Presented diploma thesis is focused on use of DGGE to analysis and identification of selected microorganisms. PCR-DGGE is a method that allows direct characterization of the microbial community in the natural environment without necessity of cultivation. A literature review is devoted to the principle of the method, current applications and its limitations too. In experimental part microbial DNA was isolated and used as a template for PCR reaction. Microbial DNA was then amplified using the universal eukaryotic primers that target the D1/D2 domain of the 26S subunit of ribosomal DNA. To improve specificity and sensitivity of detection nested PCR was chosen using outer and inner primer pairs. Generated amplicons (250 bp) were consequently separated by DGGE. The analysis of selected microorganisms by DGGE technique was performed after optimization of electrophoresis conditions (in particular the denaturing gradient extent and separation time). Despite the optimization, mutual differentiation among individual yeast strains was not possible since each reference strain was represented by several bands in the same positions. In conclusion DGGE profile obtained from wine musts is discussed. Present bands suggest the major presence of non-Saccharomyces yeasts, yeast-like strain A. pullulans is present in the minority and Saccharomyces yeasts are probably present too. The technique remains open for further optimization, particularly as regards the conditions of polymerase chain reaction.

In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).

The dinoflagellate genus Symbiodinium is a popular research topic in the coral reef molecular biology field. Primarily because these organisms serve as the coral holobiont’s primary source of energy, carrying out photosynthesis, and providing hydrocarbons to the coral host. Previous studies have shown the difficulty of isolating Symbiodinium as well as the inherent problems in trying to quantify the diversity of this genus and to qualify the distinct reactions of different Symbiodinium sp. to changing environmental conditions. The main goals of this study are: (1) to detail the relationship between the genetic classification of the organism and its physiology in regard to photosynthesis with a number of established Symbiodinium cultures; and (2) to isolate Symbiodinium from coral of the central Red Sea. To evaluate the photosynthetic physiology of Symbiodinium, a microsensor was used to measure oxygen concentrations along with a phytoplankton analyzer system that used pulse-amplitude-modulation (Phyto-PAM) to measure fluorescence. In order to identify the particular clade that the isolates belonged to, denaturing gradient gel electrophoresis (PCR-DGGE) was used to identify Symbiodinium based on their internal transcribed spacer 2 (ITS2) region. These techniques helped us to achieve our goals in the following ways: Symbiodinium sp. from a culture collection were classified to the subclade level; species-specific and clade-specific photosynthetic profiles were generated; and a Symbiodinium sp. was isolated from the central Red Sea. This study provided preliminary correlation between the photosynthetic difference and Symbiodinium genetic classification; showed the probable existence of a self-protection system inside the Symbiodinium cells by comparing the difference between the initial oxygen production at the beginning of each light step and the oxygen production after light adaptation; and confirmed the possibility of the isolation of Symbiodinium.

Real-time RT-PCR was used to assess the effects of interspecific microbial interactions on the expression of genes associated with lignin peroxidase, manganese peroxidase and alcohol oxidase production during the wood decay process. Expression levels of genes encoding the selected lignolytic enzymes were quantitated in one-, two- and multiple-organism interaction tests with the basidiomycetes Trametes elegans, Phanerochaete chrysosporium, Gloeophyllum sepiarium and Gloeophyllum trabeum. Compression strength loss was measured for each decay sample and correlated with gene expression data for each species. Soil microflora actively producing lignolytic enzymes during wood decay were also assessed and identified using degenerative PCR coupled with denaturing gradient gel electrophoresis, cloning and cycle sequencing. Differential expression was detected in three genes in the two-organism interaction tests: manganese peroxidase in T. elegans interactions, lignin peroxidase A in P. chrysosporium interactions and alcohol oxidase in G. sepiarium interactions. A positive linear correlation was observed between lignin peroxidase A expression and compression strength loss in P. chrysosporium interactions.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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L'ochratoxine A (OTA), principalement produite dans le café par les moisissures A. ochraceus et A. westerdijkiae, suscite une attention particulière pour ses effets néphrotoxiques, immunotoxiques, tératogènes et cancérigènes. La présence d'OTA dans les fèves de café peut être mise en relation avec les conditions de récolte, les conditions de traitement post-récolte et les conditions de stockage et de transport. Dans certains pays producteurs, les dommages causés sur les grains de café par des espèces fongiques sont liés à des teneurs élevées en OTA. La dynamique et la biodiversité des populations microbiennes (bactéries, levures, moisissures) lors des traitements post-récolte du café a été étudiée par une méthode d'analyse moléculaire globale des flores, la PCR-DGGE. Il a été observé une évolution et une diversité des flores microbiennes en fonction du type et des étapes des traitements utilisés, spécifiques des lieux de production. La région génomique ciblée et la proximité phylogénétique sont un obstacle à l'identification des souches ochratoxinogènes par l'approche globale utilisée. De plus, une méthode simple et rapide de différenciation moléculaire d'Aspergillus westerdijkiae et d'Aspergillus ochraceus a été mise au point et couplée avec l'analyse d'image pour permettre la « quantification » d'Aspergillus westerdijkiae. Des phénomènes de compétition/inhibition de la croissance et production d'OTA (supérieurs à 90%) ont été mis en évidence pour des souches d'Aspergillus niger et d'Aspergillus ochraceus faiblement productrices d'OTA vis-à-vis d'Aspergillus westerdijkiae qui est l'une des espèces les plus fortement productrices de la mycotoxine sur café. Les résultats obtenus au cours de ce travail sont importants pour l'amélioration des connaissances sur la dynamique des populations microbiennes au cours des procédés de transformation du café ainsi que pour de possibles applications en prévention et maîtrise de la contamination du café par l'OTA. / Ochratoxin A (OTA) is mainly produced on coffee beans by fungal species Aspergillus ochraceus and Aspergillus westerdijkiae, and is known for its impact on human health through nephrotoxic, immunotox, teratogenic and oncogenic effects.The OTA content in coffee was shown to be closely linked to harvesting conditions, post-harvest processing conditions and especially dry processing, storage and transportation conditions. In some producing countries, damaged caused on beans by fungal communities undoubtedly lead to high OTA contents in coffee. In order to understand the OTA contamination process, the dynamics and biodiversity of microbial populations (bacteria, yeast and moulds) was analyzed during post-harvest treatment by use of a global microbial ecology approach at the molecular level, so-called PCR-DGGE. Specific variations in evolution and diversity of microbial flora were observed as a function of the step and type of treatment, which were specific of the location of production. The genomic region targeted by the global approach and the genetic proximity of ochratoxigenic fungal strains made their study and identification difficult using the the global approach. In addition, a simple and rapid method for the molecular differentiation of A. westerdijkiae and A. ochraceus was established and, coupled with image analysis, allowed the quantification of A. westerdijkiae.Moreover, competition and inhibition effects on growth and OTA production (>90%) could be observed for low OTA producers A. niger and A. ochraceus species towards the high OTA producer A. westerdijkiae species. Results obtained during this study are of importance for understanding microbial population dynamics during coffee transformation processes. Moreover, it provides possible clues for prevention and control of coffee contamination by OTA.

Le compostage est une technique de valorisation des déchets organiques en un produit stable et riche en matières humiques. Certains composts « verts » se sont révélés être de 2 à 3 fois plus efficaces sur la croissance des plantes que les composts classiques. Notre étude réalisée sur un compost fabriqué à partir de déchets végétaux a permis de suivre l’évolution de la densité et de la diversité de la microflore (bactéries, champignons) au cours du processus de maturation puis de tester l’impact de ce compost sur la diversité et l’activité de la microflore tellurique. Cette analyse a été effectuée par des techniques complémentaires : biochimiques (dosages enzymatiques), microbiologiques (cultures in vitro) et de biologie moléculaire (PCR-DGGE, Séquençage). Les résultats montrent qu’au cours de sa maturation, le compost étudié présente une baisse significative de son taux d’humidité et une augmentation sensible de son pH. Sa microflore subit une complète restructuration avec apparition de souches bactériennes susceptibles de dégrader des composés polluants comme les plastiques, les pesticides et les hydrocarbures. L’ajout de ce compost à deux types de sol présentant des propriétés physico-chimiques différentes, n’a pas montré de modifications importantes et durables de la diversité microbienne et fonctionnelle de celui-ci. Les causes de l’effet remarquable de ce compost sur la croissance végétale sont discutées. / Composting is a technique of transformation organic waste in a stable product rich in organic materials. Some "green" compost proved to be from 2 to 3 times more benefit on the growth of the plants than traditional composts. The main of this study is to follow the evolution of density and diversity of the microflora (bacteria, fungi) during the process of maturation of green compost manufactured from vegetable wastes, and to investigate the impact of this compost on the diversity and the activity of the telluric microflora. This analysis was carried out by complementary techniques: biochemical (enzymatic activity), microbiological (in vitro cultures) and molecular biology (PCR-DGGE, DNA sequencing). The results show that during its maturation, the studied compost presents a significant decrease of its water content and an appreciable increase in its pH. The microflora undergoes a complete reorganization with appearance of bacterial strain suitable for degrade polluting compounds like the plastics, the pesticides and hydrocarbons. The addition of this compost with two types of soil presenting of the different physicochemical properties, did not show significant and durable modifications of the microbial and functional diversity of this one. The causes of the remarkable effect of this compost on the vegetable growth are discussed.