Frankfurt (Main), Univ., Diss., 2000. / Computerdatei im Fernzugriff.

Universiẗat, Diss., 2000--Frankfurt (Main).

No description available.

No description available.

Brevibacterium aurantiacum est une actinobactérie qui confère des propriétés organoleptiques clés aux fromages à croûte lavée lors de l’affinage. Malgré son importance industrielle, il n’existe aucun outil génétique disponible pour la modification génétique de cette espèce et seulement deux génomes complets sont disponibles à l’heure actuelle. L’acquisition de connaissances fondamentales sur le répertoire des gènes de cette espèce et sur leurs fonctions est primordiale pour comprendre son rôle dans l’affinage des fromages à croûte lavée. Lors de ce projet de maîtrise, 12 plasmides et quatre vecteurs synthétiques ont été utilisés pour transformer six souches laitières de B. aurantiacum et une souche de B. linens dans le but d’adapter l’outil génétique CRISPR-Cas9 pour ces actinobactéries. Différents protocoles de préparation de cellules électrocompétentes et de transformation par électroporation ont été testés, mais il a été impossible de transformer les souches bactériennes à l’étude. Les actinobactéries du genre Brevibacterium sont donc récalcitrantes à la transformation génétique Dans un second temps, nous avons séquencé six génomes additionnels de Brevibacterium et nous avons effectué des analyses génomiques comparatives avec les génomes publics. Nos analyses phylogénétiques ont révélé que les souches laitières précédemment considérées comme membres de l’espèce B. linens appartiennent en fait à l’espèce B. aurantiacum, mettant en évidence l’importance de cette espèce dans la production fromagère. Les génomes de B. aurantiacum sont composés de 2612 gènes de coeur et possèdent un pangénome ouvert atteignant jusqu’à 6259 gènes. Les génomes étudiés sont riches en éléments d’ADN mobiles et des transferts horizontaux de gènes (HGT) entre diverses actinobactéries d’affinage des fromages ont été observés chez tous les génomes de B. aurantiacum. Nos analyses génomiques comparatives apportent de nouvelles informations sur l’évolution et l’adaptation de B. aurantiacum à l’écosystème des fromages. / Brevibacterium aurantiacum is an orange-pigmented actinobacterium that confers key organoleptic properties to washed-rind cheeses during surface ripening. To date, only two complete and assembled genomes of B. aurantiacum are available and there is currently no genetic tool available to study this industrially relevant species. The acquisition of fundamental knowledge on the gene repertoire of this species and their functions is essential to understand its evolution and its role in cheese ripening In this study, 12 plasmids and 4 synthetic vectors were used to transform 6 B. aurantiacum dairy strains and one B. linens strain in the aim of adapting CRISPR-Cas9 tool for these bacterial species. Different electrocompetent cell preparation and electroporation methods were tested to transform various Brevibacterium strains, but no transformants were recovered with all the experiments. Therefore, it seems that Brevibacterium strains are recalcitrant to genetic transformation We sequenced six additional genomes of Brevibacterium and performed phylogenetic and pan-genome analyses. Our phylogenetic analysis revealed that cheese isolates, previously identified as B. linens, belong to the B. aurantiacum species, making this species a key player in cheese production. B. aurantiacum genomes are composed of 2612 core genes with an open pan-genome reaching now 6259 genes. Horizontal gene transfers (HGT) between cheese actinobacteria were observed in all B. aurantiacum genomes. HGT regions involved in iron acquisition were found in five B. aurantiacum genomes, which suggests cooperative evolution between smear-ripened cheese actinobacteria. Our comparative genomic analysis provides novel insights into the evolution and the adaptation of B. aurantiacum to the cheese ecosystem.

Os biossurfatantes (BS) são produtos de origem microbiana com propriedades tensoativas e emulsificantes. Estes compostos são candidatos para substituir os surfatantes sintéticos em aplicações industriais devido à sua menor toxicidade, maior biodegradabilidade, maior diversidade química e maior eficiência e efetividade em condições físicas extremas de salinidade, pressão e temperatura. O uso comercial e industrial dos BS ainda não é sustentável devido a seu alto custo de produção relacionado principalmente ao baixo rendimento. A utilização de substratos de baixo custo e ferramentas estatísticas para melhorar o rendimento de produção dos BS são duas das principais estratégias para tratar este problema. O objetivo do trabalho foi estudar a produção e recuperação do BS produzido por B. luteolum, visando o melhoramento na sua produção através do uso do planejamento fatorial e caracterizar a estrutura química do BS. A partir dos resultados, determinou-se que a adsorção em resina foi mais efetiva para a recuperação do BS comparada com a precipitação ácida. A produção do BS foi melhorada através de um planejamento fatorial 23 usando como fatores as concentrações da fonte de carbono (vaselina), a fonte de nitrogênio (nitrato de amônio) e a água do mar artificial e como resposta a tensão superficial da solução 0,1% de BS. A maior produção de BS foi obtida com 4% de fonte de carbono, 2% de fonte de nitrogênio e 20% de água do mar artificial gerando tensão superficial de 27 mNm-1. O BS foi caracterizado como uma mistura de lipopeptídeos com ácidos graxos cujo comprimento da cadeia variou entre 10-18 unidades de carbono e um conteúdo de proteína total de 5%. Três estruturas químicas foram sugeridas para os compostos ativos: dois prolina-lipídeos com os ácidos graxos C16:0 e C18:0 respectivamente e um lipopeptídeo com uma sequência peptídica Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) ligada a uma cadeia de ácido graxo C16:0. Não observou-se atividade antimicrobiana contra as cepas de S. aureus, E. coli, S. enteritidis, L. monocytogenes e S. mutans nas faixas de concentrações de BS testadas. O uso de vaselina como substrato para a produção do BS sugere que a bactéria e o BS podem ser explorados para aplicações como a biorremediação e a recuperação melhorada de petróleo (EOR). / Biosurfactants (BS) are microbial-derived molecules showing tensoactive and emulsification properties. These compounds are candidates to replace synthetic surfactants for industrial applications due to their less toxicity, greater biodegradation capacity, greater chemical diversity and greater efficiency and effectiveness under extreme physical conditions of salinity, pressure and temperature. Commercial and industrial use of BS is not sustainable due their high production cost mainly related to low production yields. The use of low cost substrates and statistical tools to enhance the production yield of biosurfactants are two of the main strategies to deal with that problem. The objective of this work was to study the production and recovery of the BS produced by B. luteolum, aiming to enhance its production through a factorial experimental design, and to characterize the chemical structure of the BS. It was found that resin adsorption was more effective than acid precipitation to recover the BS. The production of BS was enhanced through a factorial experimental design 23 using the concentrations of the carbon source (mineral oil), the nitrogen source (ammonium nitrate) and artificial seawater as the factors and the surface tension of a solution 0,1% of BS as the response. The value of factors that enhanced the production of BS were 4% of carbon source, 2% of nitrogen source and 20% of artificial sea water showing a surface tension of 27mNm-1. The BS was characterized as a mix of lipopeptides with fatty acid chains varying between 10-18 carbon units and a total protein content of 5%. Three chemical structures were proposed for the active compounds: two proline-lipids with the fatty acid chains C16:0 e C18:0 respectively and a lipopeptide with a peptide sequence Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) linked to a fatty acid chain C16:0. BS did not show antimicrobial activity against S. aureus, E. coli, S. enteritidis, L. monocytogenes and S. mutans at concentration range tested. The use of mineral oil as a substrate for the production of the BS suggests that the bacteria and the BS can be explore for applications as bioremediation and enhanced oil recovery (EOR).

Os biossurfatantes (BS) são produtos de origem microbiana com propriedades tensoativas e emulsificantes. Estes compostos são candidatos para substituir os surfatantes sintéticos em aplicações industriais devido à sua menor toxicidade, maior biodegradabilidade, maior diversidade química e maior eficiência e efetividade em condições físicas extremas de salinidade, pressão e temperatura. O uso comercial e industrial dos BS ainda não é sustentável devido a seu alto custo de produção relacionado principalmente ao baixo rendimento. A utilização de substratos de baixo custo e ferramentas estatísticas para melhorar o rendimento de produção dos BS são duas das principais estratégias para tratar este problema. O objetivo do trabalho foi estudar a produção e recuperação do BS produzido por B. luteolum, visando o melhoramento na sua produção através do uso do planejamento fatorial e caracterizar a estrutura química do BS. A partir dos resultados, determinou-se que a adsorção em resina foi mais efetiva para a recuperação do BS comparada com a precipitação ácida. A produção do BS foi melhorada através de um planejamento fatorial 23 usando como fatores as concentrações da fonte de carbono (vaselina), a fonte de nitrogênio (nitrato de amônio) e a água do mar artificial e como resposta a tensão superficial da solução 0,1% de BS. A maior produção de BS foi obtida com 4% de fonte de carbono, 2% de fonte de nitrogênio e 20% de água do mar artificial gerando tensão superficial de 27 mNm-1. O BS foi caracterizado como uma mistura de lipopeptídeos com ácidos graxos cujo comprimento da cadeia variou entre 10-18 unidades de carbono e um conteúdo de proteína total de 5%. Três estruturas químicas foram sugeridas para os compostos ativos: dois prolina-lipídeos com os ácidos graxos C16:0 e C18:0 respectivamente e um lipopeptídeo com uma sequência peptídica Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) ligada a uma cadeia de ácido graxo C16:0. Não observou-se atividade antimicrobiana contra as cepas de S. aureus, E. coli, S. enteritidis, L. monocytogenes e S. mutans nas faixas de concentrações de BS testadas. O uso de vaselina como substrato para a produção do BS sugere que a bactéria e o BS podem ser explorados para aplicações como a biorremediação e a recuperação melhorada de petróleo (EOR). / Biosurfactants (BS) are microbial-derived molecules showing tensoactive and emulsification properties. These compounds are candidates to replace synthetic surfactants for industrial applications due to their less toxicity, greater biodegradation capacity, greater chemical diversity and greater efficiency and effectiveness under extreme physical conditions of salinity, pressure and temperature. Commercial and industrial use of BS is not sustainable due their high production cost mainly related to low production yields. The use of low cost substrates and statistical tools to enhance the production yield of biosurfactants are two of the main strategies to deal with that problem. The objective of this work was to study the production and recovery of the BS produced by B. luteolum, aiming to enhance its production through a factorial experimental design, and to characterize the chemical structure of the BS. It was found that resin adsorption was more effective than acid precipitation to recover the BS. The production of BS was enhanced through a factorial experimental design 23 using the concentrations of the carbon source (mineral oil), the nitrogen source (ammonium nitrate) and artificial seawater as the factors and the surface tension of a solution 0,1% of BS as the response. The value of factors that enhanced the production of BS were 4% of carbon source, 2% of nitrogen source and 20% of artificial sea water showing a surface tension of 27mNm-1. The BS was characterized as a mix of lipopeptides with fatty acid chains varying between 10-18 carbon units and a total protein content of 5%. Three chemical structures were proposed for the active compounds: two proline-lipids with the fatty acid chains C16:0 e C18:0 respectively and a lipopeptide with a peptide sequence Phe-Al-X-X-Pro-Pro-Thr (X=Leu/Ile) linked to a fatty acid chain C16:0. BS did not show antimicrobial activity against S. aureus, E. coli, S. enteritidis, L. monocytogenes and S. mutans at concentration range tested. The use of mineral oil as a substrate for the production of the BS suggests that the bacteria and the BS can be explore for applications as bioremediation and enhanced oil recovery (EOR).

Since the catabolism of amino acids in cheese results in the formation of most volatile flavor compounds, a proper intracellular pool of amino acids must be established in order to produce a desirable flavor production in cheese. Generation of this pool of amino acids requires complex interactions among casein and its derivatives, proteolytic enzymes, and transport systems in the associated bacteria, including lactococci. In this project, we hypothesized that casein hydrolysis by the extracellular proteinases of Brevibacterium linens BL2 modulates the expression profile of proteolytic related genes in Lactococcus lactis spp. lactis IL1403. In order to monitor the global gene regulation patterns in L. lactis ssp. lactis IL1403, a high-throughput gene expression tool was needed to study the gene expression profiles on a genomic scale. In this project, we developed a novel oligonucleotide-based filter DNA array protocol for this purpose. The success of this oligonucleotide-based DNA array was dependent on technical innovations including polyI tailing, indirect high density biotin labeling, careful probe design, and integrated computational data analysis. The utility and validity of this protocol were demonstrated by profiling the expression of 375 metabolically related genes in L. lactis ssp. lactis IL1403 during heat, acid, and osmotic stresses. Subsequently the DNA macroarray was used to profile the gene expression changes of L. lactis spp. lactis IL1403 growing in a peptide-limited medium, in a casitone-based peptide-rich medium, and in a casein hydrolyte by B. linens BL2 proteolytic enzymes. L. lactis ssp. lactis IL1403 experienced nitrogen starvation even with an abundance of peptide resources because of lack of expression of peptide transporter genes. Conversely, a peptide pool generated by B. linens BL2 proteolytic activities was sufficient to sustain the growth of L. lactis ssp. lactis IL1403. The repression of the peptide transporter and other peptidase genes of L. lactis ssp. lactis IL1403 was relieved in this medium. Interestingly, the Opt system, a di-tripeptide transporter, was used as a primary peptide transporter, instead of the Opp system whose genes were not actively transcripted in IL1403. We also conducted additional experiments to further describe the protease in B. linens BL2 responsible for the peptide pool generation. This enzyme was secreted as a non-active zymogen and matured into the active protease. Both proteolysis and maturation processes were regulated. Collectively, this work demonstrated that a unique protease of B. linens BL2 generated a pool of pep tides transportable by L. lactis IL1403 and induced changes in gene expression in L. lactis IL1403. Consequently, this body of work demonstrated the hypothesis to be true.

Brevibacterium aurantiacum est l'un des principaux micro-organismes utilisés dans la production de fromages à croûte lavée dans le monde. L’utilisation de cette bactérie est dû à sa richesse métabolique, car elle produit des composés soufrés volatils, des pigments caroténoïdes et des enzymes lipolytiques et protéolytiques, qui sont nécessaires à la maturation d’une variété de fromages. Des souches de cette espèce bactérienne sont inoculées à la surface de fromages au cours de l'affinage et sont sensibles à des infections virales. Les bactériophages (phages), virus qui infectent les bactéries, sont omniprésents dans divers écosystèmes. Dans l'industrie laitière, ils sont reconnus pour perturber les procédés de production lors de l'infection de ferments lactiques, mais leur implication sur des fromages présentant des défauts de couleur et de saveur reste à démontrer. Ces anomalies de maturation de fromages à croûte lavée ont conduit à cette thèse. Le premier objectif de cette thèse de doctorat consistait à analyser le génome de la souche industrielle B. aurantiacum SMQ-1335 et qui est aussi sensible à des phages. Le deuxième objectif de la thèse visait à étudier les phages virulents infectant cette souche. D’ailleurs, cette étude rapporte la première description et caractérisation de phages infectant cette espèce bactérienne. Malgré la similitude entre ces phages, des répétitions en tandem d'ADN ont été identifiées dans des génomes viraux et une analyse approfondie a montré que ces segments d'ADN sont répandus parmi les phages. Le troisième objectif visait à étudier l'interaction phage-hôte via l’analyse du génome de souches mutantes insensibles aux phages. En étudiant ces souches mutantes, des gènes potentiellement nécessaires pour l'infection phagique ont été identifiés. Enfin, le quatrième et dernier objectif visait à évaluer l'impact des phages de B. aurantiacum dans la production de fromages à croûte lavée et ce, à l'aide des caillé modèles. À noter que le reclassement de la souche SMQ-1335, avant identifiée auparavant comme Brevibacterium linens, est décrit en annexe de cette thèse. Malgré des décennies d'études sur les phages laitiers, les phages de B. aurantiacum étaient encore inconnus. Mes travaux auront permis le développement d’un protocole reproductible pour isoler ces phages. Ces travaux ont également apporté de nouvelles connaissances sur les interactions phage-hôte et de leur impact dans les fromages affinés en surface. / Brevibacterium aurantiacum is one of the key players in the production of washed rind cheeses produced worldwide. The importance of this bacterium to the dairy industry is due to its metabolic richness, as it produces volatile sulfur compounds, carotenoid pigments, and lipolytic and proteolytic enzymes, which play roles in the maturation of washed rind cheeses .As strains of this species are regularly inoculated on the cheese surface during ripening, there is a significant risk of viral attacks. Bacteriophage (phages), viruses that infect bacteria, are ubiquitous in the cheese environment. In the dairy industry, virulent phages have long been known to disrupt cheese processes by infecting lactic acid bacteria. The recent observations of color and flavor defects in washed rind cheeses suggested that phages may also infect strains of B. aurantiacum. These observations led to this thesis.The first objective of this PhD dissertation was to study the genomics of B. aurantiacum SMQ-1335, an industrial strain used in the production of washed rind cheeses. The second objective of the thesis was to study the diversity and biology of virulent phages infecting this industrial strain. This study was the first report of phages infecting B. aurantiacum. Despite the low diversity of the isolated B. aurantiacum phages, DNA tandem repeats were found in an intragenic region of the viral genomes and extended analysis showed that these DNA segments are widespread among phages. The third objective was to investigate phage-host interactions through the genome analyses of bacteriophage insensitive mutants, which were selected by challenging SMQ-1335 with phage AGM1. Host genes likely necessary for phage infection were identified and may explain why some of these mutants are phage-resistant. Finally, the fourth objective of this thesis evaluated the impact of virulent phages on the production of washed rind cheeses using model curds. Of note, the reclassification of the strain SMQ-1335, long believed to be Brevibacterium linens, is described in the annex of the thesis. Despite decades of studies on dairy phages, B. aurantiacum phages were still unknown. Here, a reproducible protocol to isolate these phages was developed, which may allow the isolation of new phages. This work also led to increased knowledge on phage-host interactions as well as on and their roles in surface-ripened cheeses.

L’objectif de ce travail était de mieux comprendre les mécanismes moléculaires de l’adaptation microbienne à l’environnement fromager par des approches de génomique fonctionnelle via le modèle de Brevibacterium, un genre bactérien largement utilisé en technologie fromagère, mais dont l’implantation est parfois difficile à maîtriser.L’analyse génomique comparative de 23 souches de Brevibacterium, dont 12 issues de fromages, a révélé des différences en déterminants génétiques impliqués dans la capacité à croître à la surface du fromage. Parmi ces différences, plusieurs sont corrélées à la phylogénie des souches, et d’autres résultent de transferts horizontaux, notamment dans le cas des gènes liés à l’acquisition du fer et à la biosynthèse de bactériocines. Nous avons identifié des îlots génomiques correspondant à des transferts de gènes d’acquisition du fer entre des souches fromagères de Brevibacterium et des bactéries d’affinage appartenant à d’autres genres. Nous avons également mis en évidence un transposon conjugatif codant pour la synthèse de bactériocines présent chez des souches de Brevibacterium d'origine fromagère mais aussi chez une souche fromagère du genre Corynebacterium.L’étude fonctionnelle des interactions biotiques entre Brevibacterium et Hafnia alvei, une autre bactérie d’affinage du fromage, a été menée dans un modèle fromager développé au cours de ce travail. En couplant des analyses microbiologiques, biochimiques et transcriptomiques (RNA-seq), nous avons mis en évidence l’existence de différents mécanismes d’interaction entre ces bactéries. Ceux-ci concernent notamment l’acquisition du fer, la protéolyse, la lipolyse, le métabolisme soufré et le catabolisme du D-galactonate. Nos résultats suggèrent que dans la relation mutualiste observée entre certaines souches de Brevibacterium et H. alvei, cette dernière sécrète des sidérophores qui sont utilisés par Brevibacterium pour capter le fer plus efficacement, stimulant ainsi sa croissance. En contrepartie, Brevibacterium sécrète des lipases et des protéases qui dégradent les caséines et triglycérides du fromage en constituants énergétiques favorisant la croissance de H. alvei. Ce type d’interaction est intéressant à considérer pour la formulation des ferments d'affinage car il en résulte une meilleure capacité de tous les partenaires à coloniser le fromage, et ainsi à générer les propriétés technologiques recherchées. / The objective of this study was to better understand the molecular mechanisms of microbial adaptation to the cheese habitat by functional genomic approaches using Brevibacterium as a model microorganism. This bacterium is widely used for the manufacturing of cheese but its growth on the cheese surface is sometimes difficult to control.Comparative genomic analysis of 23 Brevibacterium strains, including 12 strains isolated from cheeses, revealed differences in genetic determinants involved in the growth on the cheese surface. Some of them are correlated to strain phylogeny and others are the result of gene transfers, especially those involved in iron acquisition and bacteriocin biosynthesis. We identified genomic islands corresponding to transfers of genes involved in iron acquisition between cheese-associated Brevibacterium strains and cheese-associated strains belonging to other genera. We also detected a conjugative transposon encoding bacteriocin production, which is present in cheese-associated Brevibacterium strains as well as in a cheese-associated Corynebacterium strain.Functional study of biotic interactions between Brevibacterium and Hafnia alvei, another cheese-ripening bacterium, was performed in a model cheese developed in this study. By coupling microbial, biochemical and transcriptomic (RNA-seq) analyses, we revealed several interaction mechanisms between these bacteria. These concern, in particular, iron acquisition, proteolysis, lipolysis, sulfur metabolism and D-galactonate catabolism. Our findings suggest that in the mutualistic relationship between some Brevibacterium strains and H. alvei, the latter stimulates Brevibacterium growth by the secretion of siderophores, which can be used by Brevibacterium to capture iron more efficiently. In return, Brevibacterium secretes lipases and proteases, which degrade cheese caseins and triglycerides into energetic substrates that stimulate H. alvei growth. This type of interaction is interesting to consider in the formulation of ripening cultures because it results in a better ability of all partners to colonize the cheese, and thus to generate the desired technological properties.