXr deletion in the presence of this an-tibiotic, while the effect of hflXr could be directly visualized forerythromycin. Lincomycin belongs to the lincosamide antibioticfamily, whose members bind the ribosome at the peptidyl-transfer center and inhibit peptide bond formation. Erythromy-cin is a macrolide antibiotic that also binds at the vicinity of thepeptidyl-transfer center, in the peptide exit tunnel channel,nearby to the lincomycin target site. Both antibiotics preventtranslation at early stages of elongation (32). The remainingstalled ribosomes need to be recycled to start a new round oftranslation. We noted that overexpression of HflXr in theΔlmo0919 strain (Δlmo0919-const) partially restores resistanceto lincomycin (SI Appendix, Fig. S3). These data led us to thinkthat Lmo0919 and HflXr act independently to recycle the ribo-some and restart translation, that Lmo0919 is of primary im-portance for lincomycin resistance, and that HflXr can partiallyrescue lmo0919 deletion. Our careful analysis has shown that the

lmo0919 gene encodes an “ABC-F transporter” (SI Appendix,Fig. S11), correcting our previous assumption that it encodes anantibiotic efflux pump (13). Recent studies have shown thatantibiotic resistance genes annotated as ABC-F transportershave the capability of displacing ribosome-bound antibioticsin vitro (24, 26). Interestingly, Lmo0919 is only produced in thepresence of lincomycin (13) and is not involved in erythromycinresistance (SI Appendix, Fig. S3), while other ABC-F trans-porters such as MsrA confer macrolide resistance (26). Thus, wehypothesize that HflXr could act in concert with Lmo0919 in thepresence of lincomycin: HflXr would split the ribosome, whileLmo0919 would displace the antibiotic, thus recycling the ribo-some to restart translation. In the presence of erythromycin, HflXrcould act either alone or in combination with a protein with mac-rolide displacement activity to rescue stalled ribosome and restarttranslation. This hypothesis is presented in Fig. 5.hflX transcription is regulated by an attenuation mechanism

that relies on the upstream regulatory RNA, rli80, which foldsinto alternative structures and contains a small ORF that harborsthe RLR motif. This attenuation mechanism involves the paus-ing of antibiotic-stalled ribosomes on the ORF, which in turnprevents the formation of the terminator hairpin structure, thuspermitting the transcription the full-length hflXr mRNA. Thisallows the bacteria to fine tune the expression of hflXr in responseto two antibiotics that block translation after incorporation of afew amino acids. As a consequence, the regulation also works as afeed-forward loop, by shutting down the expression of HflXr whenthe antibiotic is cleared. Indeed, in the absence of drug, the ri-bosome does not pause on the regulatory region, and this in turnprevents hflXr transcription. These findings are recapitulated inour model (Fig. 5). Such an attenuation mechanism involvingRNA structures and a small ORF has been found for differentantibiotic resistance genes e.g., the ermC gene (4, 30). It is in-teresting to note that in this latter example, a translation attenu-ation modulates the availability of the ribosome-binding site of theresistance gene, while our regulation mechanism modulates thetranscription of lmo0762.Overall, we described here an antibiotic resistance mechanism

in L. monocytogenes, that uses HflXr to recycle ribosomes in thepresence of antibiotics. The hflXr gene seems widely spread

5’ 3’lmo0762

HflXr

+ lincomycin

HflXr

Lmo0919

splitting

drug

displacement

new round of translation

70S 70S50S

30S

5’ 3’lmo0762

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+ erythromycin

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another

factor

splitting70S 70S

50S

30S

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ribosome stalled drug

displacement

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uORF

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UUUUU

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short transcript

long transcript

long transcript

no HflXr

production

no ribosome stalling

on the ORF

STOP

attenuation

new round of translationstalled ribosomesattenuation

rli805’ 3’

Fig. 5. Model of the combined action of Lmo0762 and Lmo0919 to protectbacteria against lincomycin and erythromycin.

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across species, and it is to be expected that hflXr genes areemployed in other bacterial species for antibiotic resistance inthe environment and within bacterial communities.

Materials and MethodsA detailed materials and methods section can be found in SI Appendix.

RNA-Seq Library Preparation and Analysis. RNA was extracted and DNasetreated and chemically fragmented. Strand-specific RNA-seq libraries wereprepared and sequencing was performed using the Illumina NextSEq 500.Data were deposited in the European Nucleotide Database (ENA) under ac-cession no. PRJEB25942. Sequencing reads were mapped to the NC_003210L. monocytogenes EGD-e RefSeq genome.

Minimum Inhibitory Concentration. Six to eight colonies were resuspended inBHI, diluted at OD600 = 0.001 in 96-well plates, and incubated in presenceof increasing concentrations of antibiotics for 48 h at 37 °C without shaking.The MIC was determined as the lowest concentration that fully inhibitsgrowth.

Polysome Profiling. In bacterial culture, erythromycin (0.18 μg/mL) was addedor not (untreated condition) at OD600 = 0.6 and the growth was pursued for1 h. Chloramphenicol was quickly added to every culture (2 min at 5 mMfinal concentration) to stabilize polysomes, and bacterial pellet was washedand flash frozen. Cellular content were extracted using a FastPrep appara-tus. An equal amount (15–35K OD260) of cell lysates were loaded on sucrosegradient (5–50%), ultracentrifuged, and separated on a Biocomp instru-ment. Absorbance was read at 260 nm at a speed of 0.12 mm/s.

Homology Analysis. A total of 9,078 complete genomes retrieved fromNCBI RefSeq representing 3,226 species of prokaryotes were analyzed. The

composition of Lmo0762 and Lmo1296 in protein domains was analyzedusing PFAM.Weused these domains to search the database of genomes usinghmmsearch.

Phylogenic Trees. First we reduced the redundancy of the dataset usingmmseqs2 to cluster the proteins in clusters of 80% identity and 60% identity.When necessary, we added manually to the databases, the genomes thatcontained at least two copies of the protein to ensure that the phylogeneticreconstruction contained the two copies of each pair of duplicates. We madea phylogenetic reconstruction for each of the two datasets: wemademultiplealignments with MAFFT v7.407, we collected the informative sites in multiplealignment using trimAl v1.2rev59, and made a phylogenetic reconstructionwith IQtree v1.6.7. The dataset_60 reconstruction was used in the studybecause it had higher ultrafast bootstrap results and fewer taxa. However,the key results were common between the two reconstructions.

ACKNOWLEDGMENTS. We thank Stefano Marzi and Pascale Romby foradvice; Claire Poyart, Grégory Boël, and Olivier Dussurget for useful discus-sions; Nora Vazquez-Laslop for critical input into the article; Hugo Varetfrom the C3BI-Hub (Institut Pasteur) for his valuable help on data analysisand statistics; Laurence Maranghi, Julienne Blondou, and Youssouf Boina foressential technical support; and our referees for their comments, which havesignificantly increased the quality of this work. This work was supported, inpart, by grants (to P.C.) [European Research Council (ERC) Advanced GrantBacCellEpi (670823), Agence Nationale de la Recherche Investissementd’Avenir Programme (10-LABX-62-IBEID), the Fondation le Roch les Mous-quetaires]; and, in part, by grants (to R.S.) [Israel Science Foundation per-sonal Grant (1360/16) and I-CORE Grant (1796/12), ERC Grant (ERC-CoG681203), the German Research Council priority program SPP 2002 (GrantSO 1611/1-1), and the Pasteur–Weizmann council]. P.C. is a Senior Interna-tional Research Scholar of the Howard Hughes Medical Institute.

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