The purpose of this paper is to present a comparative study of marine bacteria, molds and actinomycetes in regard to their ability to degrade certain pure and mixed complex compounds possibly occurring in the lagoon waste traps of the Texas Gulf Coast. This comparison was made using a differential oxygen uptake as the index of specific compound utilization.

10 Abstract Actinobacteria are important bacterial group participating in various ecosystem processes particularly in the decomposition of complex organic compounds. Their abilities enable them to surviving in harsh conditions of oligotrophic habitats like lakes, deserts, cave walls or recalcitrant and resistant litter in soil, where Actinobacteria often dominate. Although certain biotic and abiotic factors were recognized to modulate Actinobacteria incidence in such habitats, the influence of anthropogenic pressure on their communities is scarcely known. The main objective of this thesis is therefore to determine differences of Actinobacteria communities under the direct (the human visitors changing microenvironment of caves, part 1) and indirect (climate change factors like altered precipitation or plant litter quality, part 2) anthropogenic influence in two habitats, plant litter in soil and cave walls, where Actinobacteria play important roles and dominate. In a first part of the thesis we monitored Actinobacteria communities in French limestone caves walls differently affected by humans (pristine versus anthropized caves). For identification of important species like potential pathogens or pigments producing Actinobacteria using amplicon sequencing of environmental DNA (Illumina MiSeq), we firstly used...

Há uma simbiose quadripartida no ecossistema das formigas cultivadoras de fungos entre três mutualistas (Formiga da tribo Attini, jardim fúngico e actinomicetos simbiontes) e um parasita (fungo patogênico especializado Escovopsis sp). As actinobactérias associadas à formiga hospedeira produzem metabólitos secundários para inibir este patógeno, mas não o fungo mutualista. Produtos naturais interessantes foram relatados a partir destas bactérias com um amplo espectro de atividades biológicas. Portanto, várias actinobactérias foram isoladas do exoesqueleto e do jardim das formigas agricultoras para isolar compostos ativos contra diferentes alvos como Leishmania donovani e Escovopsis. Os antibióticos e compostos citotóxicos conhecidos griseorhodina A (1), griseorhodina C (2), griseorhodina G (3) e a dinactina (4) foram produzidos em cultivo sólido de ISP-2 por Streptomyces puniceus AB10, que foi isolada da formiga cortadeira Acromyrmex rugosus rugosus. As configurações absolutas de 1 e 2 foram inequivocamente estabelecidas como 6S,6aS,7S,8S e 6R,6aS,7S,8R, respectivamente, usando dicroísmo circular vibracional (VCD) e cálculos da Teoria do Funcional de Densidade (DFT). A bactéria Streptomyces puniceus AB10 produziu em meio-A líquido apenas uma familia de antibióticos como a dinactina (4). O composto 4 mostrou inibição contra Escovopsis e uma atividade maior contra L. donovani em promastigota e amastigota intracelular que a miltefosina. Dois estereoisômeros, strepchazolina A (5) e strepchazolina B (6), os antibióticos streptazolina (7), seu isômero-E (8), e o composto inorgânico octa-enxofre (9) foram produzidos em cultivo sólido de ISP-2 por Streptomyces chartreusis AC70, que foi isolada do jardim fúngico da formiga cortadeira Acromyrmex subterraneus brunneus. O composto 9 mostrou atividade antagonista contra o fungo patogênico especializado Escovopsis sp. Este é o primeiro relato de 8 como produto natural. As configurações absolutas de 5 e 6 foram inequivocamente estabelecida como 5S,6S,9R e 5S,6S,9S, respectivamente, usando dicroísmo circular vibracional (VCD) e cálculos da Teoria do Funcional de Densidade (DFT). A bactéria Candidatus Streptomyces philanthi ICBG292, isolada do exoesqueleto de operária de colônia de formiga Cyphomyrmex, produziu os antibióticos Mer-A2026B (10), piericidina-A1 (11) e nigericina (12). Os compostos 10-12 mostraram atividade contra Escovopsis sp e contra L. donovani. O composto 12 mostrou uma atividade maior contra L. donovani em promastigota e amastigota intracelular que a miltefosina. O composto 10 também foi ativo contra o fungo Trichoderma sp. Streptomyces sioyaensis ICBG311, isolada de machos alados de colônia de formiga Cyphomyrmex, produziu uma nova naftoquinona chamada cyphoquinona (13), dois novos compostos antifúngicos denominados cyphomycina (14) e epoxicyphomycina (15), e o antifúngico conhecido GT-35 (16). Os compostos 14-16 mostraram atividade contra diferentes linhagens de Escovopsis sp e Candida albicans K1 com MIC de 1.0, 0.5 e 0.25 ?g/mL, e uma atividade maior contra L. donovani em promastigota e amastigota intracelular que a miltefosina, enquanto 13 apresentou atividade baixa contra L. donovani. A cyphomycina (14) também mostrou uma potente atividade in vitro contra os patógenos humanos resistentes Aspergillus fumigatus 11628 (resistente à equinocandina), C. glabrata 4720 (resistente ao triazol), e C. auris B11211 (resistente à echinocandina, ao triazol, e à anfotericina B), com MIC de 0.5, 0.5 e 4 ?g/mL, respectivamente. Um estudo de dose única de cyphomycina (14) no modelo de camundongos neutropênicos de candidíase disseminada exibiu uma dose-resposta iv com um log de redução de 0.56 e 0.66 do carga infecciosa quando é tratado com 20 e 40 mg/kg da cyphomycina (14), respectivamente, e epoxicyphomycina (15) exibiu um log de redução de 0.53 com 40 mg/kg, demonstrando relevância clínica e eficácia de 14 e 15 neste modelo padrão da indústria de infecção por Candida. Por outro lado, GT-35 (16) matou os ratos 1 hora após a dose de 40 mg/kg. / There is a quadripartite symbiosis in the fungus-growing ant ecosystem between three mutualist (Attine ant, fungal garden and symbiotic actinomycetes) and one parasite (specialized pathogenic fungus Escovopsis sp). The actinobacteria associated to the ant host produce secondary metabolites to inhibit this pathogen but not the crop fungus. Interesting natural products have been reported from these bacteria with a wide spectrum of biological activities. In this thesis, several actinobacteria were isolated from the exoskeleton and garden of fungus-growing ants to isolate active compounds against different targets such as Leishmania donovani and Escovopsis. The known antibiotic and cytotoxic compounds griseorhodin A (1), griseorhodin C (2), griseorhodin G (3) and dinactin (4) were produced in solid ISP-2 culture by Streptomyces puniceus AB10, which was isolated from the leaf-cutter ant Acromyrmex rugosus rugosus. The absolute configurations of 1 and 2 were unambiguously established as 6S,6aS,7S,8S and 6R,6aS,7S,8R, respectively, using vibrational circular dichroism (VCD) and density functional theory (DFT) calculations. The bacterium Streptomyces puniceus AB10 produced in broth A-medium only one family of antibiotics as dinactin (4). Compound 4 showed inhibition against Escovopsis and a higher activity against L. donovani promastigotes and intracellular amastigotes than miltefosine. Two stereoisomers strepchazolin A (5) and strepchazolin B (6), the antibiotic streptazolin (7), its E-isomer (8), and the inorganic compound cyclooctasulfur (9) were produced in solid ISP-2 culture by Streptomyces chartreusis AC70, which was isolated from the fungal garden of the leaf-cutter ant Acromyrmex subterraneus brunneus. Compound 9 showed antagonist activity against the specialized pathogenic fungus Escovopsis sp. This is the first report of 8 as natural product. The absolute configurations of 5 and 6 were unambiguously established as 5S,6S,9R and 5S,6S,9S, respectively, using vibrational circular dichroism (VCD) and density functional theory (DFT) calculations. The bacterium Candidatus Streptomyces philanthi ICBG292, isolated from the exoskeleton of a worker of a Cyphomyrmex colony, produced the antibiotics Mer-A2026B (10), piericidin-A1 (11) and nigericin (12). Compounds 10-12 showed activity against Escovopsis sp and against L. donovani. Compound 12 showed higher activity against L. donovani promastigotes and intracellular amastigotes than miltefosine. Compound 10 was also active against the fungus Trichoderma sp. Streptomyces sioyaensis ICBG311, isolated from winged male ants of Cyphomyrmex colonies, produced a new naphtoquinone named cyphoquinone (13), two new antifungal compounds named cyphomycin (14) and epoxycyphomycin (15), and the known antifungal GT-35 (16). Compounds 14-16 displayed activity against several strains of Escovopsis sp and Candida albicans K1 with a MIC of 1.0, 0.5 and 0.25 ?g/mL, and a higher activity against L. donovani promastigotes and intracellular amastigotes than miltefosine, while 13 a weak activity against L. donovani. Cyphomycin (14) also showed potent in vitro activity against the resistant human pathogens Aspergillus fumigatus 11628 (echinocandin resistance), C. glabrata 4720 (triazole resistance), and C. auris B11211 (echinocandin, triazole, and amphotericin B resistance), with MIC of 0.5, 0.5 and 4 ?g/mL, respectively. A single-dose study of cyphomycin (14) in a neutropenic mouse disseminated candidiasis model exhibited a dose-like response with 0.56 and 0.66 log reduction of infectious burden when treated with 20 and 40 mg/kg cyphomycin (14), respectively, and epoxycyphomycin (15) exhibited 0.53 log ii reduction with 40 mg/kg, demonstrating clinical relevance and effectiveness of 14 and 15 in this industry-standard model of Candida infection. On the other hand, GT-35 (16) killed the mice 1 hr post dose at 40 mg/kg.

Neste trabalho foi explorado o potencial das actinobactérias como produtoras de agentes antimicrobianos contra bactérias causadoras da mastite bovina. Para este fim, foram otimizados dois diferentes bioensaios um de antagonismo e o outro de antibiose, contra 13 bactérias Gram-positivas e Gram-negativas relacionadas a mastite bovina. Através do bioensaio de antagonismo 120 actinobactérias foram triadas, sendo 55% delas consideradas ativas. Para o ensaio de antibiose foram triados 70 extratos brutos e 60% mostram a formação de halos de inibição. Os extratos brutos Caat 1-54, Caat 2-63 e Caat 8-25 foram considerados os mais promissores dentre todos os extratos estudados. Esses extratos brutos foram submetidos a estudos de desreplicação empregando LC-PAD-MS/MS e isolamento guiado por bioensaio para a identificação de seus metabolitos ativos. Os extratos Caat 1-54 e Caat 2-63 foram ativos principalmente contra as linhagens de bactérias Gram-positivas e a ação antimicrobiana observada foi atribuída a presença da lisolipina I e actinomicina D e V, respectivamente. O extrato Caat 8-25 foi ativo tanto contra bactérias Gram-positivas como para as Gram-negativas, sendo a oxitetraciclina associada a atividade contra as linhagens Gram-positivas e a herbicidina B ativo contra linhagens Gram-negativas. O estudo de desreplicação por LC-MS/MS também possibilitou a identificação de outros metabolitos secundários presentes nos diferentes extratos estudados. Para o extrato Caat 2-63 foi identificada a presença de outros peptídeos provenientes da via biossintética da actinomicina D e, no extrato bruto Caat 8-25 também foi identificado herbicidina F. Um estudo de mudança no metabolismo também foi realizado para a actinobactérias Caat 1-54 produtora de lisolipina. Através da suplementação de íons brometo no meio de cultivo em diferentes períodos da fermentação foi observada a produção de um análogo da lisolipina com bromo e da lisolipina clorada e bromada. Estudos de DESI-TLC-MS associado a bioautografia em TLC também foram aplicados para análise do extrato bruto Caat 2-63 e mostraram a potencialidade desta metodologia para acelerar a identificação de compostos ativos, pois a análise por espectrometria de massas na região da placa cromatográfica correspondente ao halo de inibição revelou a presença de actinomicina D e V, as quais já haviam sido associadas a atividade antimicrobiana deste extrato bruto. Assim os resultados obtidos nesta tese confirmam o grande potencial das actinobactérias como fonte promissoras de compostos bioativos e que a espectrometria de massas empregada nos seus diferentes modos de análise acelera o processo de identificação desses compostos. / In this work were used two different bioassays to evaluate the antimicrobial potential of some actinomycetes against bacteria that cause bovine mastitis. For this propose an antagonism and antibiose assays were optimized against 13 Gram-positive and Gram-negative bacteria related with bovine mastitis. Through antagonism bioassay 120 actinomycetes were screened and 55% were considered active. In disk diffusion bioassay, 70 crude extracts were screened and 60% showed inhibition zones. The crude extracts Caat 1-54, Caat 2-63 and Caat 8-25 were considered the most promising of all the extracts. Dereplication studies and fractionation guided by bioassay were procedure with this extracts to identify the actives metabolites. The extracts Caat 1-54 and Caat 2-63 were active against strains of Gram-positive bacteria and the antimicrobial effect was related to lisolipin I and actinomycin D and V, respectively. The extract Caat 8-25 was active against both Gram-positive and Gram-negative bacteria. Oxytetracycline were associated with activity against Gram-positives strains and a herbicidin B was active against Gram-negative bacteria. Using dereplication strategies by LC-MS/MS others compounds were characterized in different crude extracts. The extract Caat 2-63 was also identified the presence of other peptides from the same biosynthetic pathway of actinomycin D and for crude extract Caat 8-25 were identified herbicidin F. In addition, studies of the shift in secondary metabolism carried out for actinobacteria Caat 1-54 (lysolipin producer). Culture medium supplementation by bromide ions at different stages of fermentation induce the production of bromine lysolipin analog and a chloro-bromine lysolipin. DESI-MS TLC studies associated with TLC bioautography were used to analyze a crude extract Caat 2-63. This study showed how this method could be used to accelerate the identification of active compounds, because mass spectrometry analysis in situ at bioautigraphy inhibition zone showed the presence of actinomycin D and V, which had been associated with antimicrobial activity of this crude extract. Therefore, the results of this thesis confirm the potential of actinobacteria as bioactives compounds source and the importance of mass spectrometry experiments to accelerate the identification of these compounds.

Mes travaux de recherche ont eu pour but d'analyser la diversité des microorganismes des trois domaines du vivant présents dans des biofilms phototrophes exposés à l'air, se développant sur des substrats minéraux divers, afin d'essayer, d'une part, de répondre à des questions de diversité et de biogéographie et, d'autre part, d'étudier le processus de colonisation par le biais d'expériences d'exposition contrôlées.J'ai ainsi caractérisé, essentiellement par des approches moléculaires basées sur l'analyse des banques des gènes d'ARNr de la petite sous-unité (SSU rDNAs) et sur des analyses d'empreintes communautaires, la diversité microbienne (procaryote et eucaryote) formant des biofilms matures (exposés depuis plusieurs années) dans plusieurs sites géographiques en Irlande du Nord, en France et en Ukraine, dans la région de Chernobyl. Dans ces biofilms soumis à forte pression sélective, nous avons mis en évidence beaucoup de microorganismes hétérotrophes et phototrophes, mais avec une diversité relativement restreinte en comparaison à d'autres milieux comme les sols ou les systèmes aquatiques. Les archées étaient absentes. Les conditions environnementales auxquelles ce type de biofilm est constamment exposé comme l'irradiation, la dessiccation et la limitation des nutriments sélectionnent des microorganismes qui développent des stratégies pour s'adapter comme, entre autres, la production de pigments. Ce sont des microorganismes fréquemment retrouvés dans des milieux désertiques extrêmes et résistants aussi aux radiations ionisantes qui ont ainsi été identifiés, notamment des Deinococcales et des Actinobacteria, ou encore des champignons ascomycètes (Ascomycota). Parmi les organismes phototrophes, nous avons dénombré des Cyanobacteria, des algues vertes (Chlorophyta) et des Streptophyta. Nous avons mis en évidence que les facteurs environnementaux influencent la composition des biofilms. Toutefois, tandis que la composition de la communauté bactérienne est fortement dépendante de la nature du substrat ou elle se développe, la composition des communautés microbiennes eucaryotes dépend de la distance géographique. Nous avons également mené des expériences de colonisation en exposant un même substrat minéral dans trois sites géographiques en Irlande du Nord et en France. L'analyse de la diversité microbienne lors du processus de colonisation a révélé des changements importants dans la composition des communautés, que ce soit pour les procaryotes ou pour les eucaryotes avec, cependant, des comportements différents de ces deux groupes de microorganismes. Dans le cas des bactéries, on observe une transition des Gammaproteobacteria, qui dominent les temps 0-6 mois et qui correspondent vraisemblablement aux cellules inactives en dispersion, vers des Betaproteobacteria, Bacteroidetes, Alphaproteobacteria et Actinobacteria dans des phases successives de formation du biofilm. Par contre, dès leur détection sur le substrat minéral, les eucaryotes sont massivement dominés par des champignons ascomycètes et basidiomycètes, des algues vertes ainsi que d'autres composantes minoritaires comme des ciliés, étant détectées dans des stades plus tardifs. Nos résultats montrent que les organismes hétérotrophes sont pionniers dans la formation de ces biofilms, ce qui permet d'émettre l'hypothèse qu'ils facilitent l'installation des cyanobactéries et surtout des algues vertes. Ils montrent aussi que le processus d'assemblage des communautés bactériennes dépend du temps de colonisation, alors que le site géographique détermine celui des microorganismes eucaryotes. Ces différences majeures de comportement pourraient être expliquées par des modes de vie différents entre les organismes de ces deux grands groupes.

Neste trabalho foi explorado o potencial das actinobactérias como produtoras de agentes antimicrobianos contra bactérias causadoras da mastite bovina. Para este fim, foram otimizados dois diferentes bioensaios um de antagonismo e o outro de antibiose, contra 13 bactérias Gram-positivas e Gram-negativas relacionadas a mastite bovina. Através do bioensaio de antagonismo 120 actinobactérias foram triadas, sendo 55% delas consideradas ativas. Para o ensaio de antibiose foram triados 70 extratos brutos e 60% mostram a formação de halos de inibição. Os extratos brutos Caat 1-54, Caat 2-63 e Caat 8-25 foram considerados os mais promissores dentre todos os extratos estudados. Esses extratos brutos foram submetidos a estudos de desreplicação empregando LC-PAD-MS/MS e isolamento guiado por bioensaio para a identificação de seus metabolitos ativos. Os extratos Caat 1-54 e Caat 2-63 foram ativos principalmente contra as linhagens de bactérias Gram-positivas e a ação antimicrobiana observada foi atribuída a presença da lisolipina I e actinomicina D e V, respectivamente. O extrato Caat 8-25 foi ativo tanto contra bactérias Gram-positivas como para as Gram-negativas, sendo a oxitetraciclina associada a atividade contra as linhagens Gram-positivas e a herbicidina B ativo contra linhagens Gram-negativas. O estudo de desreplicação por LC-MS/MS também possibilitou a identificação de outros metabolitos secundários presentes nos diferentes extratos estudados. Para o extrato Caat 2-63 foi identificada a presença de outros peptídeos provenientes da via biossintética da actinomicina D e, no extrato bruto Caat 8-25 também foi identificado herbicidina F. Um estudo de mudança no metabolismo também foi realizado para a actinobactérias Caat 1-54 produtora de lisolipina. Através da suplementação de íons brometo no meio de cultivo em diferentes períodos da fermentação foi observada a produção de um análogo da lisolipina com bromo e da lisolipina clorada e bromada. Estudos de DESI-TLC-MS associado a bioautografia em TLC também foram aplicados para análise do extrato bruto Caat 2-63 e mostraram a potencialidade desta metodologia para acelerar a identificação de compostos ativos, pois a análise por espectrometria de massas na região da placa cromatográfica correspondente ao halo de inibição revelou a presença de actinomicina D e V, as quais já haviam sido associadas a atividade antimicrobiana deste extrato bruto. Assim os resultados obtidos nesta tese confirmam o grande potencial das actinobactérias como fonte promissoras de compostos bioativos e que a espectrometria de massas empregada nos seus diferentes modos de análise acelera o processo de identificação desses compostos. / In this work were used two different bioassays to evaluate the antimicrobial potential of some actinomycetes against bacteria that cause bovine mastitis. For this propose an antagonism and antibiose assays were optimized against 13 Gram-positive and Gram-negative bacteria related with bovine mastitis. Through antagonism bioassay 120 actinomycetes were screened and 55% were considered active. In disk diffusion bioassay, 70 crude extracts were screened and 60% showed inhibition zones. The crude extracts Caat 1-54, Caat 2-63 and Caat 8-25 were considered the most promising of all the extracts. Dereplication studies and fractionation guided by bioassay were procedure with this extracts to identify the actives metabolites. The extracts Caat 1-54 and Caat 2-63 were active against strains of Gram-positive bacteria and the antimicrobial effect was related to lisolipin I and actinomycin D and V, respectively. The extract Caat 8-25 was active against both Gram-positive and Gram-negative bacteria. Oxytetracycline were associated with activity against Gram-positives strains and a herbicidin B was active against Gram-negative bacteria. Using dereplication strategies by LC-MS/MS others compounds were characterized in different crude extracts. The extract Caat 2-63 was also identified the presence of other peptides from the same biosynthetic pathway of actinomycin D and for crude extract Caat 8-25 were identified herbicidin F. In addition, studies of the shift in secondary metabolism carried out for actinobacteria Caat 1-54 (lysolipin producer). Culture medium supplementation by bromide ions at different stages of fermentation induce the production of bromine lysolipin analog and a chloro-bromine lysolipin. DESI-MS TLC studies associated with TLC bioautography were used to analyze a crude extract Caat 2-63. This study showed how this method could be used to accelerate the identification of active compounds, because mass spectrometry analysis in situ at bioautigraphy inhibition zone showed the presence of actinomycin D and V, which had been associated with antimicrobial activity of this crude extract. Therefore, the results of this thesis confirm the potential of actinobacteria as bioactives compounds source and the importance of mass spectrometry experiments to accelerate the identification of these compounds.

Les patrons de distribution chez les micro-organismes reposeraient sur leurs capacités à disperser dans le temps et dans l'espace, en lien avec des facteurs abiotiques comme les propriétés du sol, le climat, et des interactions biotiques, notamment avec l'hôte dans le cas des symbiontes, mais aussi sur les traits d'histoire de vie propres aux micro-organismes, telle que la capacité à sporuler. Frankia sp. est une actinobactérie sporulante et fixatrice d'azote à la biogéographie complexe, car vivant à la fois de façon saprophytique dans le sol, en symbiose racinaire (nodosité) avec les plantes actinorhiziennes dont les aulnes (Alnus, Betulaceae). Deux types de souches de Frankia génétiquement différentes ont été décrites dont la distinction phénotypique majeure réside dans la capacité à maintenir (Sp+) ou non (Sp-) leur sporulation in planta. Cette sporulation endophytique est à notre connaissance unique dans un contexte symbiotique et son implication dans la biogéographie de Frankia, reste peu connue. Ces travaux de thèse intègrent à la fois des approches descriptives et expérimentales, sur le terrain et au laboratoire, afin d'accroître la compréhension du rôle écologique de la sporulation in planta de Frankia. Dans un premier temps, nous avons étendu la description de la phylobiogéographie des souches de Frankia Sp+ afin de tester la validité du patron de distribution centré sur les milieux froids des zones de haute altitude et de haute latitude de l'hémisphère nord. Un intérêt tout particulier a été porté sur les aires géographiques où une plus forte diversité de Frankia était attendue, dans la zone d'origine de l'aulne et ses refuges glaciaires. Dans un second temps, nous avons étudié l'influence du partenaire végétal dans la distribution observée des Frankia Sp+ et l'implication du trait Sp+ dans la capacité d'association à l'hôte. Des croisements expérimentaux ont été réalisés au laboratoire afin de découpler les effets de l'espèce-hôte et du climat, et tester les implications du trait Sp+ en termes d'infectivité, compétitivité et spectre d'hôte. Enfin, nous avons étudié les conséquences écosystémiques de l'expansion subalpine du complexe symbiotique Alnus/Frankia, au niveau de la diversité microbienne et du fonctionnement du cycle de l'azote, en fonction du phénotype de sporulation des souches associées. Des analyses pédologiques, en association avec des mesures de nitrification, dénitrification et fixation d'azote, ainsi que des analyses de diversité microbienne (globale et fonctionnelle), ont été réalisées dans différentes aulnaies Sp+, Sp- ou mixte, à différents stades de colonisation de l'aulne. Les résultats obtenus démontrent une prédominance des souches Sp+ associées aux espèces d'aulne des milieux froids sur les 3 continents de la zone Holarctique, avec une diversité nouvelle dans l'aire d'origine et les zones refuges de l'aulne. Les croisements effectués révèlent une infectivité et compétitivité plus forte des Sp+ par rapport aux Sp-. De plus, contrairement aux Sp- à spectre d'hôte très large, les Sp+ présentent un spectre limité entraînant des incompatibilités d'association suggérant une dépendance forte à une espèce-hôte donnée. Les modifications des communautés microbiennes du sol en réponse à l'expansion du complexe symbiotique Alnus/Frankia ont été démontrées, en lien avec la stimulation du cycle de l'azote dans les milieux sub-/alpins. Les premiers résultats sur l'efficience comparée de la fixation d'azote in natura des souches Sp- par rapport aux Sp+ suggèrent que 100% de l'azote de l'aulne est obtenu par le biais de la fixation. Aucun patron n'est mis en évidence entre souches Sp+ et Sp-, suggérant un effet plus complexe de la saisonnalité, de l'âge de l'arbre et de celui de la nodosité. Les résultats obtenus nous permettent de mieux appréhender les facteurs guidant la biogéographie de Frankia et de discuter de l'évolution de ces patrons de distribution en réponse au réchauffement climatique / Microbial biogeography would be based on the ability of microorganisms to disperse across time and space, as a function of abiotic factors such as soil properties, climate, and of biotic interactions, in particular with the host in the case of symbionts, but also on life history traits such as the ability to sporulate. Frankia sp. is a spore-forming and nitrogen-fixing actinobacterium that has a complex biogeography given its abilities for both saprophytic life and root symbiotic interaction with actinorhizal plants such as alders (Alnus, Betulaceae). Two distinct groups of Frankia lineages have been described according to a major phenotypic divergence, based on the presence (Sp+) or the absence (Sp-) of spores in planta.. To the best of our knowledge, this endophytic sporulation is an original trait in a symbiotic context and very little is known about its incidence in Frankia biogeography. This work integrates descriptive and experimental approaches on both field and laboratory areas, in order to improve the understanding of the ecological role of Frankia in planta sporulation. First, we have extended the description of the phylobiogeography of Sp+ Frankia strains to validate the previously proposed distribution pattern focused on cold environements at high altitude or high latitude. A phylogeny has been computed using a large number of nodular strains coming from the 3 continents of the Northern Hemisphere and 10 different Alnus species. Special attention was paid to geographic areas where a higher diversity was expected, in Asia, and in its glacial refuges. Second, we studied the influence of the host-plant on the distribution of Fankia Sp+ and the incidence of Sp+ in the symbiotic interaction. Experimental crosses have been performed to disentangle host and climate effects and to test the incidence of the Sp+ trait in terms of infectivity, competitiveness and host-range. Finally, we studied the ecological consequences of the Alnus/Frankia symbiotic complex, on the microbial diversity and on the nitrogen cycle functionning, with respect to the sporulation of Frankia and to the Alnus expansion on sub-/alpine grasslands. Soils analyses were performed in association with measures of nitrification and denitrification, as well as global and functional microbial diversity analyses, in Sp+, Sp- or mixed alder stands and at different colonization stages. In each part of this work, alder ectomycorhizae were analyzed to compare the distribution pattern between the two symbionts and to highlight potential interactions with the Sp+ trait of Frankia. Our results show the dominance of Sp+ strains in nodules of alder species from cold environments over the 3 continents of the Holarctic zone, with original diversity patterns in alder area of origin and in glacial refuges. Even if these strains are genetically homogenous, host-specific clusters were observed in the phylogeny. Crosses revealed that Sp+ strains were more infective and competitive than Sp- strains. Moreover, unlike Sp- strains that harbor a wide host-range, Sp+ strains have a narrower specificity leading to association’s incompatibilities and suggesting strong host dependence. For the first time, modifications of microbial communities were revealed in response to the Alnus-Frankia symbiotic complex colonization and were linked to a stimulation of the nitrogen cycle in the sub-/alpine grasslands. The first comparative results of nitrogen fixation between Sp+ and Sp- strains in natura suggest a maximal efficiency of fixation, representing almost 100% of the alder nitrogen. However, unlike previous reports in literature, no pattern was observed between Sp+ and Sp- strains, suggesting a complex effect of seasonality, alder age as well as that of nodules. Altogether, the previous results contribute to a better understanding of the Frankia biogeography drivers and allow us to discuss the expected evolution of distribution pattern in response to the global warming

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...

Indiana University-Purdue University Indianapolis (IUPUI) / Eagle Creek Reservoir (ECR), located in the Midwestern U.S., is a freshwater limnic system plagued by seasonal Harmful Algal Blooms (HABs) which generate water-fouling Geosmin (GSM) and 2-Methylisoborneol (MIB) Taste and Odor (T&O) compounds. Past investigations of T&O event dynamics have identified Actinomycetes as responsible for MIB production and several genera of cyanobacteria for GSM production. During 2018, a temporally and spatially expansive sampling regimen of the reservoir was carried out and a battery of biological, chemical, physical, and hyperspectral experiments performed. The resulting data was analyzed using time series, cross-correlation, lag time, and multivariate analyses as well as machine learning algorithms to pick apart and interrogate any relationships between HABs, T&O events, and environmental parameters. The results show that local weather and watershed conditions exert significant control over the state of the reservoir and the behavior of the algal community. GSM and MIB peaked during early May under well-mixed, cold, and nutrient-rich water column conditions, then declined under summer thermal stratification before making a small resurgence during late season mixing. Bloom die-off and decay was effectively ruled out as a mechanism controlling T&O concentrations, and no links were found between T&O concentrations and algal biomass. Strong evidence was found that GSM/MIB concentrations were a response by bloom microbes to changing nutrient conditions within the reservoir, and it was determined that nutrient fluxes from the watershed 30-40 days prior to peak T&O concentrations are likely instrumental in the development of the slow- ix growing microbes characteristic of the reservoir. Attempts were made to assess spatial and temporal variability but no significant spatial differences were identified; differences between sampling sites were far smaller than differences between different sampling dates. The findings here add to the growing body of literature showing T&O and HAB dynamics are more closely linked to the relative abundance and speciation of nutrients than other parameters. Additionally, these findings carry important implications for the management of ECR and other similar freshwater reservoirs while highlighting the importance of reducing watershed eutrophication.

Metagenomics has revealed that the marine microbial biosphere is immensely more diverse than originally considered, and is an almost untapped reservoir for the potential discovery of microbial natural products. Despite numerous advances in culturing, biosynthetic engineering and genomic-based screening efforts to uncover much of this diversity in relatively accessible environments, a high rediscovery rate has resulted in the investigation of unique, relatively unexplored ecosystems harboring phylogenetically diverse communities of marine organisms. The focus of this research was to establish a culture repository of microorganisms collected from the Red Sea and from deep-sea hydrothermal vents, and to assess their biosynthetic potential for the production of new chemical scaffolds. Cultivation of marine cyanobacteria from the Red Sea has led to the identification of five new cyclic depsipeptides, apratoxin H, grassypeptolides D and E, Ibu-epidemethoxylyngbyastin 3 and leptochelin, the latter possessing a unique chemical scaffold capable of binding metals. A collection of deep-sea hydrothermal vent sediment and microbial mat samples led to the isolation of 64 unique bacterial strains, with eight assigned as members of the order Actinomycetales. Importantly, these isolates, along with a collection of deep-vent invertebrates and microbes, have led to the development of methods for the collection, culturing and biological screening of organisms from this extreme environment for future natural products research. / Graduation date: 2013