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E. faecalis est une bactérie à gram positif, responsable d’infections nosocomiales. Dans cette étude, nous avons identifié par RNA-seq 65 ARN régulateurs potentiels induits en conditions de stress et/ou d’infection chez ce pathogène opportuniste. Parmi ceux-ci, trois ARN surexprimés in vivo, en présence de sels biliaires et à pH acide ont fait l’objet d’une étude approfondie.Le premier, SRC65, s’est avéré être un petit ARN (sARN) agissant probablement en trans. Il présenterait des fonctions redondantes avec son homologue SRC90. Différentes cibles potentielles ont été identifiées et des expériences de physiologie ont révélé un rôle de SRC65 dans le déclenchement de la phase exponentielle de croissance.Le 2ème ARN étudié est une région 5’ régulatrice non traduite (5’UTR), appelée 5’1515. Elle formerait un atténuateur et agirait de manière répressive sur le gène ef1515. EF1515 est un antiterminateur de la famille BglG/SacY capable de se fixer sur 5’1515 pour réguler son expression et celle du gène ef1516 localisé en aval et codant un système de transport des sucres de type PTS. L’antiterminateur EF1515 contrôle aussi l’expression du gène ef3023 codant une protéine impliquée dans la virulence d’E. faecalis.Le dernier ARN régulateur étudié est également une 5’UTR. Celle-ci participerait à la régulation d’une hélicase à motif DEAD (CshA) codée en aval de la 5’UTR. Sa caractérisation s’inscrit dans une étude plus large concernant les éléments du métabolisme des ARN, impliquant les ribonucléases et les autres hélicases à motif DEAD d’E. faecalis. CshA aurait un rôle prépondérant pour la bactérie, en étant impliquée dans la réponse au stress, le fitness et la virulence. L’identification de l’interactome de CshA a notamment permis d’identifier l’énolase comme partenaire privilégié. / E. faecalis is a gram-positive bacterium responsible for nosocomial infections. Using RNA-seq, we identified 65 putative regulatory RNA induced under stress and/or during infection. Of these, three RNA overexpressed in vivo, in the presence of bile salts and at acidic pH have were more deeply studied.The first, SRC65, was found to be a small RNA (sRNA) probably acting in trans. It would present redundant functions with its homologous sRNA SRC90. Different potential targets were identified and physiology experiments revealed a role for SRC65 in triggering the exponential growth phase.The 2nd studied RNA is a 5’ untranslated region (5'UTR), called 5'1515 which would form an attenuator and act repressively on the ef1515 gene. EF1515 is an antiterminator of the BglG/SacY family capable of binding at 5'1515 to regulate its expression and that of the downstream gene ef1516 encoding a PTS-type sugar transporter. The EF1515 antiterminator also controls the expression of the ef3023 gene encoding a protein involved in E. faecalis virulence.The last regulatory RNA studied is also a 5'UTR. It would participate in the regulation of a DEAD-box helicase (CshA) encoded downstream of the 5'UTR. Its characterization is part of a broader study of the elements of RNA metabolism, involving ribonucleases and other DEAD-box helicases of E. faecalis. CshA would have a prominent role for the bacteria, being involved in stress response, fitness and virulence. The identification of the CshA interactome made it possible to identify enolase as a preferred partner.

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Lambdoid phages suppress transcription termination to fully express their genes. Antitermination of early gene expression in most lambdoid phages is mediated by an interaction between the N protein and a number of host-encoded factors. Bacteriophage HK022 does not rely on a protein for antitermination. To promote full expression of early phage genes, the transcripts of the HK022 put sites interact directly with RNA polymerase to convert it to a termination resistant form. Bacteriophage HK639 also uses RNA-mediated antitermination. However, it only possesses a single put-like element in its left operon. Because most lambdoid phages, including HK022, have antiterminator elements in each of their early operons, the presence of a single antitermination site in HK639 was unexpected. We have shown that host genes involved in promoting protein-mediated antitermination are not required for HK639 growth. We have also shown that expression of the left operon is essential for lytic growth. Replacement of the left operon promoter, PL, and the putL antitermination sequence prevented HK639 phage release. A similar construct that only replaced putL also prevented phage release. These results suggest that antitermination is required for HK639 excision and/or lytic growth. To distinguish between a defect in phage excision versus a defect in lytic growth, the mutations were crossed onto lytically growing phage. Recombinant phages could not be recovered which suggests a defect in lytic growth is preventing phage release. Additional replacements of left operon sequences suggest that antitermination is not the only requirement for lytic growth. A 2,161bp deletion (HK639 genome coordinates 30,888-33,048) and a 1,736bp deletion (HK639 genome coordinates 29,152- 30,887) downstream of the HK639 putL site also prevented phage release, whereas a 1,746bp deletion (HK639 genome coordinates 29,151-27,406) did not. These results suggest that the deleted HK639 left operon sequences are required for lytic growth. BLAST analysis did not provide insight into the function of the deleted genes. Although the function of many of the HK639 left operon genes is unknown, their importance in phage growth can now be verified by complementation analysis. Our results suggest that HK639 may use a novel mechanism to control the expression of its early genes.

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