Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.

A produção de etanol nas destilarias brasileiras é baseada na atividade fermentativa da levedura Saccharomyces cerevisiae que utiliza o caldo da cana-de-açúcar e/ou o melaço como substrato. Bactérias contaminantes da fermentação alcoólica competem com as leveduras pelos açúcares, afetando o rendimento do sistema produtivo e, consequentemente, causando perdas econômicas significativas às usinas. Biofilmes formados nos tanques de fermentação alcoólica agem como reservatórios de bactérias, contribuindo para contaminações persistentes e de difícil controle. Os biofilmes proporcionam aos seus habitantes certo grau de proteção contra diversas ameaças do meio, incluindo a ação dos antibióticos. Desta forma, o conhecimento da comunidade bacteriana dos biofilmes é fundamental para as medidas que visam o controle das contaminações na produção do bioetanol. No primeiro estudo, a composição e dinâmica da comunidade bacteriana foram determinadas pela análise de sequências do gene 16S rRNA de biofilmes com diferentes períodos de crescimento, correspondentes aos estágios iniciais de estabelecimento destes biofilmes dentro dos tanques de fermentação alcóolica Os resultados mostraram que estas comunidades foram compostas predominantemente pelas bactérias ácido-lácticas (LAB), com destaque para o gênero Lactobacillus. A visualização da estrutura dos biofilmes por microscopia eletrônica de varredura evidenciou que estes são formados por bactérias e leveduras (biofilmes mistos). No segundo estudo, a suscetibilidade aos antimicrobianos (monensina, virginiamicina e beta-ácido derivado do lúpulo) e a capacidade de formação de biofilmes em culturas puras foram avaliadas para isolados de Lactobacillus spp. provenientes de biofilmes (células sésseis) e de vinho bruto (células planctônicas) coletados dos tanques de fermentação. A partir dos resultados foi possível observar que as diferenças na suscetibilidade aos antimicrobianos e na habilidade de formar biofilmes foram estirpe-dependentes e que, em alguns casos, o perfil apresentado para algumas espécies mostrou-se relacionado à fonte de isolamento. Este foi o primeiro estudo sobre biofilmes contaminantes da fermentação alcoólica, em escala industrial, para a produção de etanol a partir da cana-de-açúcar / Bioethanol production in Brazilian distilleries is based on fermentative activity of the yeast Saccharomyces cerevisiae which uses sugarcane juice and/or molasses as a substrate. Bacterial contaminants of alcoholic fermentation compete with yeasts for sugars, affecting ethanol yield and consequently causing relevant economic losses to the fuel ethanol industry. Biofilms formed into fermentors act as bacterial reservoirs, contributing to persistent contaminations that are difficult to control. Biofilms provide a certain degree of protection for their inhabitants against some environmental threats, including antibiotics. Thus, understanding bacterial community within biofilms is essential for actions to control contaminations in bioethanol production. In the first study, composition and dynamic of bacterial community were determined by 16S rRNA gene sequences analysis of biofilms with different growth periods, corresponding to initial stages of biofilm establishment in fermentation tanks. Results showed that these communities were dominated by lactic acid bacteria (LAB), mainly of the genus Lactobacillus. Visualization of biofilm structure by scanning electron microscopy revealed a mixed-species biofilm composed by bacteria and yeasts. In the second study, susceptibility to antimicrobials (monensina, virginiamicina and beta-acids from hops) and capacity to form biofilm in pure culture were evaluated for Lactobacillus spp. isolated from biofilms (sessile cells) and wine (planktonic cells) collected from fermentors. The results showed that differences in the susceptibility to antimicrobials and the ability to form biofilms were strain-specific and, in certain cases, the response of some species was related to the isolation source. This was the first investigation of contaminant biofilms from sugarcane-based alcoholic fermentation on an industrial scale

O presente trabalho teve por objetivo avaliar a eficiência de remoção do surfactante aniônico alquilbenzeno linear sulfonado (LAS) em reator anaeróbio de leito granular expandido - EGSB (1,5 litros) com recirculação e alimentação com meio mineral. Além de caracterizar filogeneticamente a diversidade de bactérias na presença do surfactante. O sistema foi operado em condição mesofílica em 4 etapas: (I), (II) e (IV) com TDH de 32 horas, e (III) com TDH de 26 horas. Em todas as etapas a DQO foi em média de 609 \'+ OU -\' 137 mg/L e 14 \'+ OU -\' 1,71 mg/L de LAS afluente. As maiores remoções de LAS foram verificada nas etapas II e IV, com valores de 73,6 \'+ OU -\' 5,6% e 63,6 \'+ OU -\' 6,17%, respectivamente de. Na etapa III essa remoção foi de 47,8 \'+ OU -\' 6,2%. Por meio do balanço de massa constatou-se que 56,6% do total de LAS adicionado foram removidos compreendendo 48,4% por biodegradação e 8,2% por adsorção. A remoção de matéria orgânica não foi afetada com a adição do LAS e nem pela exposição prolongada a esse surfactante. Entretanto, a estrutura do grânulo foi comprometida quando da adição do surfactante, observado pelo aumento da concentração de sólidos totais efluente de 0,049 g/L na etapa I (sem LAS), 0,128 g/L na etapa II, 0,064 g/L na etapa III e 0,038 g/L na etapa IV, quando da adição de 14 \'+ OU -\' 1,71 mg LAS/L. Além disso, foi notada diminuição do diâmetro médio dos grânulos no decorrer da operação do reator de 0,36 cm nas etapas I e III para 0,34 cm na etapa IV. Por meio da técnica de tubos múltiplos (NMP) foi constatado aumento das bactérias anaeróbias totais e diminuição das arqueias metanogênicas, em função do tempo de operação do reator. As bactérias redutoras de ferro representaram 8% da biomassa anaeróbia na etapa IV. Por meio do seqüenciamento da região 16S do RNAr para o domínio Bacteria da biomassa da extremidade superior do reator e da biomassa do leito, foi verificado semelhança com os seguintes filos Proteobacteria, Firmicutes e Synergistetes. Notou-se diferença significativa entre as bibliotecas de clones para essas duas amostras. / This study aimed to evaluate the efficiency of removal of linear alkylbenzene sulfonate (LAS) in expanded bed reactor (1.5 liters) using granular sludge (EGSB) with recirculation and feed with mineral medium modified. The system was operated at mesophilic condition in four stages: (I) (II) and (IV) with HRT of 32 hours, and (III) with HRT of 26 hours. At all stages the COD averaged 609 \'+ OR -\' 137 mg/L and 14 \'+ OR -\' 1.71 mg/L LAS influent. The higher removals of LAS were found in stages II and IV, respectively, 73.6 \'+ OR -\' 5.6% and 63.6 \'+ OR -\' 6.17%. In stage III this removal was 47.8 \'+ OR -\' 6.2%. Through mass balance was found that 56.6% of total LAS added were removed by biodegradation comprising 48.4% and 8.2% by adsorption. The organic matter removal was not affected by the addition of LAS and not by prolonged exposure to this surfactant. However, the granule structure was compromised after the addition of surfactant, the observed increase in effluent total solids concentration of 0.049 g/L in stage I (no LAS), 0.128 g/L in stage II, 0.064 g/L in stage III and 0.038 g/L in stage IV when adding 14 \'+ OR -\' 1.71 mg/L. Furthermore, it was noticed significant decrease in mean diameter of the granules during the operation of the reactor of 0.36 cm in stages I and III to 0.34 cm in stage IV. Through the multiple tube method (MPN) was found to increase the total anaerobic bacteria and methanogenic archaea decreased depending on the time of reactor operation. Iron-reducing bacteria accounted for 8% of anaerobic bacteria total in step IV. By sequencing the 16S rRNA for the domain Bacteria biomass from the upper end of the reactor and the biomass of the bed, was found similar to the following phyla Proteobacteria, Firmicutes and Synergistetes. Significant difference was noted between the clone libraries for these two samples.

Coral diseases were first noted in the 1960s and 1970s and have had major impacts globally on coral reef community structures. In the Caribbean, a major outbreak of white band disease has been considered responsible for the drastic decline of Caribbean Acroporids since the 1970s. In addition to white band disease, another more recently described condition known as rapid tissue loss (RTL) has had major impacts on Acropora cervicornis populations, specifically offshore Broward County Southeast Florida. While these diseases have contributed to the population decline, determining their etiologies has been elusive. Coral diseases have been characterized by shifts in their microbial counterparts within many levels of the coral host. While some coral diseases have had specific pathogens identified, research has not been able to determine pathogens for most. Evidence points toward bacterial causes for many diseases, but due to the complexity of the coral holobiont and the interaction with the environment, elucidating the causes has proven difficult. Many studies have examined the microbiomes of specific diseases and determined some potential pathogens or at least taxa playing important roles in the disease, although none have looked at RTL. Recognizing the local affect of RTL on A. cervicornis, this study set out to gain a baseline understanding of the healthy and RTL affected microbiome of A. cervicornis. 16S rRNA gene sequencing was used to examine the microbiome of completely healthy colonies, healthy regions of diseased colonies, and the disease margin of diseased colonies. Analysis of four microbial diversity metrics revealed marked increases in diversity with respect to declining health states. Additionally, community dissimilarity analysis and analysis of differentially abundant taxa exhibited distinct microbial community structures due to coral health. Several highly abundant (Rickettsiales, Rhodobacteraceae) and a few low abundance (Bdellovibrionales) taxa were identified as primary drivers of the differences. Additionally, Piscirickettsiaceae, a known fish pathogen, was consistently associated with RTL and warrants further investigation. All of the taxa identified with in RTL have been associated with other Acroporid and non-Acroporid diseases throughout the Caribbean and the rest of the world. The consistent IV association of similar taxa for coral diseases around the world, including those found in this study, supports the recent ideas of non-specific primary pathogens. While most disease studies, coral and otherwise, aim to determine a single pathogen for a single disease, this study and others suggest there could be a multitude of organisms responsible for the disease. Therefore understanding the interactions of the coral holobiont and the environment is important to understanding coral disease. While this study reveals significant changes in the bacterial community associated with RTL as well as some potential pathogens, the relationships appear complex and perhaps at a functional level rather than merely taxonomic. Furthermore, this study did not examine viruses, fungi, or protists, which could be possible pathogens. Therefore, to further develop an understanding of RTL and many other coral diseases it will be necessary to consider additional none-bacterial members of the holobiont as well as the bacterial functions and taxa coupled with the roles of environmental factors.

Microbial enzymes implicated in plant cell hydrolysis may have several potential aplications such as biomass degradation biocatalysts or with biofuel production. Bark beetles establish symbiosis with several microbial strains which play different roles benifitting the beetle, as the production of hydrolytic enzymes to degrade the ingested wood, the protection against mirobial antagonist or the detoxification of the environment. Fungal symbionts have been traditionally the best studied, but several recent research with bacterial symbionts of several bark beetle species show that bacterial also display important functions for the host. In this study, the bacterial communities of the bark beetle species Cryphalus piceae and Pithophtorus pithophtorus, collected in the Czech Republic from pine and fir trees, respectively, were isolated and 55 out of 89 samples were identified by 16S rRNA gene amplification and sequencing. Members of the genera Erwinia, Pantoea, Curtobacterium, Yersinia, Pseudomonas and Staphylococcus were detected. The isolates were object of study for their possible biotechnological potential in (ligno)cellulose materials degradation by screening several enzymes implicated in plant cell hydrolysis, as cellulases, xylanases, amylases, laccases, as well as their capability for colorant...

Le microbiote humain est défini par l’ensemble des microbes habitant un site corporel en particulier. Les différents microbiotes de l’Homme, notamment le microbiote intestinal qui est le plus étudié, peuvent moduler de nombreux mécanismes biologiques dont le métabolisme et la réponse immunitaire. Un débalancement du microbiote au niveau des espèces qui le composent, ou dysbiose, a été associé à plusieurs maladies inflammatoires comme le diabète, l’obésité, mais aussi divers types de cancer. De plus, il a été démontré que les bactéries pouvaient avoir un impact sur la réponse des patients aux thérapies contre le cancer. Le cancer du sein est le cancer le plus mortel chez la femme. Or, l’étiologie de la maladie reste incertaine. Récemment, il a été montré que des bactéries pouvaient infiltrer les tissus plus profonds comme le tissu mammaire, formant un microbiote local. Considérant l’impact que la dysbiose peut avoir sur la réponse immunitaire antitumorale et la réponse aux traitements, nous avons émis comme hypothèse qu’une présence bactérienne intratumorale similaire, en composition et en quantité, à celle du tissu normal non-cancéreux affecte la progression du cancer du sein ainsi que le devenir clinique des patientes. La présence du microbiote intratumoral du sein a donc été validée par la détection de plusieurs composants bactériens sur des coupes tumorales à l’aide de marquages moléculaires. Puis, nous avons évalué le rôle potentiel de ce microbiote en quantifiant et identifiant les espèces bactériennes présentes dans les tumeurs et les tissus normaux adjacents des patientes de notre cohorte du cancer du sein. Nos résultats montrent une abondance moins élevée de l’ADN bactérien dans les tumeurs du sein comparativement aux tissus normaux adjacents appariés, suggérant qu’une altération du microbiote mammaire est associée au cancer. De plus, les patientes ayant un signal bactérien très faible dans leur tumeur avaient un nombre de récidives plus élevé. Cette influence de la quantité apparente de bactéries sur le devenir clinique a été observée principalement chez les patientes ayant une tumeur avancée, soit un grade ou un stade élevé, et de sous-types moléculaires Luminal HER2+, HER2+ (non-luminal) et Luminal B. Aucune relation n’a été observée entre la composition bactérienne du microbiote intratumoral mammaire et la récidive. Nos travaux suggèrent une implication pronostique et thérapeutique de la charge bactérienne du microbiote associé aux tumeurs mammaires. / The human microbiota is defined by all the microbes inhabiting a specific body site. The different human microbiota, and in particular the intestinal microbiota which is the most studied, can modulate many biological mechanisms, including metabolism and the immune response. An imbalance in the bacterial species that compose the microbiota, or dysbiosis, has been associated with several inflammatory diseases such as diabetes, obesity, but also various types of cancer. Additionally, bacteria have been shown to impact the response of patients to cancer therapy. Breast cancer is the deadliest cancer in women. However, the etiology of the disease remains uncertain. Recently, it has been shown that bacteria can infiltrate deeper tissues like breast tissue, forming a local microbiota. Considering the impact that dysbiosis can have on the anti-tumor immune response and the response to treatments, we hypothesized that an intratumoral bacterial presence similar in composition and quantity to that of normal non-cancerous tissue affects the progression of breast cancer, as well as the clinical outcomes of patients. The presence of the intratumoral breast microbiota was therefore validated by the detection of several bacterial components on whole tumor sections using molecular staining. Then, we evaluated the potential role of this microbiota by quantifying and identifying the bacterial species present in tumors and adjacent normal tissues of patients in our breast cancer cohort. Our results show a lower abundance of bacterial DNA in breast tumors compared to adjacent paired normal tissues, suggesting that an alteration of the mammary microbiota is associated with breast cancer. In addition, patients with a very low bacterial signal in their tumor had a higher number of recurrences. This influence of the apparent quantity of bacteria on the clinical outcomes has been observed mainly in patients with an advanced tumor, either a high grade or a high stage, and of the Luminal HER2+, HER2+ (non-luminal) and Luminal B molecular subtype. No relationship has been observed between the bacterial composition of the breast intratumoral microbiota and the recurrence. Our work suggests a prognostic and therapeutic implication of the bacterial load of the microbiota associated with breast tumors.

The cell wall provides a complex and rigid structure to the plant for support, protection from environmental factors, and transport. It is mainly composed of polysaccharides, proteins, and lignin. Arabinoxylan (AX), pectin (P), and cellulose (C) are the main components of cereal cell walls and are particularly concentrated in the bran portion of the grain. Cereal arabinoxylans create networks in plant cell walls in which other cell wall polysaccharides are imbedded forming complex matrices. These networks give an insolubility profile to plant cell wall. A previous study in our lab showed that soluble crosslinked arabinoxylan with relatively high residual ferulic acid from corn bran provided advantageous <i>in vitro </i>human fecal fermentation products and promoted butyrogenic gut bacteria. In the present work, arabinoxylan was isolated from corn bran with a mild sodium hydroxide concentration to keep most of its ferulic acid content. Highly ferulated corn bran arabinoxylan was crosslinked to create an insoluble network to mimic the cereal grain cell wall matrices. Firstly, arabinoxylan film (Cax-F), pectin film (P-F), the film produced by embedding pectin into arabinoxylan networks (CaxP-F), and cellulose embedding arabinoxylan matrices (CaxC-F), and embedding the mixture of cellulose and pectin into arabinoxylan networks (CaxCP-F) were fabricated into simulated plant cell wall materials. Water solubility of films in terms of monosaccharide content was examined and revealed that Cax-F was insoluble, and P-F was partially insoluble, and nanosized pectin and cellulose were partially entrapped inside the crosslinked-arabinoxylan matrices. In a further study, these films were used in an <i>in vitro </i>human fecal fermentation assay to understand how gut microbiota access and utilize the different simulated plant cell walls to highlight the role of each plant cell wall component during colonic fermentation. <i>In vitro </i>fecal samples, obtained from three healthy donors were used to ferment the films (Cax-F, P-F, CaxP-F, CaxC-F, and CaxCP-F) and controls (free form of cell wall components -Cax, P and C). The fabricated films that were compositionally similar to cell walls were fermented more slowly than the free polysaccharides (Cax and P). Besides, CaxP-F produced the highest short chain fatty acids (SCFA) amount among the films after 24 hour <i>in vitro </i>fecal fermentation. Regarding specific SCFA, butyrate molar ratio of all films was significantly higher than the free, soluble Cax and P. 16S rRNA gene sequencing explained the differences of the butyrate proportion derived from specific butyrogenic bacteria. Particularly, some bacteria, especially in a butyrogenic genera from Clostridium cluster XIVa, were increased in arabinoxylan films forms compared to the native free arabinoxylan polysaccharide. However, no changes were observed between P and P-F in terms of both end products (SCFA) and microbiota compositions. Moreover, CaxP-F promoted the butyrogenic bacteria in fecal samples compared with pectin alone, arabinoxylan alone, and the arabinoxylan film. Differences in matrix insolubility of the film, which was high for the covalently linked arabinoxylan films, but low for the non-covalent ionic-linked pectin film, appears to play an important role in targeting Clostridial bacterial groups. Overall, the cell wall-like films were useful to understand which bacteria degrade them related to their physical form and location of the fiber polymers. This study showed how fabricated model plant cell wall films influence specificity and competitiveness of some gut bacteria and suggest that fabricated materials using natural fibers might be used for targeted support of certain gut bacteria and bacterial groups.

Des études ont récemment mis en évidence le rôle des lipopolysaccharides (LPS) des bactéries à Gram négatif de la flore intestinale dans le processus de l’inflammation conduisant à l’obésité et au diabète de type 2.Le présent travail est réalisé dans le cadre d’une collaboration entre les équipes du Dr. Caroff (U. Paris-Sud, Orsay) et du Pr. Zhao (U. Jiao Tong, Shanghai). Les expériences présentées ont été réalisées lors de séjours dans les deux laboratoires.Il a été démontré en Chine que des bactéries Sulfato-réductrices (SRB) à Gram négatif étaient présentes en plus forte proportion dans la flore intestinale chez les souris suivant un régime gras. Les mêmes résultats ont été observés chez l'homme. L’hypothèse selon laquelle des SRB seraient à l’origine de grandes quantités d’endotoxines chez les obèses et les patients diabétiques a été émise. Plusieurs souches de SRB isolées de la flore intestinale humaine d’un sujet sain et d’un sujet diabétique ont été cultivées en Chine. Des études de relation structure/activité des LPS isolés de ces bactéries ont été réalisées dans le laboratoire Français pour déterminer leur rôle dans le développement des maladies métaboliques. Les souches isolées des deux sujets ont pu être classées dans le genre Desulfovibrio. Les LPS correspondants ont été extraits et purifiés par des méthodes mises au point dans l’équipe d’Orsay. La structure chimique a été élucidée par les méthodes suivantes : Electrophorèse, Chromatographie sur couche mince, Chromatographie en phase gazeuse et Spectrométrie de masse MALDI. C’est ainsi que des spectres de masse ont été obtenus et que la structure des lipides A, principes actifs des LPS, isolés de SRB a été décrite pour la première fois. Les activités biologiques testées (TNFα, IL-6) varient en fonction du nombre d’acides gras présents. Les LPS de SRB du patient sain ont une structure variable (Smooth versus Rough) en fonction de la quantité de fer présent dans le milieu, et ceux isolés du patient diabétique présentent des structures atypiques qui ne sont pas toutes inflamogènes. Une molécule membranaire inconnue, que nous avons nommée « Glycosyl’X » était co-extraite avec les LPS. Elle joue apparemment un rôle important dans la croissance des SRB et a été étudiée après des étapes de purification complexes. Les structures et le pouvoir inflammatoire de ces molécules dont la structure varie avec les souches, et qui chélatent le fer, ont été étudiées. Elles sont de nature principalement osidique et fixées à la membrane. La proportion de ces molécules par rapport aux LPS varie avec la quantité de fer disponible dans le milieu. Un milieu riche en fer favorise la croissance des Desulfovibrio portant les Glycosyl’X qui n’ont pas de pouvoir inflammatoire eux-mêmes, mais entrent en compétition avec les LPS, modulant ainsi indirectement l’activité de ces derniers. L’augmentation du nombre de Desulfovibrio conduisant à l’augmentation des molécules Glycosyl’X pourrait aussi moduler positivement (par présentation) ou négativement (par élimination des bactéries) l’adsorption du fer dans les intestins dont l’équilibre est essentiel pour l’homéostasie métabolique.Par ailleurs, la croissance des Desulfovibrio augmente la production d’Hydrogène Sulfuré connu pour son action délétère sur les cellules. Nous favorisons l’hypothèse selon laquelle son action sur la disjonction des cellules épithéliales permettrait le passage des différents LPS relargués par la flore Gram-négative intestinale, et même des bactéries entières, vers la circulation sanguine. / Recent studies have highlighted the role of lipopolysaccharide (LPS) in the intestinal flora (gut microbiota) which could contribute to the inflammation process leading to obesity and type 2 diabetes. This thesis is part of a collaborative project between the laboratories of Dr. Caroff (U. Paris -Sud, Orsay, France) and Prof. Zhao (U. Jiao Tong , Shanghai, China). It has been shown by Pr.Zhao’s team in 2010 that the Sulfate -Reducing Bacteria (SRB) were presented in greater proportion in the intestinal mice flora following a fat diet compared to mice following a normal diet. The same results were observed in humans. The starting hypothesis was that SRB could produce a large amount of endotoxin in obese and diabetic patients and play a role in the development of metabolic diseases. Several SRB strains isolated from the human intestinal flora of a healthy subject and of a diabetic subject were grown in the Chinese laboratory. Studies of their LPS structure / activity relationships were carried out in the French laboratory. The aim of this study was to determine their roles in the development of metabolic diseases.Strains isolated from the two subjects could be classified in the Desulfovibrio genus. The corresponding LPS were extracted and purified by the methods developed in the French laboratory. The chemical structure was elucidated by the following methods: Electrophoresis, Thin layer chromatography, Gas chromatography and MALDI mass spectrometry. The mass spectra were obtained and the structure of lipid A, the active part of LPS isolated from SRB was described here for the first time. The biological activities test (TNFα, IL-6) vary depending on the number of fatty acids present in their lipid A structure. The LPS of SRB isolated from the healthy patient had a variable structure (Smooth versus Rough) depending on the amount of iron present in the medium, and those isolated from diabetic patients had atypical structures are not all inflamogenic .An unknown membrane molecule, which we named "Glycosyl'X" was co-extracted with the LPS. It apparently plays an important role in the growth of SRB was investigated after complex purification steps. The structures and the inflammatory power of these molecules variying with strains chelating iron were studied. They are mainly of glycosidic nature and linked to the bacterial membrane.The proportion of these molecules relatively to LPS varies with the amount of iron in the medium. An environment rich in iron promotes the growth of Desulfovibrio Glycosyl'X, molecules but competes with LPS and indirectly modulates the activity of the latter. The increase number of Desulfovibrio leading to increased Glycosyl'X molecules may also modulate positively (by presentation) or negatively (by killing bacteria) the absorption of iron in the intestines which balance is essential for metabolic homeostasis.Furthermore, the growth of Desulfovibrio increasing the production of Hydrogen Sulfide is known for its deleterious effects on the cells. We favor the hypothesis that its action on the separation of epithelial cells favors the passage of different LPS released by the Gram- negative of intestinal flora and even whole cell bacteria into the bloodstream.

Morphological diversity of 61 Anabaena populations of 13 morphospecies was described under the field conditions of Czech fishponds and reservoirs. Polyphasic approach was then applied in classification of 45 clonal strains isolated from those populations. Detailed morphological analyses were performed and partial 16S rRNA gene sequences were obtained for 33 of the strains, and secondary metabolite production was evaluated in 20 strains. Plasticity of morphological characteristics under varied conditions of light, temperature, nitrogen and phosphorus was studied in selected strains, as well as their temperature and light growth requirements. The results were then discussed with respect to the delimitation of single Anabaena morphospecies. A new genus Sphaerospermum was defined for the morphospecies Anabaena kisseleviana, A. reniformis and Aphanizomenon aphanizomenoides, whose phenotypic and genotypic features differed considerably from all other Anabaena morphospecies. Unique information was provided on the occurrence and distribution of A. reniformis and Aph. aphanizomenoides in the Czech Republic.