Silencing of natural transformation by an RNA chaperone and a multitarget small RNA Laetitia Attaiecha,b,c,d,e,f,g, Aïda Boughammouraa,b, Céline Brochier-Armaneth, Omran Allatifc,d,e,f,g, Flora Peillard-Fiorentea,b, Ross A. Edwardsi, Ayat R. Omari, Andrew M. MacMillani, Mark Gloveri, and Xavier Charpentiera,b,c,d,e,f,g,1 aCNRS UMR5240, Microbiologie Adaptation et Pathogénie, 69622 Villeurbanne, France; bUniversité Claude Bernard Lyon 1, 69100 Villeurbanne, France; cInternational Center for Infectiology Research (CIRI), International Center for Infectiology Research, Université de Lyon, 69364 Lyon, France; dINSERM U1111, 69364 Lyon, France; eEcole Normale Supérieure de Lyon, 69364 Lyon, France; fUniversité Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, 69364 Lyon, France; gCNRS UMR5308, 69007 Lyon, France; hLaboratoire de Biométrie et Biologie Evolutive, CNRS UMR5558, Université de Lyon, Université Lyon 1, F-69622 Villeurbanne, France; and iDepartment of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada Edited by Ralph R. Isberg, Howard Hughes Medical Institute/Tufts University School of Medicine, Boston, MA, and approved May 26, 2016 (received for review January 29, 2016) A highly conserved DNA uptake system allows many bacteria to The concerted expression of the DNA uptake system during actively import and integrate exogenous DNA. This process, called competence invariably relies on transcriptional activation (1). In natural transformation, represents a major mechanism of horizon- Gram-positive bacteria, it results from the action of a transcriptional tal gene transfer (HGT) involved in the acquisition of virulence and activator (e.g., ComK in Bacillus subtilis)orsigmafactors(e.g.,σX in antibiotic resistance determinants. Despite evidence of HGT and Streptococcus pneumoniae, σH in Staphylococcus aureus). In the the high level of conservation of the genes coding the DNA uptake Gram-negative Haemophilus influenzae and Vibrio cholerae,it system, most bacterial species appear non-transformable under involves the transcription activator TfoX/Sxy. However, a number of laboratory conditions. In naturally transformable species, the DNA species that are naturally transformable lack these known compe- uptake system is only expressed when bacteria enter a physiological tence activators. One such species is the Gram-negative pathogen state called competence, which develops under specific conditions. Legionella pneumophila, which was found to develop competence Here, we investigated the mechanism that controls expression under microaerophilic growth, exposure to DNA-damaging agents, of the DNA uptake system in the human pathogen Legionella or suboptimal growth temperature (11–13). Possible competence pneumophila. We found that a repressor of this system displays regulatory elements in L. pneumophila were revealed by a genetic a conserved ProQ/FinO domain and interacts with a newly charac- screen that identified a gene annotated “proQ, activator of osmo- terized trans-acting sRNA, RocR. Together, they target mRNAs of the ” genes coding the DNA uptake system to control natural transforma- protectant ProP as a repressor of natural transformation (14). The tion. This RNA-based silencing represents a previously unknown reg- proQ gene was initially identified in Escherichia coli as an activator ulatory means to control this major mechanism of HGT. Importantly, of the proline transporter ProP (15), but recent evidence suggests these findings also show that chromosome-encoded ProQ/FinO that this effect is indirect (16). More significantly, ProQ bears the domain-containing proteins can assist trans-actingsRNAsandthatthis conserved PFAM domain 04352 (named the ProQ/FinO domain), class of RNA chaperones could play key roles in post-transcriptional which has been functionally and structurally studied in the F plas- gene regulation throughout bacterial species. mid-encoded FinO protein (17, 18). FinO is an RNA chaperone that down-regulates the conjugative transfer of IncF plasmids natural transformation | RNA chaperone | non-coding RNA | Legionella pneumophila | ProQ/FinO Significance atural transformation is a common mode of horizontal gene Natural transformation is a major mechanism of horizontal gene Ntransfer in bacteria. It results from the intrinsic capacity of transfer (HGT) by which bacteria take up exogenous DNA di- bacteria to import exogenous DNA and integrate it by recombination rectly in their environment and integrate it in their genome. in their chromosome (1). Active acquisition of random parts of the Acquiring new genetic information may confer an adaptive ad- genetic material released by phylogenetically close organisms pro- vantage but an uncontrolled uptake of foreign DNA may be duces genetic polymorphism and functions as “localized sex” in ref- harmful. We document a previously unsuspected means to erence to the function of sex in eukaryotic organisms (2). In addition, control HGT by natural transformation in the human pathogen evidence suggests that genetic transformation can also occur with Legionella pneumophila. We found that the DNA uptake system distantly related and even damaged DNA (3, 4). Depending on the required for natural transformation is subjected to silencing. A bacteria’s biotope, the constant and random acquisition of ge- member of the widespread ProQ/FinO domain-containing pro- netic material could be as harmful as it is beneficial, and in most tein family acts as an RNA chaperone and allows the targeting of transformable species, natural transformation is a strictly regu- the mRNAs of the genes coding the DNA uptake system by a lated process (1, 5). Natural transformation only occurs when newly identified trans-acting small RNA. bacteria enter a specialized physiological state, called compe- tence, during which a DNA uptake system is set up (6). This Author contributions: L.A., A.B., C.B.-A., A.M.M., M.G., and X.C. designed research; L.A., A.B., C.B.-A., F.P.-F., R.A.E., A.R.O., and X.C. performed research; O.A. contributed new system generally involves a type IV pilus exposed at the cell reagents/analytic tools; O.A. performed statistical analyses; L.A., A.B., C.B.-A., R.A.E., A.R.O., MICROBIOLOGY surface and a transporter associated to the cytoplasmic mem- A.M.M., M.G., and X.C. analyzed data; and L.A., C.B.-A., M.G., and X.C. wrote the paper. brane (1, 6). The type IV pilus is thought to initially interact with The authors declare no conflict of interest. DNA and convey it to the small ComEA protein that binds This article is a PNAS Direct Submission. double-strand DNA (dsDNA) (7). The captured dsDNA is then Data deposition: RNA-seq data were deposited to the MINSEQE-compliant public ArrayExpress converted into single-strand DNA (ssDNA) and transported database, https://www.ebi.ac.uk/arrayexpress (accession no. E-MTAB-4094) and brokered to the across the cytoplasmic membrane through a transmembrane European Nucleotide Archive, www.ebi.ac.uk (accession no. ERP013398). channel formed by ComEC (8). The ssDNA entering the cyto- 1To whom correspondence should be addressed. Email: [email protected]. plasm is rapidly loaded with ssDNA binding proteins SsbB and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DrpA and can recombine with the chromosome (9, 10). 1073/pnas.1601626113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1601626113 PNAS | August 2, 2016 | vol. 113 | no. 31 | 8813–8818 Downloaded by guest on September 30, 2021 between Enterobacteriaceae by controlling the function of an anti- reads mapping on lppnc0692 correspond to a 66-nt sequence be- sense RNA, FinP (19). FinO facilitates the interaction between ginning at the previously mapped transcription start site (26) and FinP and the complementary 5′ untranslated region (UTR) of the terminating at a predicted Rho-independent transcription termi- mRNA of traJ (20, 21). Sense–antisense pairing prevents translation nator (Fig. S2A). The RIP-seq data suggest that Lpp0148 nearly of the TraJ transcriptional activator of the plasmid tra operon and exclusively binds in vivo a 66-nt sRNA expressed from the lppnc0692 thereby inhibits plasmid conjugation. Although finO is restricted to feature (Fig. S2A). Interestingly, this enriched 66-nt sequence is IncF plasmids, genome-encoded proteins with a ProQ/FinO do- present in all sequenced Legionella species with a sequence identity main have been identified, but their function remains elusive of over 95%, indicating a functional constraint (Fig. S3). Indeed, (19). As the L. pneumophila ProQ represses natural transformability RNA fold predicts that this putative sRNA adopts a stable sec- (14) and to avoid any unfounded inference with proline metabolism, ondary structure with two strong stem-loop structures (SL1 and SL3) we will refer to it by its locus tag name in the Paris strain, Lpp0148. and a weak one (SL2) (Fig. 1A). Northern blot analysis confirmed We characterize here Lpp0148 and describe its biological the expression of this 66-nt sRNA in the wild-type strain (Fig. 1B). function in interaction with the first trans-acting sRNA-negative Importantly, deletion of the coding region for this sRNA resulted in regulator of competence. We document an entirely novel mech- anism of competence regulation by post-transcriptional silencing, an increase in comEA expression similar to that observed in the diverging from the current notion that competence strictly de- lpp0148TAA mutant (Fig. 1B) as well as a dramatically