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Propionibacterium Freudenreichii Flavia Figueira Aburjaile Mécanismes moléculaires de la survie à long terme chez Propionibacterium freudenreichii Flavia Figueira Aburjaile To cite this version: Flavia Figueira Aburjaile. Mécanismes moléculaires de la survie à long terme chez Propionibac- terium freudenreichii. Alimentation et Nutrition. Agrocampus Ouest, 2015. Français. NNT : 2015NSARB273. tel-01697901v2 HAL Id: tel-01697901 https://tel.archives-ouvertes.fr/tel-01697901v2 Submitted on 1 Feb 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. RÉSUMÉ ABSTRACT Mécanismes moléculaires de la survie à long terme chez Propioni- Molecular mechanisms of long-term survival in Propionibacterium bacterium freudenreichii freudenreichii Propionibacterium freudenreichii est une bactérie très utili- Propionibacterium freudenreichii is a dairy bacterium belonging sée par l’industrie laitière. Elle appartient aux Actinomycètes to the Actinobacteria group, which is known to survive for long connus pour leur survie pendant de longues périodes, dans periods in harsh environmental conditions. In order to investi- des conditions environnementales défavorables. Pour mieux gate the long-term survival phenomenon in P. freudenreichii, comprendre ce phénomène, la caractérisation phénotypique 8 strains were phenotypically characterized for a period of 11 de 8 souches de P. freudenreichii a été réalisée sur 11 jours days in nutrient shortage condition. Bacterial survival rate was dans un milieu en carence nutritionnelle. Le taux de survie assessed by optical density, CFU counting and live-dead cellular bactérienne a été mesuré par densité optique, par énuméra- viability. In addition, the absence of cell lysis was evaluated by tion et évaluation de la viabilité cellulaire. En outre, l’absence quantitative PCR. P. freudenreichii growth phases were classifi ed de lyse cellulaire a été évaluée par PCR quantitative. La crois- as exponential, stationary, late stationary and long-term survival. sance de P. freudenreichii a été décrite en phases exponen- Moreover, it was observed that bacterial viability was maintained tielle, stationnaire, stationnaire tardive et survie à long terme. during long-term survival. Phenotypical characterization indica- Dans nos conditions expérimentales pendant la période de ted that P. freudenreichii CIRM-BIA138 was more resistant to survie à long terme, les bactéries sont restées viables. La nutrient shortage being able to enter into a viable but noncultu- freudenreichii Propionibacterium caractérisation phénotypique a montré que P. freudenreichii rable dormant state. In addition, functional studies of this strain CIRM-BIA138 était la plus résistante à la carence nutrition- were conducted by RNA-Seq on cultures sampled in exponential nelle et entrait dans un état viable mais non-cultivable. Cette and stationary growth phases. Concomitantly, several bioche- souche a été utilisée pour une étude fonctionnelle par RNA- mical analyses were carried out on the culture supernatant. An Seq ainsi que pour des analyses biochimiques sur les surna- integrative approach of metabolomic and transcriptomic data geants de culture, en phases exponentielle et stationnaire. allowed us to infer strategies associated with the survival of this L’association de ces données transcriptomiques et métabolo- bacterium, such as preparation for the dormant state, slow down miques a permis de déduire les stratégies impliquées dans la of metabolic activity and utilization of alternative sources of ener- survie de cette bactérie. La préparation à l’état de dormance, gy, which altogether might allow P. freudenreichiiCIRM-BIA 138 la diminution du métabolisme et l’utilisation de sources alter- to adapt and persist through nutrient shortage for long periods. natives d’énergie semblent impliquées dans l’adaptation et la persistence de P. freudenreichii CIRM-BIA138 en carence nutritionnelle durant de longues périodes. Mots-clés : P. freudenreichii, survie à long terme, RNA-Seq, Keywords: Propionibacterium freudenreichii, long-term survival, métabolome, VBNC. RNA-Seq, metabolomics, viable but nonculturable. Flavia FIGUEIRA ABURJAILE • 9 décembre 2015 Thèse AGROCAMPUS OUEST ÉCOLE DOCTORALE • Vie-Agro-Santé (VAS) sous le label de l’Université Européenne de Bretagnepour pour LABORATOIRE D’ACCUEIL • UMR 1253 INRA - AGROCAMPUS obtenir le titre de OUEST Science et Technologie du Lait et de l’Œuf (STLO) DOCTEUR D’AGROCAMPUS OUEST THÈSE EN COTUTELLE • UFMG, Belo Horizonte, Brasil Spécialité Biochimie, Biologie moléculaire et cellulaire Mécanismes moléculaires de la survie chez à long terme Michel GAUTIER lavia Mécanismes moléculaires Professeur, AGROCAMPUS OUEST, UMR INRA AO STLO / AGROCAMPUS OUEST Institut supérieur des sciences agronomiques, • président agroalimentaires, horticoles et du paysage de la survie à long terme chez 65 rue de Saint-Brieuc – CS84215 – F-35042 Rennes Cedex Muriel COCAIGN-BOUSQUET Tél. : 02 23 48 50 00 Directrice de recherche, INRA Toulouse / rapporteur FIGUEIRA F Propionibacterium freudenreichii www.agrocampus-ouest.fr Artur SILVA • Professeur, université fédérale de Parà, Brésil / rapporteur Marie-France PILET 2015-28 Maître de conférences, ONIRIS, Nantes / examinatrice — Vasco Ariston de CARVALHO AZEVEDO Professeur, UFMG Belo Horizonte, Brésil / directeur de thèse B-273 Yves LE LOIR Directeur de recherche, UMR INRA-AO STLO / directeur de thèse Thèse Thèse Hélène FALENTIN Ingénieure de recherche, UMR INRA-AO STLO / co encadrante INRA 2012 THESE / AGROCAMPUS OUEST Sous le label de l’Université Européenne de Bretagne pour obtenir le diplôme de : DOCTEUR DE L'INSTITUT SUPERIEUR DES SCIENCES AGRONOMIQUES, AGRO-ALIMENTAIRES, HORTICOLES ET DU PAYSAGE Spécialité : Spécialité Biochimie, Biologie moléculaire et cellulaire Ecole Doctorale : « Vie Agro Santé » présentée par : Flavia Figueira Aburjaile Mécanismes moléculaires de la survie à long terme chez Propionibacterium freudenreichii soutenue le 9 décembre 2015 devant la commission d’Examen Composition du jury : Rapporteurs : Muriel Cocaign-Bousquet (Directrice de recherche, INRA, Toulouse, France) Artur Silva (Professeur, UFPA, Belém, Brésil) Membres : Marie-France Pilet (Maître de conférences, ONIRIS, Nantes; Examinatrice) Michel Gautier (Professeur, AGROCAMPUS OUEST, Rennes, France; Président) Directeurs de Thèse : Vasco Ariston de Carvalho Azevedo (Professeur, UFMG, Belo Horizonte, Brésil) Yves Le Loir (Directeur de recherche, INRA, Rennes, France) Co-encadrante de Thèse : Hélène Falentin (Ingénieur de recherche, INRA, Rennes, France) UNIVERSIDADE FEDERAL DE MINAS GERAIS INSTITUTO DE CIÊNCIAS BIOLÓGICAS PROGRAMA INTERUNIDADES DE PÓS-GRADUAÇÃO EM BIOINFORMÁTICA Tese Mecanismos moleculares de sobrevivência em longo prazo em Propionibacterium freudenreichii Orientada: Flavia Figueira Aburjaile Orientadores: Prof. Dr. Vasco Ariston de Carvalho Azevedo Dr. Yves Le Loir Rennes 2015 2 Flavia Figueira Aburjaile Mecanismos moleculares de sobrevivência em longo prazo em Propionibacterium freudenreichii Tese apresentada ao Programa Interunidades de Pós-Graduação em Bioinformática da Universidade Federal de Minas Gerais, como requisito parcial para obtenção do título de Doutor em Bioinformática. Orientadores: Prof. Dr. Vasco Azevedo Dr. Yves Le Loir Rennes 2015 3 “Foi o tempo que dedicaste à tua rosa que a fez tão importante”. « C'est le temps que tu as perdu pour ta rose qui fait ta rose si importante ». Antoine de Saint-Exupéry 4 AGRADECIMENTOS - Agradeço a Deus por ter me dado persistência e determinação durante estes quatro anos de trabalho seguindo em frente com os meus objetivos e não desanimando com as dificuldades; - Ao meu orientador brasileiro, Vasco Azevedo, por todo o aprendizado e ensinamento durante estes anos; - Ao meu orientador francês, Yves Le Loir, pelo recebimento em seu Instituto de Pesquisa; - Aos membros da banca pela disponibilidade em avaliarem este trabalho de Tese; - A Hélène Falentin e aos membros do INRA de Rennes, pela ajuda e disponibilidade; - A Anne Pinto por todo o acolhimento, paciência e ajuda durante a redação da Tese; - Paulette e Jessica, técnicas, pela preparação de todos os meios de cultura e de materiais essenciais utilizados nos experimentos; - À Pós-graduação em Bioinformática, por todo o suporte na parte administrativa do processo desde o início da seleção de doutorado; - As agências de fomento CNPq, CAPES e CAPES-COFECUB pelo auxílio financeiro concedido no Brasil e na França durante este período de Tese; - A todos os “LGCMistas”, pelos aprendizados dentro e fora do laboratório, pelas amizades e pelas conquistas. Não vou descrever nomes, pois vocês todos são muito especiais! - Aos meus colegas e amigos que fiz durante a minha caminhada em Rennes, estrangeiros e brasileiros, aos laços que se criaram e que continuarão, com certeza, nos próximos anos. E a frase que resume toda esta trajetória, está em meu livro favorito, O pequeno príncipe: “Tu te tornas eternamente responsável por aquilo que cativas (Antoine de Saint-Exupéry)”; - Aos meus queridos amigos e amigas de infância, de colégio, de faculdade e de estágio, que me apoiaram em todos
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