Dissertations / Theses on the topic 'Actinomycetales'

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Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde
Casos de infecções invasivas por corinebactérias, colonizadoras do ambiente e da microbiota humana ou de animais, tem sido crescentes em decorrência de melhor sobrevivência de indivíduos imunocomprometidos. Além da fenotipagem, métodos moleculares tem sido fundamentais na identificação de bastonetes Gram positivos irregulares(BGPI). O presente estudo teve como objetivo a caracterização fenotípica e genotípica de estirpes de corinebactérias isoladas de sítios intravenosos de pacientes internados em um hospital universitário no Rio de Janeiro. Neste sentido, 60 estirpes de microrganismos Gram positivos foram analisadas por metodologia enotípica convencional, sistemas API Coryne e Vitek 2 (bioMérieux) e análise das sequências dos genes 16S rRNA e rpoB, utilizada como metodologia de referência para avaliação dos sistemas fenotípicos. Os dados indicaram o isolamento de Corynebacterium striatum (44,68 %), Corynebacterium amycolatum (31,91 %), Corynebacterium jeikeium (8,51 %), Corynebacterium urealyticum (6,39 %), Corynebacterium diphtheriae (4,26%), Corynebacterium simulans (2,12%) e Corynebacterium minutissimum (2,12%) do sangue de pacientes fazendo o uso ou não de dispositivos invasivos. As espécies predominantes C. striatum e C. amycolatum apresentaram 8 e 9 perfis de resistência aos agentes antimicrobianos, respectivamente. O perfil de resistência com sensibilidade apenas à tetraciclina, linezolida e vancomicina, foi prevalente durante um surto epidêmico de C. striatum ocorrido em 2010. Este mesmo perfil foi observado para C. amycolatum, C. jeikeium e C. urealyticum. A identificação definitiva da maioria das estirpes de C. striatum, C. amycolatum, C. jeikeium, C. simulans e C. minutissimum só foi possível pela genotipagem. Interessantemente, a análise das sequências do gene 16S rRNA permitiu a identificação de outros microrganismos como Abiotrophia defectiva (6,67%), Arthrobacter (1,67%), Brevibacterium (11,67%) e Microbacterium (1,67%).
Cases of invasive infections corynebacteria , colonizing the environment and human and animal microbiota, has been increasing due to better survival of immunocompromised individuals. Besides phenotyping, molecular methods have been of great value in the identification of Gram positive irregular rods (BGPI). The present study aimed to phenotypic and genotypic characterization of isolates from corynebacteria isolated of intravenous sites of patients at a university hospital in Rio de Janeiro. Thus, 60 isolates of Gram positive microrganisms were analyzed by conventional phenotype methodology, and Vitek and API Coryne 2 systems (bioMérieux) and by sequence analysis of 16S rRNA and rpoB genes. The genotypic identification was used as reference methods for evaluation of phenotypic systems. The data indicated the isolation of Corynebacterium striatum (44,68 %), Corynebacterium amycolatum (31,91 %), Corynebacterium jeikeium (8,51%), Corynebacterium urealyticum (6,39 %), Corynebacterium diphtheriae (4,26%), Corynebacterium simulans (2.12%) and Corynebacterium minutissimum (2,12%) from the blood of patients making use or not of invasive devices. The predominant species C. striatum and C. amycolatum presented 8 and 9 profiles of resistance to antimicrobial agents, respectively. The resistance profile with sensitivity just to tetracycline, linezolid and vancomycin, was prevalent during an outbreak of C. striatum occurred in 2010. The same profile was observed for C amycolatum, C. jeikeium and C. urealyticum. The definitive identification of most strains of C. striatum, C. amycolatum, C. jeikeium, C. simulans and C. minutissimum was possible only by genotyping . Interestingly, the sequence analysis of the 16S rRNA gene allowed the identification of other microorganisms such as Abiotrophia defectiva (6,67%), Arthrobacter (1,67 %), Brevibacterium (11.67 %) and Microbacterium (1.67 %). In conclusion, BGPI isolates from invasive infections should not be neglected and sequence analysis of 16S rRNA and rpoB genes can contribute to the definitive identification of the species of Gram positive microorganisms, including corynebacteria involved in these infections.

The Gram-positive bacterium, Rhodococcus sp. 33, was selected for further study to identify the characteristics conferring it with high tolerance to concentrations of benzene. Since most organic solvents, like benzene, are lipophilic, they tend to accumulate within lipid membranes where they express toxicity. The mechanisms conferring this Rhodococcus with resistance to benzene were hypothesised to be located within the subcellular region of this bacterium - cell wall, membrane, and cell-bound polymer. Therefore, this investigation was instigated to identify these mechanisms. To accomplish this, the development of methodologies to isolate highly purified cell wall and membrane fractions, from the organism, were required. To corroborate this investigation, a total of 6 benzene-sensitive mutants were prepared from Rhodococcus sp. 33 and their characteristics compared to those of the parent wild-type strain. 1-D PAGE analysis of proteins revealed various benzene-induced wall, membrane, and cytoplasmic proteins in the w-t. A protein band, with an approximate molecular weight of 58 kDa, was identified to be absent in the most sensitive mutant isolated (mutant M2b). Interestingly, much of this research showed that the benzene-catabolising enzymes played an insignificant role in tolerating the benzene. Gas chromatography and mass spectrometry of whole cell-derived fatty acids revealed that benzene induced an increase in the ratios of saturated/unsaturated fatty acids. Moreover, protein determinations revealed that benzene induced an increase in the concentration of total membrane protein. These increases are suggestive as possible mechanisms to decrease the fluidity of the cell membrane. This was further supported by the observed increase in the generalised polarisation (GP) of laurdan fluorescence in the membranes during growth of the organism with benzene, which is correlated with a decrease in membrane fluidity. The organism was also found to synthesise hexadecenoic acid, 16:1w6c (11 - 13% of total fatty acids), an uncommon fatty acid and previously unreported in other Rhodococcus spp. Analysis of the organism's cell-bound extracellular polymer revealed it to be composed of polysaccharide with biosurfactant-like properties. Its function is proposed to act as a surfactant layer outside the cell, concentrating the benzene within its matrix and reducing benzene's contact with the cell membrane.

Bibliography: pages 147-177.
Nocardioforms are Gram-positive, aerobic actinomycetes and are a metabolically diverse group which produce antibiotics, useful enzymes, are important in the biotransformation of organic compounds and the decomposition of organic wastes and are important medically. A gene cloning vector designated pLR591 was constructed from the broad host range, multicopy Streptomyces plasmid pIJ702 and the positive selection Escherichia coli plasmid pEcoR251. This plasmid has useful features for the construction of actinomycete genomic libraries. Cloning of DNA into the unique Bg1II endonuclease site of pLR591 inactivated the lethal EcoRI gene derived from pEcoR251, thereby selecting for recombinant plasmids containing inserted DNA. The thiostrepton resistance gene derived from pIJ702 was shown to be functional in Streptomyces lividans enabling selection of recombinant pLR591 plasmids containing foreign DNA in S. lividans. The vector pLR591 therefore functions as a positive selection Streptomyces-E. coli shuttle vector facilitating construction of actinomycete genomic libraries in E. coli and subsequent transfer of recombinant plasmids into S. lividans.

Phenolic residues which accumulate in the environment as a result of agro-industrial practices has resulted in the need to find and use Eco-Friendly techniques, rather than the traditional methods of burning or burying this kind of waste. Bioremediation and bioconversion are attractive alternatives using whole cell or enzyme-based systems. The aims of this project were to isolate and uses thermophilic Actinomycetes, which produce thermo-tolerant oxidoreductase enzymes, which can be used to bioconvert a model industrial phenolic waste commonly genersated in the wine-making industry of South Africa. Current research in bioconversion and bioremediation focuses on mesophilic microbes in that their enzymes can catalyse reactions at higher temperatures without affecting its activity and lower contamination levels. Three novel Actinomycete isolates were isolated (RU-A0l , RU-A03 and RU-A06) from a compost site and characterized using a combination of conventional identification techniques and 16S rDNA methodology to identity the three isolates. All three isolates belong to the Streptomyces clade. In addition, five known Actinomycetes were selected from an internation culture collection and also screened for oxidoreductase activity in comparision to the three novel isolates. Although the five isolates were selected based on their ability to produce oxidoreductase enzymes, unexpectedly, no activity was detected. Screening assays for peroxidase, polyphenol oxidase and laccase on RU-AO 1, RU-A03 and RU-A06, showed that all three isolated produced peroxidases and peroxidases but no laccase. Substrate specificity studies revealed that the most suitable substrates to determine peroxidase and polyphenol oxidase activity on these isolates were catechol for polyphenol oxidase, 2,4-dichlorophenol for peroxidases and veratryl alcohol for lignin peroxidases. Previous studies have indicated that peroxidases and polyphenol oxidases are produced in Actinomycetes during the primary stage of growth. This was the case with RU-AOI , RU-A03 and RU-A06. Growth rates were higher that other Actinomycetes, with maxImum biomass being reached at 36 hours for the isolates RU-AOI and RU-A06 and 48 hours for isolate RUA03. pH studies showed that the three isolates were adaptable and could grow over a broad pH range. Catabolism studies of phenolic model compounds showed that the three isolates were capable of catabolizing the model phenolic compounds within a period of 24 hours. Further studies were carried out to determine the effect of these microbes and their enzymes in whole cell and enzyme-based systems on a model phenolic waste, graoe waste consisting of compressed grape skins, pips and stalks. Whole cell studies showed that the isolates were capable of bioconverting the waste at a maximum concentration of 30% grape waste (vol:vol). Peroxidase and polyphenol oxidase activity increased indicating induction of these enzymes in the presence of phenolic compounds, with a maximum increase of up to 15.9 fold increase in extracellular lignin peroxidase activity in RU-AO1. HPLC and phenolic determination assays indicated that bioconversion of the phenolic grape waste had occurred in the presence of the three isolates. Attempts were made to isolate and identify a peroxidase or phenol oxidase gene from one the isolates. As bacteria, Actinomycetes are amendable to gene manipulation making them suitable candidates for methods such as site directed evolution in comparison to fungi. Two clones were selected for sequencing based on positive activity results when assayed for peroxidase activity. However the resultant sequences did not identify a functional gene sequence. Southern Blotting was then carried out to determine the nature of the peroxidase gene. Previous studies have been focused on the catalase-peroxidase gene (CalC gene) found Actinomycetes and other bacteria. A probe was developed from the CalC gene. No hybridization occurred with any of the enzyme restricted DNA from the three isolates. The implications of these results are that the peroxidase genets in the three isolates are in fact lignin peroxidase in nature. This project has the potential in the bioconversion of phenolic wastes and is the first description of the use of thermophilic Actinomycetes in the bioconversion of an industrial phenolic waste.

O lixo urbano tem sido apontado como um dos maiores poluentes ambientais. O lixo plástico chega a representar 20% do volume do lixo doméstico. Como alternativa aos plásticos petroquímicos, produtos plásticos menos agressivos ao meio ambiente e mais biodegradáveis têm sido estudados, entre eles os polihidroxialcanoatos (PHA). Os PHA são poliésteres biodegradáveis acumulados como material de reserva por inúmeras bactérias e que possuem aplicabilidade comercial bastante abrangente. Recentemente, os actinomicetos passaram a ser estudados para a produção destes polímeros. Em trabalho prévio, 53 novas linhagens de actinomicetos produtoras de biopolímeros foram isoladas de solo. Neste trabalho foi feita a seleção das bactérias quanto aos polímeros produzidos em diferentes de carbono. Das quatro linhagens selecionadas, duas foram analisadas quanto à produção de um novo polímero. Nas outras duas linhagens foram amplificados e estudados os genes sintetizadores dos polímeros. Em todas as linhagens foram feitas análises taxonômicas e cultivos em rejeitos industriais.
The urban waste has been described as one of the largest environmental pollutants. The plastic garbage can represent up to 20% of the volume of household waste. As an alternative to petrochemical plastics, plastic products less damaging to the environment and more biodegradable have been studied, among them polyhydroxyalkanoates (PHA). The PHA is a biodegradable polyester material accumulated as a reserve material by many bacteria and they have very broad commercial applicability. Recently, the actinomycetes have been studied for the production of polymers. In previous work, 53 new strains of actinomycetes producers of polymers were isolated from soil. In this work the bacterial the selection of bacteria was made concerning the polymers production on different carbon. Of the four strains selected, two were analyzed for the production of a new polymer. In the other two strains were amplified and studied the genes of polymers synthases. In all lineages were analyzed taxonomically and in cultivation on industrial waste.

A espécie Araucaria angustifolia, pertencente ao fragilizado Bioma Mata Atlântica, foi, durante décadas, uma das maiores fontes de madeira no Brasil. Essa espécie é de grande importância sendo fonte de alimento e matéria prima para a produção de móveis, polpa de celulose e vernizes. Devido à grande importância econômica e ambiental da araucária, estudos voltados à preservação e manejo da espécie se tornaram necessários. Este trabalho teve como finalidade isolar actinobactérias, presentes na rizosfera de araucária, antagônicos aos fungos Fusarium sp .e Armillaria sp. Estes patógenos são responsáveis por danos às raízes e sementes causando podridão e perda de mudas. Além disso, verificou-se também o efeito das actinobactérias sobre a germinação de esporos de Gigaspora rosea. Após a seleção dos melhores inibidores, identificação morfológica e seqüenciamento do gene 16S rRNA, verificou-se o efeito destes sobre o crescimento de Pinus taeda, na presença e na ausência do fungo ectomicorrízico (Suillus brevipes). Os isolados ainda foram avaliados quanto à produção de enzimas como quitinase, lipase, fosfatase, celulase, amilase e protease. Para o isolamento foram coletadas raízes de 15 árvores adultas presentes na mata nativa. Dez gramas de raízes frescas com resíduos de solo aderidos firmemente foram agitados por 30 minutos em solução salina 0,85 % padronizando como solo rizosférico aquele que soltou das raízes. Neste isolamento foram utilizadas duas metodologias: por plaqueamento e por separação física pela utilização de membrana de 0,45 micrômetros. Foram obtidos 33 possíveis actinobactérias. Os isolados foram testados quanto à capacidade antagônica pelo método de contato direto, inoculando-os a 30 mm de distância do fungo fitopatogênico Fusarium sp., em placas de petri contendo meio ISP2. Os halos de inibição foram medidos após 3, 5, 7 e 10 dias. De todos os isolados testados 6 mantiveram a inibição por 10 dias com manutenção de halos de inibição de até 4 mm contra um Fusarium sp. isolado de sementes de milho e 2 foram eficientes na inibição de Fusarium sp, patógeno de Pinus sp. A análise de inibição do fungo Armillaria sp, foi feito em meio líquido medindo o crescimento em mg/dia após 30 dias na presença de extratos de actinobactérias e também pela contagem de rizomorfas em placa de petri após 20 dias de incubação em contato direto com as actinobactérias. Verificou-se que de 28 isolados testados 24 foram capazes de inibir a produção de rizomorfas, destacando-se o isolado A43 que foi capaz de inibir ambos os fungos, Fusarium e Armillaria. Os esporos de Gigaspora rosea tiveram a taxa de germinação avaliada na presença de actinobactérias a partir da técnica de dupla camada. Todos os seis isolados testados foram capazes de estimular a germinação dos esporos desse fungo micorrizico. Nenhum dos seis isolados (A43, A43b, PNA, A64, A75 e A93) foi capaz de produzir fosfatases e lipases. Porém os isolados A93, A75, A64 e PNA produziram protease, quitinase e amilase. Em casa de vegetação, foi testado o efeito de seis isolados, na presença e não ausência de ectomicorriza (Suillus brevipes), sobre plântulas de Pinus taeda. Analisou-se o diâmetro, a altura, a massa seca da raiz e da parte aérea e também o fósforo da parte aérea. Destaca-se o isolado A43 que, quando na ausência de ectomicorriza, estimulou o desenvolvimento da planta em relação ao controletambém sem ectomicorriza, provocando aumento de 100 % para massa seca da parte aérea e da raiz. Os resultados encontrados neste trabalho poderão levar ao desenvolvimento de novas tecnologias, visando à identificação de novos metabólitos e novas técnicas de manejo, voltados ao controle de doenças de plantas, especialmente as espécies arbóreas.
The tree Araucaria angustifolia, belonging to the endangered Atlantic Forest biome, for many decades was the source of Brazilian wood. This species is also very important in providing food and feed, as well as raw material for joinery, cellulose pulp and varnish. Due to the economic and environmental importance of A. angustifolia, research projects involving the preservation and management of this species are becoming more urgent and necessary. The aim of this work was to isolate Araucaria rhizosphere actinobacteria with antagonic effects against the plant pathogens Fusarium sp. and Armillaria sp. These fungi cause root rot and seed damage, with the consequent loss of seedlings. Moreover, the effect of these actinobacteria on Gigaspora rosea spore germination was studied. After the selection of the best pathogen inhibitors, we also tested the effect of these microorganisms on Pinus taeda growth, in the presence or absence of the ectomycorrhizal fungus Suillus brevipes. The production of protease, chitinase, lipase, phosphatase, cellulase and amylase of these bacteria in culture media was also investigated. For the isolation of rhizosphere bacteria, we collected roots of 15 adult trees in a native forest. Ten grams of fresh roots with soil residues adhered to the surface were shaken in 0,85 % salt solution for 30 minutes. Two techniques were used, the dilution plate method and the coverage of the medium, utilizing a 0,45 µm membrane to separate these filamentous bacteria. About 33 actinobacteria were isolated. After isolation the actinobacteria were tested against the plant pathogenic fungi Fusarium spp., utilizing dual culture techniques and ISP2 medium. The inhibition halo was measured after 3, 5, 7 and 10 days. Six of our isolates maintained an inhibition zone measuring at least 4 mm against Fusarium sp. isolated from corn seed and 2 mm against the Fusarium, which causes root root of Pine trees. For the inhibition test of Armillaria, in liquid medium with the addition of culture extracts of actinobacteria, the growth in mg/day was measured after thirty days growth, and the number of rizomorphs produced in culture dishes after twenty days in dual culture with the actinobacteria was counted. Six bacteria proved to be antagonistic (A43, A43b, A64, PNA, A93 e A75), and only one had no effect. Possibly the elevation of the pH value played also a role in this situation. About 24 of 28 isolates inhibited the rizomorph production, especially the isolate A43 that showed a double antagonism against Fusarium and Armillaria. The dual layer test was used to investigate the reaction of the arbuscular mycorrhizal fungus Gigaspora rosea spore germination to the presence of actinobacteria. All the six actinobacteria stimulated the germination, but the germ tube did not grow straight forward as in the control. This result may indicate a negative effect against this arbuscular mycorrhizal fungus. None of the six isolates tested (A43, A43b, PNA, A64, A75 and A93) produced phosphatases and lipases, but A93, A75, A64 and PNA produced protease, amylase and chitinase. Isolates A43 and A43b did not produce any of the enzymes tested. This fact suggests that there is production of an antibiotic acting against the pathogenic fungi. Pinus taeda seedlings were grown under green-house conditions. After three months the stem diameter, shoot height, root and shoot dry weight and shoot phosphorus content were evaluated. Plants with ectomycorrhiza presented a significant growth promotion in comparison with the nonmycorrhizal ones. Among the actinobacteria in the absence of mycorrhiza only the isolate A43 produced a 100% growth enhancement in comparison with the control plant without ectomycorrhiza. The results presented in this dissertation could lead to the development of new technologies and new management techniques, with regard to the control of plant diseases, especially in tree species.

Les PLP qui assurent la polymérisation finale du peptidoglycane sont les cibles des β-lactamines. Un nouveau mécanisme de résistance à ces antibiotiques par activation d'une autre voie de synthèse (L,D-transpeptidation) a été récemment caractérisé. L'existence et l'impact de la voie de L,D-transpeptidation ont éte�� étudiés chez C. Jeikeium et M. Tuberculosis. Leur L,D-transpeptidases utilisent un substrat tétrapeptidique et sont inhibées par les carbapénèmes. Chez C. Jeikeium la voie des PLP est prédominante. La disponibilité du substrat tétrapeptidique généré par la Pbp4 est le facteur limitant la L,D-transpeptidation. La Pbp2C a été identifiée comme responsable de la résistance aux β-lactamines. Chez M. Tuberculosis, le peptidoglycane des formes non réplicatives comporte très majoritairement des ponts générés par L,D-transpeptidation suggérant que les L,D-transpeptidases et les carbapénèmes représentent une cible et une famille d'antibiotiques pour le traitement de la tuberculose
The PBP that catalyze the last cross-linking step of peptidoglycan synthesis are the targets of β-lactams. A novel mechanism of β-lactam resistance due to activation of another pathway (L,D-transpeptidation) has been recently characterized. The aim of the study was to investigate the impact of the L,D-transpeptidation pathway in C. Jeikeium and M. Tuberculosis. Their L,D-transpeptidases use substrates containing a tetrapeptide stem and are inhibited by carbapenems. In C. Jeikeium, the PBP pathway is predominant. The supply in tetrapeptide substrate generated by Pbp4 is the limiting factor for L,D-transpeptidation pathway. Pbp2C was identified as the cross-linking enzyme responsible for β-lactam resistance in C. Jeikeium. In M. Tuberculosis, the peptidoglycan of non-replicating cells predominantly contains crosslinks generated by L,D-transpeptidation. L,D-transpeptidases and carbapenems may represent a target and a drug family relevant to the eradication of persistent M. Tuberculosis

Orientador : Prof. Dr. Paulo Henrique Gorgatti Zarbin
Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Exatas, Programa de Pós-Graduação em Química. Defesa: Curitiba, 19/02/2016
Inclui referências : f. 151-166
Área de concentração : Química Orgânica
Resumo: Este trabalho descreve aspectos relacionados à ecologia química de três espécies distintas (uma espécie de inseto e duas de bactérias), abordando desde a criação dos organismos em condições de laboratório com o objetivo de realizar a extração de voláteis e posterior identificação estrutural, síntese, estudos biossintéticos e de determinação de bioatividade. Desta maneira, o trabalho está dividido em dois capítulos. O primeiro capítulo discute de aspectos relacionados à identificação e síntese do feromônio de agregação do besouro Homalinotus depressus (Coleoptera: Curculionidae), cuja população tem crescido constantemente na região norte do Brasil, levando a perdas financeiras de grandes proporções na produção de coco. Devido a aspectos biológicos da espécie, acredita-se que o uso de feromônios no controle da praga é promissor. Respostas comportamentais de H. depressus a extratos de aeração de coespecíficos sugerem a presença de um feromônio de agregação produzido pelos machos. As análises destes extratos em GC revelaram a presença de quatro compostos macho-específicos. O conjunto de dados analíticos sugeriu que o componente minoritário da mistura estaria relacionado à estrutura da isoforona, que foi confirmada através da co-injeção com um padrão analítico. Algumas reações foram propostas a partir da isoforona, com o objetivo de identificar os demais componentes da mistura. A reação de epoxidação resultou na epóxi-isoforona, a qual coelui com um dos compostos macho-específicos. Através das análises em GC-FTIR e GC-MS e da redução da epóxi-isoforna com NaBH4, o composto majoritário foi identificado como 4,4,6-trimetil-7-oxa-biciclo[4.1.0]heptan-2-ol (homalinol). A preparação diastereoseletiva dos estereoisômeros syn e anti do homalinol e a comparação de seus respectivos perfis cromatográficos permitiu a determinação da configuração relativa do composto majoritário como syn. Ambos os enantiômeros do syn-homalinol foram obtidos em bons excessos enantioméricos via biocatálise e a configuração absoluta do homalinol natural foi determinada como (1R,2R,6S). Bioensaios em olfatômetro-Y e em campo demonstraram que o componente majoritário sintético puro ou em mistura com os demais compostos macho-específicos é atrativo para H. depressus. A segunda parte do trabalho relata a identificação, síntese e biossíntese de nitrilas voláteis emitidas por culturas das bactérias Pseudomonas veronii e Micromonospora echinospora. Os voláteis produzidos por culturas de P. veronii R02 e M. echinospora DSM 43816 foram coletados por aparato de trapeamento em circuito fechado (CLSA) e analisados por GC-MS. Análises prévias à realização deste trabalho permitiram a identificação de diversos compostos presentes nos extratos de ambas as espécies, assim como a detecção de nitrilas de cadeia longa, não identificadas. Foram detectados seis compostos nos extratos de P. veronii R02 e onze compostos nos extratos de M. echinospora DSM43816 contendo a função nitrila. Através da análise dos respectivos índices de retenção, espectros de massas e dados oriundos de derivatização com disulfeto de dimetila (DMDS) foram elaboradas propostas estruturais para as nitrilas presentes nos extratos de ambas as espécies. As propostas foram confirmadas através da síntese de padrões. Em P. veronii R02 foram identificadas as seguintes estruturas: (Z)-tetradec-7-enonitrila, tetradecanonitrila, pentadecanonitrila, (Z)-hexadec-9-enonitrila, hexadecanonitrila e (Z)-octadec-11- enonitrila. Em M.echinospora DSM43816 foram identificadas as seguintes estruturas: (Z)-12-metiltridec-3-enonitrila, (E)-12-metiltridec-3-enonitrila, (Z)-tetradec-3- enonitrila, (E)-tetradec-3-enonitrila, (Z)-13-metiltetradec-3-enonitrila, (Z)-12- metiltetradec-3-enonitrila, (E)-13-metiltetradec-3-enonitrila, 13- metiltetradecanonitrila, (Z)-pentadec-3-enonitrila, (Z)-14-metilpentadec-3-enonitrila e (Z)-hexadec-3-enonitrila. Através da síntese e aplicação de compostos isotopicamente marcados às culturas foi possível elaborar propostas biossintéticas para a obtenção das nitrilas naturais em ambas as espécies. Estudos de atividade antimicrobiana preliminares demonstraram que a E/Z-pentadec-3-enonitrila é capaz de inibir o crescimento de culturas de Staphylococcus aureus.
Abstract: This project describes aspects related to the chemical ecology of three different species (one insect and two bacteria species), approaching since the organism rearing in laboratory conditions, aiming the extraction of volatiles followed by sctructural elucidation, synthesis, biosynthetic studies, and bioactivity determination. In this way this thesis is divided in two distinct chapters. The first chapted describes aspects related to the identification and synthesis of the aggregation pheromone produced by the beetle Homalinotus depressus (Coleoptera: Curculionidae), whose population has constantly increased on the Brazilian Northern region, leading to heavy financial losses on coconut production. Due to biological aspects of the species, the use of pheromones in pest management programs is promising. The behavioral responses of H. depressus to conspecifics aeration extracts suggested the presence of a maleproduced aggregation pheromone. GC analyses of these extracts revealed the presence of four male-specific compounds. Analytical dataset suggested the identity of the minor component as isophorone which was confirmed by co-injection with an analytical standard. Several reactions were proposed starting from isophorone in order to identify the remaining components. The epoxydation reaction resulted in epoxyisophorone, which co-eluted with one of the male-specific compounds. By GCFTIR and GC-MS analyses and NaBH4 reduction of epoxyisophorone the major compound was identified as 4,4,6-trimetil-7-oxa-biciclo[4.1.0]heptan-2-ol (homalinol). Diastereoselective preparation of syn and anti stereoisomers of homalinol and the comparison of their respective chromatographic profiles allowed the determination of the relative configuration of the major compound as syn. Both enantiomers of syn-homalinol were obtained in high enantiomeric excesses by biocatalysis and the absolute configuration on natural homalinol was determined as (1R,2R,6S). Y-tube olfactometer and field bioassays demonstrated that the synthetic major compound pure or as a mixture of all the male specific compounds is attractive to H. depressus. The second chapter describes the identification, synthesis and biosynthesis of volatile nitriles released by bacterial cultures of Pseudomonas veronii and Micromonospora echinospora. Volatiles produced by P. veronii R02 and M. echinospora DSM 43816 cultures were collected by closed loop striping apparatus (CLSA) and analyzed by GC-MS. Previous analyses allowed the identification of several compounds in extracts from both species and the detection of unidentified long chain nitriles. Six nitriles were detected on extracts from P. veronii R02 and eleven from M. echinospora DSM43816. Structural proposals for the nitriles contained on extracts from both species we performed through the analysis of the respective retention indexes, mass spectra and dimethyl disulfide (DMDS) derivatisations data. The proposed structures were confirmed by synthesizing standards. The following structures were identified on P. veronii R02: (Z)-tetradec-7-enenitrile, tetradecanenitrile, pentadecanenitrile, (Z)-hexadec-9-enenitrile, hexadecanenitrile e (Z)-octadec-11-enenitrile. The following structures were identified on M.echinospora DSM43816: (Z)-12-methyltridec-3-enenitrile, (E)-12-methyltridec-3-enenitrile, (Z)- tetradec-3-enenitrile, (E)-tetradec-3-enenitrile, (Z)-13-methyltetradec-3-enenitrile, (Z)- 12-methyltetradec-3-enenitrile, (E)-13-methyltetradec-3-enenitrile, 13- methyltetradecanenitrile, (Z)-pentadec-3-enenitrile, (Z)-14-methylpentadec-3- enenitrile and (Z)-hexadec-3-enenitrile. It was possible to propose biosynthetic routes to the natural nitriles through synthesis and application of isotopically labeled compounds to the cultures. Preliminary antimicrobial activity studies showed that pentadec-3-enenitrile is active against growth of Staphylococcus aureus cultures

Un calcul theorique ayant montre que la culture de aspergillus niger sur milieu solide d'amidon de manioc etait limitee par la disponibilite de l'eau dans le substrat, l'introduction de 20% d'un support lignocellulosique a forte capacite de retention d'eau a permis d'augmenter l'activite de l'eau du milieu et, ainsi, d'accelerer et ameliorer le processus fermentatif. L'extension de la notion de support a ensuite conduit a pratiquer des cultures de a. Niger sur un milieu liquide dissous absorbe sur une phase solide. Dans un tel systeme, l'activite de l'eau de la solution d'impregnation, la taille des particules du support et la quantite d'inoculum de spores ont ete les parametres importants de la croissance du champignon. Ce type de culture a permis d'utiliser des milieux liquides glucoses tres concentres (400 g/l) qui sont utilises pour la croissance en 40h. Une etude microcalorimetrique a mis en evidence l'existence d'une phase exothermique situee entre la germination et la croissance exponentielle

Algumas actinobactérias habitantes da rizosfera são produtoras de substâncias capazes de combater micro-organismos patogênicos às plantas, o que as torna potenciais agentes de controle biológico, passíveis de serem utilizadas como princípio ativo de inoculantes de sementes e mudas. O presente trabalho teve como objetivo isolar e avaliar o potencial de isolados de actinobactérias no controle de doenças causadas por fungos nas espécies arbóreas Araucaria angustifolia (Araucária) e Pinus elliottii (Pinus). Além disso, foi iniciado o estudo do processo de elaboração de inoculante a base de actinobactérias antagônicas. Foram isoladas 215 estirpes de actinobactérias do rizoplano da Araucária, das quais 13 apresentaram, em testes in vitro, potencial como antagonistas contra os fungos fitopatogênicos Fusarium oxysporum, Cylindrocladium candelabrum, C. pteridis e Armillaria sp., e apenas os isolados Ac136 e Ac202 apresentaram os maiores valores de inibição nos testes com os quatro patógenos. Na avaliação de produção de algumas substâncias antimicrobianas, apenas celulases, quitinases e sideróforos foram produzidas pelos isolados, sendo este último o mais frequente. Nos testes de interação com organismos foi verificado que, embora os isolados de actinobactéria tenham inibido a germinação de esporos do fungo micorrízico arbuscular (FMA) Gigaspora rosea em teste in vitro, no experimento in vivo, em que foi utilizado o milho como planta hospedeira para inoculação com o FMA e os isolados A43, Ac136 e Ac202, os isolados Ac136 e Ac202 estimularam a colonização das raízes pelo FMA. Estes mesmos isolados também estimularam a germinação de sementes e desenvolvimento inicial de plântulas de Pinus, porém prejudicaram a germinação e desenvolvimento inicial de Araucária. Estes mesmos isolados foram capazes de reduzir a mortalidade de plântulas de Pinus em cerca de 25%, e esta diminuição foi atribuída à inibição de Fusarium sp.. No teste de viabilidade dos isolados em diferentes veículos o isolado que mostrou maior sobrevivência foi o Ac202, mantendo um número de propágulos viáveis correspondente a 5,48 log UFC mL-1, enquanto que o veículo mais apropriado para a elaboração de inoculante com actinobactérias foi a glicerina. A análise molecular mostrou que os isolados mais promissores apresentaram maior similaridade com S. kasugaensis. Dentre todos isolados Ac202 (S. kasugaensis) foi o mais promissor obtido neste trabalho para o uso como agente de biocontrole de doenças causadas por fungos, apresentando forte antagonismo contra os quatro patógenos testados, promovendo a germinação e desenvolvimento inicial de Pinus e aumentando a sobrevivência de plântulas contaminadas com fungos fitopatogênicos.
Some actinobacteria that inhabits the rhizosphere are producers of substances that are capable of combating plant-pathogenic microorganisms, what makes them potential biological control agents, which can be used as the active ingredient of seeds and seedlings inoculants. This study aimed to isolate and evaluate the potential of actinobacteria isolates in controlling diseases caused by fungi in Araucaria angustifolia and Pinus elliottii. In addition, the study initiated the process of elaboration of an actinobacteria-based inoculant. 215 actinobacterial strains were isolated from the Araucarias rhizoplane, and 13 of them showed potential as antagonists against the phytopathogenic fungi Fusarium oxysporum, Cylindrocladium candelabrum, C. pteridis and Armillaria sp. in in vitro tests, and only the Ac136 and Ac202 strains showed the highest inhibition in the tests against the four pathogens. Among the antimicrobial substances tested, only cellulases, chitinases and siderophores were produced, with the latter being the most frequent. In the interaction tests with other organisms it was found that although the actinobacterial strains have inhibited the germination of the arbuscular mycorrhizal fungi (AMF) Gigaspora rosea spores on the in vitro test, the in vivo experiment, with maize as host plant, inoculated with the AMF and A43, Ac136 and Ac202 actinobacterial strains, Ac136 and Ac202 stimulated root colonization by AMF. These strains also stimulated Pinus seed germination and seedling early development, but hindered the germination and early development of Araucaria. In addition, the same strains were able to reduce the mortality of pine seedlings by about 25%, and this decrease was attributed to the inhibition of Fusarium sp.. In the viability test of the strains in different vehicles the strain that showed the greatest shelf-life was Ac202, with 5.48 log CFU mL-1, and the most appropriate vehicle for the actinobacteriabased inoculant development was glycerin. Molecular analysis showed that the most promising isolates showed the greatest similarity with S. kasugaensis. Among all strains Ac202 (S. kasugaensis) was the most promising one for the use as biocontrol agent of fungal diseases, exhibiting a strong antagonism against the four pathogens tested, promoting germination and early development of Pinus and increasing survival of seedlings infected with pathogenic fungi.

Ce travail porte sur la spécificité d'association entre le genre actinorhizien le plus primitif, Myrica, et l'actinomycète fixateur d'azote Frankia. La diversité génétique et la phylogénie des souches de Frankia nodulant naturellement 5 espèces de Myrica à distribution très large ou restreinte (espèces insulaires, endémiques) a été étudiée par PCR-RFLP et séquençage du gène rrs et de l'IGS 16S-23S. La phylogénie des plantes-hôtes (12 espèces représentatives de la distribution mondiale du genre) a été étudiée par séquençage du gène chloroplastique rbcL et de l'ITS 18S-26S nucléaire. Les résultats montrent que M. Gale est une espèce à spécificité étroite, associée à des souches phylogénétiquement proches des souches d'Alnus. Les autres espèces testées, associées à des souches de Frankia phylogénétiquement distantes et à spectre d'hôte varié, présentent une spécificité large. L'hypothèse d'une coévolution des 2 partenaires est discutée sur la base de l'ensemble des résultats.

Gordonia jacobaea es una bacteria bacilar, no formadora de esporas, catalasa positivo, que producen pigmentos carotenoides de interés industrial, pero que también pertenece al grupo CNM (actinomicetos) donde encontramos géneros de interés clínico como Mycobacterium, Nocardia y Corynebacterium. Estos microorganismos poseen una pared extremadamente hidrofóbica por lo que es de gran interés tanto a nivel industrial como clínico, poder estudiar las proteínas formadoras de canal que en ella se encuentra, ya que con un mejor conocimiento de éstas supone conocer mejor las posibles vías de entra hasta el microorganismo, de sustancias antibióticas. Hasta el momento se han descrito proteínas formadoras de canal, o porinas, en la mayoría de los géneros de este grupo, pero en el género Gordonia somos los primeros en describir esta actividad. Debido a esta ausencia de publicaciones previa, hemos tenido que adaptar los protocolos existentes y presentados para el estudio otros géneros. El objetivo de esta tesis ha sido extraer y estudiar la actividad formadora de canales de las proteínas de membrana de Godonia jacobaea, así como secuenciar su genoma con el objetivo de buscar la presencia de secuencias codificantes con alta similitud a secuencias de porinas previamente descritas en el grupo de los micolata. Para ello se han puesto en práctica distintas técnicas de extracción de proteínas de pared, así como electroforéticas. Las proteínas obtenidas han sido analizadas mediante la técnica de Black Lipid Bilayer, identificándose tres extractos proteicos con actividad formadora de canal. Hemos identificado una primera proteína de pared de 100 kDa aparentes en G. jacobaea, que al ser hervida muestra dos subunidades de 40 y 35 kDa aparentes. Esta proteína forma canales llenos de agua, con una conductancia de 0.8 nS en soluciones de 1M KCl pH7, con selectividad para los aniones y no dependiente de voltaje. En el extracto activo para esta proteína formadora de canales identificamos una proteína de 558 aminoácidos, que muestra una hélice transmembrana entre los aminoácidos 22 y 42, posibles secuencias de hélice alfa más abundantes en la zona amino terminal y láminas beta distribuidas a lo largo de toda la secuencia, pero más abundantes en la región carboxilo terminal. Así mismo también se identificó una región entre los aminoácidos 160 y 276 con similitud a secuencias conservadas de proteínas integrales de membrana (familia CRBC) y una familia de toxinas que forma poros (RTX). Esta proteína no muestra homología con ninguna de las porinas publicadas, ni con otras proteínas con funciones conocidas. Hemos identificado una segunda proteína de pared de 60 kDa aparentes que forma canales llenos de agua con selectividad por los cationes, una conductancia mayoritaria de 1 nS en 1M KCl, y conductancias minoritarias de 2 y 3 nS, lo que parece indicar una conformación trimérica. Y hemos identificado una tercera proteína de pared de 20 kDa aparentes que muestra una conductancia de 2 nS en solución de 1M KCl pH7. Dentro del genoma de G. jacobaea se identifican 4 secuencias codificantes que identificamos como pertenecientes a la familia de porinas MspA. Las secuencias están emparejadas, dos de ellas son de 228 y 229 aminoácidos y las dos restantes de 214 y 239 aminoácidos. Los alineamientos, así como las longitudes de las proteínas parecen indicar que forman parte de la familia MspA. Estas secuencias coinciden con la longitud de la proteína de 20 kDa identificada en este microorganismo como formadora de canales.
The aim of this thesis was to extract and study the proteins with channel-forming activity of Godonia jacobaea,an organism belonging to the micolata group (Mycobacterium, Corynebacterium, Nocardia) and shed some light on the actual role of the porins located in the cell wall with such activity. In order to do so, wall protein extraction techniques and electrophoretic determination methods have been used, as well as Edman sequencing, mass spectrometry, bioinformatics techniques, massive genome sequencing (pyrosequencing), MS/MS, etc. Proteins obtained from wall extracts were analyzed by the Black Lipid Bilayer technique, allowing us to identify three proteins with pore-forming activity. The first protein, of ~ 100 kDa showed a conductance of 0.8 nS in 1M KCl, selectivity for anions and not voltage-dependent. The second of ~ 60 kDa, displayed a conductance of 1 nS in 1M KCl and selectivity for cations. Finally, the third protein of ~ 20 kDa had a conductance of 2 ns in 1M KCl. The 100 kDa protein was also studied by mass spectrometry (MS / MS) and Edman sequencing, enabling the identification of its gene, which resulted in a 558 amino acid protein showing a transmembrane region between amino acids 22 and 42, potential alpha helical sequences -more abundant in the amino terminal region- and beta sheets, distributed throughout the entire sequence, being more abundant in the carboxyl terminal region. This protein shows no homology with any of the previously published porins, nor with other proteins of known functions. At the same time, the whole genome of G. jacobaea has been sequenced, and published in the NCBI database. It was possible to identify four sequences of proteins in the genome of G. jacobaea belonging to the families MspA, a family of channel-forming proteins (porins) belonging to the actinomyces group.

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Cento e dezessete actinomicetos, sendo 96 obtidos de diferentes amostras de rizosfera e rizoplano de tomateiro e 21 culturas pré-selecionadas por Moura (1996), pela ação contra Ralstonia solanacearum, foram avaliados no controle biológico de Pseudomona syringae pv. tomato e Alternaria solani através microbiolização de sementes de tomateiro (Santa Cruz 'Kada'), seguindo-se o plantio em solo não-tratado e inoculação como os patógenos. A seleção massal, realizada em casa de vegetação, permitiu selecionar, pela contagem do número de lesões, o antagonista mais promissor (RD-01). Paralelamente, foram conduzidos teste de colonização de raízes de tomateiro em tubos contendo ágar- água, não inclinados, e testes de antibiose com os 117 antagonistas contra os patógenos Pseudomonas syringae pv. tomato, Xanthomonas campestris pv. vesicatoria e Alternaria solani, pelo método da sobre-camada. Cerca de 88% dos antagonistas não tiveram nenhum efeito inibitório contra os patógenos testados apesar de 70% destes terem sido capazes de colonizar raízes em condições gnotobióticas. Em testes para o controle de Corynespora cassiicola, Stemphylium solani, Xanthomonas campestris pv. vesicatoria e Ralstonia solanacearum em casa de vegetação, o isolado RD-01 demonstrou incapacidade em reduzir sintomas incitados pelo patógeno de solo, Ralstonia solanacearum, mas apresentou potencial como agente de biocontrole dos patógenos foliares. Através de um ensaio de promoção de crescimento, realizado em casa de vegetação com os cento e dezessete isolados, pode-se concluir a inexistência de uma relação entre o efeito de promoção de crescimento vegetal para os isolados testados com os resultados de biocontrole observados em experimento anterior. Em experimento de campo para o controle de Phytophthora infestans e Alternaria solani, dois actinomicetos pré-selecionados (RD-01 e SON-17) foram aplicados através da microbiolização de sementes de tomateiro e pela colonização do filoplano, respectivamente. Apesar de o controle químico ter sido mais efetivo que os actinomicetos, estes revelaram sua potencialidade como medida passível de utilização em procedimentos de manejo integrado, a qual precisa ser mais explorada.
One hundred and seventeen actinomycetes were used in the microbiolization of tomato seeds, followed by the sowing in non-treated soil. From these, 96 were isolated from samples of tomato rizosfere, and 21 cultures had been pre-selected by Moura (1996) for their action against Ralstonia solanacearum. At the same time, root colonization tests with tomato grown in test tubes containing water-agar and antibioses tests were conducted with the antagonists against the pathogens Pseudomonas syringae pv. tomato, Xanthomonas campestris pv. vesicatoria and Alternaria solani. Nearly 88% of the antagonits had no inhibitory effect against the tested pathogens despite 70% of these had been able to colonize the roots in gnotobiotic conditions. Mass selection, to control P. s. pv. tomato and A. solani, allowed the selection of the most promising antagonist (RD-01), though the reduction of the number of lesions. In a greenhouse test for the control of Corynespora cassiicola, Stemphylium solani, X. c. pv. vesicatoria e R. solanacearum, the isolate RD-01 was unable to reduce symptoms incited by the soilborne pathogen, R. solanacearum, but presented potential as biocontrol agent of the foliar pathogens. A greenhouse essay was conducted to evaluate the plant-growth promotion by the 117 rhizobacteria, and no correlation could be observed between the effect of growth promotion and the biocontrol effect observed in a previous experiment. In a field experiment to control Phytophthora infestans and A. solani, two pre- selected actinomyces (RD-01 e SON-17) were applied through tomato seed microbiolization and phylloplan colonization, respectively. Even though the chemical control was more effective than the actinomycetes, these showed their potential of use in integrated disease management, which needs to be more explored.
Dissertação importada do Alexandria

Tropheryma whipplei est détectée avec une prévalence variable dans les selles et de salive. Pour déterminer les facteurs épidémiologiques qui peuvent influencer l'histoire naturelle de la bactérie, nous avons réalisé des études sur l'ensemble de la population de 2 villages au Sénégal (Dielmo et Ndiop), chez les personnes sans domicile fixe (SDF) et dans les familles en France. Dans ces différentes populations, la prévalence du portage de T. whipplei dans les selles était respectivement de 31.2% (139/446), 12,9% (21/162) et 37,5% (24/64). En ce qui concerne les résultats des études phylogénétiques, nous avons identifié au Sénégal 22 génotypes, dont 16 étaient nouveaux. Un seul génotype (53) était commun aux deux villages. Parmi les génotypes spécifiques, l'un (n ° 52) était épidémie à Dielmo (15/28, 53,4%, p <10-3) et l'autre (n ° 49) à Ndiop (27,6%, p = 0,002). Deux génotypes, le génotype 3 et le génotype 85, circulent plus fréquemment chez les SDF par rapport à d'autres groupes personnes positives pour T. whipplei. Au Sénégal, la séroprévalence était estimée à 72,8% (291/400). Dans les familles, la séroprévalence était plus élevée chez les membres (23/30, 77%) par rapport à la population générale (143/300, 48%). Nos résultats montrent que T. whipplei est une bactérie fréquente et contagieuse qui est contractée très tôt dans l'enfance. La mise en évidence de génotypes épidémiques associée à l'absence de la bactérie dans des échantillons d'eau, chez les arthropodes vecteurs; la très faible présence (<1%) dans les selles des animaux domestiques et dans les écouvillons de poussière suggèrent une transmission interhumaine du T whipplei
Tropheryma whipplei is detected with variable prevalence in stool and saliva. To investigate the epidemiological factors which influences the natural history of the bacterium; we performed studies in entire population of 2 villages in Senegal (Dielmo and Ndiop) in homeless people and in family in Marseille-France. In these populations, the prevalence of T. whipplei in stool was respectively 31.2% (139/446), 12.9% (21/162) and 37.5% (24/64).Regarding findings from phylogenetic studies we identified in Senegal 22 genotypes, 16 of which were new. Only one genotype (#53) was common to both villages. Among the specific genotypes, one (#52) was epidemic in Dielmo (15/28, 53.4%, p<10-3) and another (#49) in Ndiop (27.6%, p=0.002). Two genotypes, the genotype 3 and the genotype 85, circulate more frequently in the homeless people compared to other people positive for T. whipplei and are epidemic. The same circulating genotype was significantly more common in families compared to other people. In Senegal, the seroprevalence was estimated at 72.8% (291/400). In family study, the seroprevalence was higher in the relatives (23/30, 77%) compared to the general population (143/300, 48%). Our findings show that T. whipplei is a common and contagious bacterium that is contracted early in childhood. Epidemic genotypes associated with absence of the bacterium in water samples, arthropods vector; almost no presence (< 1%) in domestic animals and dust suggest a human transmission of T whipplei

La mucoviscidose est une maladie génétique autosomique causée par une mutation dans le gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Mon travail s’est décomposé en deux parties principales : d’une part j’ai réalisé une revue de la littérature sur l’analyse des génomes bactériens isolés de patients mucoviscidosiques comparativement aux génomes des mêmes espèces isolées dans d’autrescontextes et d’autre part j’ai analysé les génomes de trois espèces bactériennes (Microbacterium yannicii, Chryseobacterium oranimense et Haemophilus parahaemolyticus). L’analyse exhaustive des génomes bactériens issus de patients atteints de mucoviscidose a révélé une extraordinaire évolution de ces génomes en fonction du temps et des traitements reçus par ces patients qui témoigne de la capacité qu’ont ces bactéries à s’adapter à leur écosystème notamment par l’acquisition de nouveaux gènes par transfert latéral de gènes. Ce travail montre l’extraordinaire plasticité des génomes bactériens dans un milieu donné et à ce titre le poumon de patients atteints de mucoviscidose représente un modèle unique pour comprendre l’évolution des génomes bactériens. De plus, notre travail a permis d’identifier leurs mécanismes moléculaires de résistance aux antibiotiques. Les travaux à venir sur l’étude des métagénomes de prélèvements chez ces patients pourrait permettre de répondre à ces questions dans le futur. La découverte de nouvelles espèces et / ou émergentes va nous permettre d’avoir une image plus complète de la mucoviscidose qui pourrait conduire à une meilleure connaissance de la maladie et donc à une meilleure prise en charge thérapeutique
Cystic fibrosis is an autosomal genetic disorder caused by a mutation in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. Pulmonary infection is the major problem faced by patients with cystic fibrosis. My work is divided into two main parts: first I made a review of the literature on the analysis of bacterial genomes isolated from CF patients compared to the genomes of the same species isolated in autrescontextes and other part I analyzed the genomes of three species of bacteria (Microbacterium yannicii, Chryseobacterium oranimense and Haemophilus parahaemolyticus). The comprehensive analysis of bacterial genomes from cystic fibrosis patients revealed an extraordinary evolution of these genomes with time and treatment received by these patients reflects the ability of these bacteria to adapt to their particular ecosystem the acquisition of new genes by lateral gene transfer. This work shows the extraordinary plasticity of bacterial genomes in a given environment and as the lungs of patients with cystic fibrosis represents a unique model for understanding the evolution of bacterial genomes. In addition, our work has identified their molecular mechanisms of resistance to antibiotics. Future work on the study of metagenomes sampling in these patients could help to answer these questions in the future. The discovery of new species and / or emerging will allow us to have a more complete picture of cystic fibrosis which could lead to a better understanding of the disease and thus a better therapeutic management

Caves represent one of few remaining isolated planetary habitats, in terms of human impact and characterisation of microbial biodiversity. Caves are unique environments characterised by little or no light, low levels of organic nutrients, high mineral concentrations and a stable microclimate providing ecological niches for highly specialised organisms. Caves are not uniform environments in terms of geological and geochemical characteristics, as they can vary from one to the other, eg. rock type, method of formation, length, depth, number of openings to the surface, presence or absence of active streamways, degree of impact by human visitation etc. Furthermore, on a smaller scale, various microhabitats, with vast differences in community structure can exist within caves. Culture studies point to the dominance of actinomycetes in caves and reveals great taxonomic diversity within actinomycetes isolated. However it is widely accepted that only - 1 % of microbes are cultured in the laboratory. Culture-independent methods are being increasingly used to describe the composition of microbial communities and reveal significantly broader diversity than culture-based studies. Nevertheless, to date our knowledge of bacterial communities in caves is largely due to culture studies. Based on the literature available, this study was initially aimed at examining culturable vs. non-culturable diversity of actinomycetes in Entrance and Loons Caves and to gain an increased understanding of the composition of cave microbial communities employing classical isolation and advanced molecular detection methods. As the study progressed the focus evolved as it became apparent that actinomycetes dominated only very specific habitats, the dry sediment in Entrance Cave, and represented only a minor fraction of the microbial biodiversity of most other microhabitats studied. Entrance Cave dry sediments and inactive (dry) speleothems produced a higher number of actinomycete isolates compared to saturated sediments and wet formations from Entrance and Loons Caves. This was reinforced by the actinomycetes being the second most abundant group (26.8%) detected in clone analysis of the dry Entrance sediment and low abundances (4-16%) detected in saturated sediments from both Entrance and Loons Caves. Sediment phylotypes and isolates identified in this study closely resemble species associated with oligotrophic, chemolithotrophic and heterotrophic lifestyles indicating that these communities survive by utilising a combination of metabolic pathways. Bacteria involved in the nitrogen and sulfur cycles were important members of all sediment communities along with hydrogen-oxidising bacteria. Pair-wise comparisons of sediment communities demonstrated that they were more similar to each other within individual cave systems, Entrance and Loons, rather than between microhabitat types (dry vs. wet sediment) though saturated sediment from Entrance Cave did show a higher degree of similarity in community composition to Loons Cave samples than the dry sediment from Entrance Cave. Saturated sediments were dominated by oligotrophs able to fix atmospheric gases, methanotrophs and had a high proportion of rare phylotypes most likely representing new lineages related to microbes detected in anaerobic, anoxic environments, but low abundances of heterotrophic microbes. Geornicrobiological activities are no longer underestimated since studies have shown that bacterial metabolism may lead to mineral precipitation or dissolution. Questions remain as to the identity of these microbes and whether they are actively involved in speleothem formation, or simply buried during mineral precipitation. Results demonstrated a marked difference between sediment communities and those associated with calcite speleothem and calcite mat samples. Results of ESEM and XRD analysis demonstrated that calcite speleothem samples ME3 and MXl are true calcite moonrnilk (mondmilch). Phylogenetic analyses and isolation results demonstrated the unique composition of the microbial communities associated with moonrnilk deposits, predominantly composed of nitrogen-fixing ~-Proteobacteria and psychrotrophic heterotrophic CFBs and to a lesser extent, heterotrophic actinomycetes. Despite XRD and ESEM analysis showing similar calcite composition and crystal morphology, phylogenetic results indicated that sample ME2 represented a very different rnicrohabitat to moonmilk samples, dominated by oligotrophic a.-Proteobacteria and heterotrophic actinomycetes composing 84.2% of the total diversity. Phylogenetic analyses and biodiversity indices reveal the striking similarities between moonmilk samples from both Entrance and Exit Caves and the uniqueness of the calcite mat in Entrance Cave. The one similarity in composition between all three calcite communities was the presence of members of the Pseudonocardineae in particular of the genus Saccharothrix, in all calcite samples. 165 rRNA gene sequencing of cave isolates detected high levels of diversity and novelty, particularly of moonrnilk isolates. A total of two putatively novel genera (within the CFBs and Actinobacteria) and 18 putatively novel species (of genera: Paracoccus, Actinoplanes I Couchioplanes, Micromonospora, Amycolatopsis, Saccharothrix, Bacillus, Paenibacillus, Methylobacterium, Porphyrobacter, Sphingomonas, Alcaligenes, Stenotrophomonas, Xanthomonas) were identified. This study represents the first reported culture-independent analysis of moonrnilk microbial communities globally and of cave sediment communities in the Southern Hemisphere. Information gained from this study and the discovery of actively growing microbial communities appearing to precipitate CaC03 provides focus for important future studies and represents a unique opportunity to examine the nature and extent of complex microbe-mineral interactions in the formation of speleothems and implications for cave management. The biodiversity described acts as a baseline for assessing environmental impacts and to identify factors influencing microbial biodiversity.