This work was focused on DNA isolation from cheeses for the use in polymerase chain reaction. First, there was optimised the procedure of homogenisation of different types of cheeses from commercial sources, cell lysis and DNA isolation. DNA was isolated using magnetic microspheres and phenol extraction. It was shown that the DNA was amplified in PCR for domain Bacteria after dilution. Next, there was optimised the procedure of DNA isolation from fresh cheeses and from contaminated fresh cheeses and their pickles. DNA from all samples was amplified in PCR. The presence of DNA of domain Bacteria and yeast DNA was demonstrated. In the last part of the work, there were optimised the preparation of PCR mixtures and bacterial DNA amplification in PCR with primers with clamp (F357-GC and R518). Synthetized PCR products were analysed using DGGE. It was shown that amplicons of DNA isolated from cheeses and pickles differ in positions and numbers. Larger number of bands of different intensities was detected after amplification of DNA isolated from contaminated pickles.

"Où peut-on trouver des microbes, et comment survivent-ils dans ces lieux ?" sont des questions essentielles afin de comprendre la vie sur Terre. Les populations bactériennes du sol sont connues pour jouer un rôle important dans les cycles biogéochimiques, l'entretien des sols, les effets climatiques et l'agriculture.Dans ce travail, j'ai utilisé la technique de pyroséquençage, via le produit d’une PCR d’ADNr 16S amplifiée extraite d’ADN totale, afin de révéler les populations bactériennes présentes dans quatre environnements inhabituels et oligotrophes différents:A. Les écosystèmes saumâtres sont largement distribués sur Terre et sont représentés par des systèmes aquifères salés et des sols salins. Nous avons examiné la composition bactérienne des sédiments des estuaires, sols saumâtres et des échantillons de sol sablonneux de la région de Camargue, échantillonnés pendant deux années consécutives. Les membres appartenant au phylum Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Acidobacteria et Actinobactéries ont été trouvés principalement dans les sols et sédiments. Nous avons constaté que les membres de ces groupes bactériens étaient associés principalement à des bactéries halophiles, sulfatoréductrices (SRB), nitratoréductrices et coliformes, dont leurs proportions ont probablement été affectées par la salinité et leurs localisations géographiques.B. Les bactéries associées à la rhizosphère des plantes sont connues pour jouer un rôle essentiel dans les cycles biogéochimiques, la nutrition des plantes et la lutte biologique contre les maladies végétales. Nous avons examiné les populations bactériennes de la rhizosphère du riz (Oryza sativa) en fin de croissance dans la région de la Camargue en 2013 et 2014. Les populations bactériennes les plus abondantes se sont révélées être des membres appartenant au phylum Proteobacteria, Acidobacteria, Chloroflexi et Gemmatimonadetes. Les genres bactériens auxquels appartiennent ces différents phylums sont connus pour participer dans des processus biogéochimiques du sol, tel que la nitrification, la dénitrification, l'oxydation, ainsi que comme agents de control biologique. Les proportions bactériennes trouvées varient considérablement en fonction de leur localisation géographique et selon l’année d’échantillonnage.C. Nous avons examiné les sols de surface de "Padza de Dapani" situés sur l'île de Mayotte au large de la côte est de l'Afrique, car cette région n’est pas un vrai désert, mais y ressemble due à l’érosion du sol. Les sols de Mayotte sont acides, oligotrophes et minéralisées, et leur population bactérienne principale appartient aux phylums des Actinobactéries, Proteobacteria et Acidobacteria. Un fait intéressant, les membres des genres Acinetobacter, Arthrobacter, Burkholderia et Bacillus sont prédominants dans nos échantillons, comme observé dans des déserts (asiatiques) chauds et jouant probablement un rôle dans la minéralisation des sols, expliquant la désertification.D. Les régions arides de la Terre constituent > de 30% de la surface continentale et les sols oligotrophes sont soumis à des facteurs environnementaux difficiles tels que la faible pluviométrie moyenne annuelle, l'exposition aux UV et les grandes fluctuations de température. Nous avons examiné les populations bactériennes présentes dans la rhizosphère des plantes pionnières et les sols de surface du désert de Jizan d'Arabie Saoudite. Les phylums bactériens les plus abondants appartiennent aux groupes des Bacteroidetes, Proteobacteria et Firmicutes qui diffèrent entre la rhizosphère des plantes étudiées par rapport à la surface du sol, à l'exception de la plante "Panicum Turgidum" qui contient des proportions élevées (70%) des membres appartenant au genre Flavobacterium. / “What microbes are where, and how do they live there” is now an essential question to understand life on Earth, even when comparing seemingly similar ecosystems in different locations. Soil bacterial populations are known to play important roles in biogeochemical cycles, soil maintenance, climatic effects and agriculture. I used pyrosequencing of PCR amplified 16S rDNA from total extracted DNA in order to reveal the bacterial populations living in four different unusual and oligotrophic environments: A. Saline areas are widely distributed on Earth’s and are represented by both saline lakes and saline soils. We examined the bacterial composition of estuary sediments, brackish and sandy soil samples from the Camargue region (Rhône delta in southern France) sampled in two consecutive years. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Acidobacteria and Actinobacteria phyla were found principally in saline sediment and soil samples. We found that members from these phyla were associated principally to halophilic bacteria, sulphate reducing bacteria (SRB), nitrate reducing bacteria and coliforms, and that their varying proportions were likely affected by salinity and geographical location. B. Bacterial populations associated with the rhizosphere of plants are known to play essential roles in biogeochemical cycles, plant nutrition and disease biocontrol. We examined the bacterial populations of the rhizosphere of rice (Oryza sativa) growing in the Camargue region in 2013 and 2014. The most abundant bacterial populations were found to be members belonging to the Proteobacteria, Acidobacteria, Chloroflexi and Gemmatimonadetes phyla. The genera members belong these phyla were found to participate in soil biogeochemical processes such as nitrification, denitrification, oxidation, as well as act as biocontrol agents. The bacterial populations were found to significantly vary by geographical location as well by year of collection. C. We examined the surface soils from “Padza de Dapani” on the island of Mayotte off the east coast of Africa, as this region is not a true (hot) desert, but resembles one due to extensive soil erosion. In the acidic, oligotrophic and mineralized soil samples from Mayotte, members of the Actinobacteria, Proteobacteria and Acidobacteria phyla dominated the bacterial populations. Interestingly, members of the genera Acinetobacter, Arthrobacter, Burkholderia and Bacillus were found to be predominant in our samples, as is also observed in hot (Asian) deserts and may play roles in soil mineral weathering, thus helping to understand desertification processes. D. Earth’s arid regions comprise >30% of the continental surface and the oligotrophic soils are subjected to harsh environmental factors such as low average annual rainfall, high UV exposure and large temperature fluctuations. We examined the bacterial populations present in the rhizosphere of pioneer plants and surface soils in the Jizan desert of Saudi Arabia. The most abundant bacterial phyla belonged to the Bacteroidetes, Proteobacteria and Firmicutes phyla that were different between the rhizosphere of plant versus these from surface sand, with the exception of the plant “Panicum Turgidum”, which contain in its rhizosphere high proportions (70%) of members belonging to the Flavobacterium genus.

No description available.

A plethora of new functions of non-coding RNAs have been discovered in past few years. In fact, RNA is emerging as the central player in cellular regulation, taking on active roles in multiple regulatory layers from transcription, RNA maturation, and RNA modification to translational regulation. Nevertheless, very little is known about the evolution of this \Modern RNA World' and its components. In this contribution we attempt to provide at least a cursory overview of the diversity of non-coding RNAs and functional RNA motifs in non-translated regions of regular messenger RNAs (mRNAs) with an emphasis on evolutionary questions. This survey is complemented by an in-depth analysis of examples from different classes of RNAs focusing mostly on their evolution in the vertebrate lineage. We present a survey of Y RNA genes in vertebrates, studies of the molecular evolution of the U7 snRNA, the snoRNAs E1/U17, E2, and E3, the Y RNA family, the let-7 microRNA family, and the mRNA-like evf-1 gene. We furthermore discuss the statistical distribution of microRNAs in metazoans, which suggests an explosive increase in the microRNA repertoire in vertebrates. The analysis of the transcription of non-coding RNAs (ncRNAs) suggests that small RNAs in general are genetically mobile in the sense that their association with a hostgene (e.g. when transcribed from introns of a mRNA) can change on evolutionary time scales. The let-7 family demonstrates, that even the mode of transcription (as intron or as exon) can change among paralogous ncRNA.

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.

Sustainable farming is an integrated practice of crop and livestock production system (integrated crop-livestock system; ICLS) that aims to reduce the environmental impacts of agricultural practices while maintaining the productivity and profitability. The use of one step's byproducts by another is a crucial component of this practice. The continuity and effectiveness of sustainable farming greatly rely on deep understanding of each component and good management strategy. One essential aspect involved in all farming components is the role of microorganisms in mediating the biological processes therein. Thus, understanding the composition and activities of these communities would open up ways to engineer them and optimize the respective processes for better sustainable farming practices. The research presented in this dissertation aimed to characterize the microbial metabolism involved in the ICLS with a broader goal of manipulating these systems to improve sustainable agriculture. We focused on two systems that are widely used in the United States, and employed the analysis of 16S rRNA-V4 element for this purpose. In our first system, we characterized the rumen microbiomes of beef cattle alternately grazing nontoxic MaxQ and toxic KY-31 tall fescue pasture, to understand how these cultivars shape the rumen microbiome and identify microbial species potentially capable of degrading ergot alkaloids for better feed utilization. We found that KY-31 grazing remodeled the rumen microbiome substantially at the cellulolytic and saccharolytic guilds. It suppressed the abundances of Fibrobacter, a major ruminal cellulolytic bacterium, as well as those of Pseudobutyrivibrio and Butyrivibrio, and these losses were compensated by increased occurrences of Eubacterium species. Parts of these new communities lingered once developed, and a different guild composition surfaced upon transfer to MaxQ. We also discovered that most of the observations were not evident at the whole microbiome levels but was identified by analyzing the sessile and planktonic fractions separately. Thus, it showcased the need for analyzing sessile and planktonic segments separately while interrogating a heterogenous microbiome. Finally, we identified several potential ergovaline degrading bacteria such as Paraprevotella and Coprococcus. In our second system, we studied the microbiome composition and associated transformation pathways mediating nitrogen loss in two dairy manure storage systems, the clay-lined Earthen Pit (EP) and aboveground concrete storage tank (CS) on two commercial dairy farms, to develop strategies to minimize these losses. We first developed a catalog of the archaea and bacteria that were present therein based on the 16S rRNA-V4 amplicons from manure samples collected from several locations and depths of the storages. Then, we inferred the respective metabolic capabilities via PICRUSt2 and literature curation, and developed schemes for nitrogen and carbon transformation pathways operating at various locations of EP and CS. Our results showed that the stored manure microbiome composition was more complex and exhibited more location-to-location variation in EP compared to CS. Further, the inlet and a location with hard surface crust in EP had unique consortia. With regards to nitrogen transformation, the microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to nitrate and further to gaseous compounds such as anammox and autotrophic nitrifiers. However, microbial conversion of nitrate to gaseous N2, NO, and N2O via denitrification and to stable ammonia via dissimilatory nitrite reduction (DNRA) seemed possible. Minor quantity of nitrate was present in manure, potentially originating from oxidative processes occurring on the barn floor. Higher prevalence of nitrate-transforming microbes at the near-surface locations and all depths of the inlet were found as a result of this instance. These findings suggested that ammonia oxidation to nitrate started on the barn floor and as manure is being stored in EP and CS, nitrate was lost to the environment via denitrification. For carbon transformation, hydrogenotrophic Methanocorpusculum species were the primary methane producers, and it exhibited higher abundance in EP. / Doctor of Philosophy / Sustainable farming is an integrated practice of crop and livestock production systems that aims to reduce the environmental impacts of agricultural practices while maintaining the productivity and profitability. The use of one step's byproducts by another such as the utilization of arable land to grow forages for livestock grazing or the use of manure as organic nitrogen amendments for crops is a crucial component of this practice. The continuity and effectiveness of sustainable farming greatly rely on deep understanding of each component and good management strategy. One essential aspect involved in all farming components is the role of microorganisms in mediating the biological processes therein. Thus, understanding the composition and activities of these communities would open up ways to engineer them and optimize the respective processes for a better sustainable farming practice. The research presented in this dissertation aimed to characterize the microbial metabolism involved in the integrated crop-livestock system with a broader goal of manipulating these to improve sustainable agriculture. We focused on two systems that are widely used in the United States, and employed bioinformatic analysis of a genetic marker for this purpose. In our first system, we characterized the rumen microbiomes of beef cattle grazing alternately on KY-31 tall fescue, a major grass used in Virginia that carry a toxin-producing fungi, and nontoxic MaxQ tall fescue pasture, to understand how these cultivars shape the rumen microbiome and identify potential microbial species capable of degrading the toxin for better feed utilization. We found that KY-31 grazing remodeled the rumen microbiome substantially, especially affecting microbes responsible for degrading cellulose and starch. Some of these communities lingered once developed, and a different microbial population surfaced upon transfer to MaxQ. Several potential toxin-degrading bacteria were also identified. In our second system, we studied the microbiome composition and associated transformation pathways mediating nitrogen loss in two dairy manure storage systems, the clay-lined Earthen Pit (EP) and aboveground concrete storage tank (CS), to develop strategies to minimize these losses. We first develop a catalog of the archaea and bacteria that were present in the manure samples collected from several locations and depths of the storages based on a genetic marker. Then, we inferred the respective metabolic capabilities and developed schemes for nitrogen and carbon transformation pathways operating at various locations of EP and CS. Our results showed that the stored manure microbiome exhibited more location-to-location variation in EP compared to CS. Oxygen exposure, continuous addition of fresh manure, and the presence of crust at the storage surface gave rise to these unique populations. With regards to nitrogen transformation, the microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to nitrate and further to gaseous compounds. However, microbial conversion of nitrate to gaseous N2, NO, and N2O seemed possible. These observations showcased that ammonia is stable during storage. Nitrate, on the other hand, can be converted into volatile nitrogen compounds via various processes. Thus, it is imperative to limit the level of nitrate in manure prior to placement in the storage, which is potentially originating from oxidative processes occurring on the barn floor.

There is strong crystallographic evidence that the 23S rRNA is the only catalytic entity in the peptidyl transferase center. Various mechanisms for the catalysis of peptidyl transfer have been proposed. Recently, attention has been given to the idea that the 23S rRNA simply acts to position the tRNA for spontaneous peptidyl transfer and that chemical catalysis may play only a secondary role. Conserved nucleotides U2585 and U2506 are thought to be involved in positioning the 3’ ends of A- and P-site substrates based on the crystallographic evidence, and because mutagenesis at these sites severely impairs peptide bond formation. In this study, pure populations of ribosomes with either U2585A or U2506G mutations in the 23S rRNA were analyzed to test the hypothesis that substitutions at nucleotides U2585 and U2506 in the peptidyl transferase center impair peptide bond formation by altering the position of the 3’ end of P-site tRNA relative to the 23S rRNA. Pure populations of mutant or wild-type ribosomes were obtained by an affinity tagging system and probed with 32P-labeled [2N3A76]tRNAPhe to determine how the 3’ end of tRNA interacts with the ribosomal proteins and 23S RNA at the peptidyl transferase center. Some of the data for the ribosomes with a G at position 2506 are consistent with a model suggested by Schmeing and coworkers in which nucleotide U2506 breaks from its original wobble base pair with nucleotide G2583 during A-site tRNA binding and swings towards the 3’ end of P-site tRNA, while nucleotide U2585 simultaneously moves away from the 3’ end of P-site tRNA.

Azithromycin, a macrolide antibiotic, specifically binds to the 50S ribosomal subunit of bacterial ribosomes and inhibits translation. Azithromycin also prevents 50S ribosomal subunit assembly by binding to a 50S ribosomal subunit precursor particle. When exposed to azithromycin, several ribonucleases in wild-type Escherichia coli cells degrade antibiotic-bound 50S precursor particles. Presumably, cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound precursor less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonucleaseûdeficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability, growth rates, and protein synthesis rates were measured. Degradation of 23S rRNA was examined by hybridization with a 23S specific probe. Ribonuclease II and polynucleotide phosphorylase mutants demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation, suggesting that these two ribonucleases are important for 23S rRNA turnover in azithromycin-treated Escherichia coli.

The genus Chenopodium contains many economically important species in the New World, but is relatively understudied and poorly understood, especially in terms of evolutionary relationships. A better understanding of the structure of this genus could significantly help in breeding efforts on its cultivated members, notably the tetraploid C. quinoa and also certain varieties of C. berlandieri, also tetraploid. Of special concern is determining which diploid weed species are the most likely ancestors for C. quinoa, C. berlandieri, and the other tetraploid members of subsection Cellulata. The phylogeny can be understood in part by examining the ribosomal RNA loci and observing how many copies of the 5S and 45S loci each New World species contains. In this work, the 5S and 45S ribosomal RNA loci are characterized by means of fluorescence in situ hybridization in 23 Chenopodium species collected in the New World, with the 5S locus labeled red and the 45S locus labeled green. Based on these results, the pool of most likely candidate ancestor species for C. quinoa and C. berlandieri includes C. fremontii, C. incanum, C. neomexicanum, and C. watsonii.

The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.