Eukaryotic microalgae from the Red Sea were isolated, characterized and identified with the purpose of building a culture collection that will serve future research activities in the area of industrial microbiology. Seven sampling locations were surveyed using an in-house designed isolation protocol. Microalgae enrichment was carried out in vitro using the streak plate method and fluorescence activated cell sorting approaches. Colonial and cellular microscopy, growth media preference assays, as well as temperature, pH and salinity tolerance tests were carried out to describe the isolates. DNA extraction, PCR amplification, template sequencing and in silico analyses were carried out to identify the isolates and arrange them in a proper phylogenetic description. In total, 129 isolates were obtained. From these, only 39 were selected for characterization given their increased ability of accumulating large amounts of biomass in solid and liquid media in relatively short periods of time. All of these have a green color, are unicellular, non-motile, photosynthetic organisms and have a cell size ranging from 5 to 8 µm. More than half of them showed growth preference in Walne media, followed by F/2, MN and BG-11 SW. Maximum temperature tolerance of all organisms was around 38 ºC, while optimum growth was observed close to 25 ºC. pH preference was diverse and three groups were identified: acidic (6), intermediate (8 - 9) and alkaline (> 10) growing isolates. Salinity tests showed an overall growth preference at 25 PSU, approximately 10 units lower than that found at the sampling stations. Most isolates showed diminished growth at high salinity and high pH, except for OS3S1b which grew well in both cases, and could be an interesting strain to study further. Twenty four isolates were related to Ulvophyceae sp. MBIC10591 by BLAST approaches with a maximum identity of 96 - 97%. A maximum likelihood phylogenetic tree was created for these isolates, relative to the BLAST hits and to some model eukaryotic microalgae for positioning reference. It was shown that the 24 OS isolates are related to each other with a confidence value of 84%. Differential responses of all high biomass producing isolates towards abiotic stresses might suggest that each represents a distinct, novel, unclassified marine organism.

L'exploration récente des fonds marins a révélé l'existence d'une vie microbienne abyssale bien plus diverse et abondante que l'état de nos connaissances sur les limites du vivant ne le laissait penser. Ainsi on estime que plus de 60 % de la biosphère subit des conditions de pression jugées a priori défavorable au fonctionnement de la machinerie cellulaire. Ces pressions peuvent atteindre 1000 bars dans les fosses sous-marines les plus profondes. La découverte récente de Pyrococcus yayanosii CH1, premier organisme hyperthermophile et piézophile strict, a relancé la question de l'adaptation aux fortes pressions.Au cours de cette thèse cette question de l'adaptation à la haute pression a été abordée à travers les protéines par l'étude de deux familles enzymatiques, les malate déshydrogénases et les glyoxylate hydroxypyruvate réductases, provenant d'organismes piézophiles et non piézophiles.Les études comparatives associant enzymologie, biophysique et cristallographie des protéines présentées dans cette thèse révèlent des différences de comportements significatives vis à vis de la pression, chez des protéines d'une même famille enzymatique. Nos analyses montrent que ces différences portent sur différents aspects de la dynamique fonctionnelle des protéines. Nous montrons donc ainsi que la pression peut "potentiellement" représenter un paramètre discriminant susceptible de faire l'objet d'une adaptation.Le travail réalisé a permis de poser les bases d'une méthode de comparaison exhaustive des propriétés des protéines vis à vis de la pression afin de détecter les traces d'une adaptation piézophile sur d'autres systèmes protéiques. / The recent discovery of marine biodiversity shows that a large part of the biosphere is a high-pressure environment. The existence of a specific pressure adaptation is still an open question. Recently, the first obligate piezophilic hyperthermophilic microorganism was isolated from hydrothermal vent. This finding suggests the existence of a specific enzyme adaptation with respect to high pressure.To deeper understanding protein adaptation with respect to high pressure, we examine the enzymatic properties of two family enzymes, malate deshydrogenases and glyoxylate hydroxypyruvate reductases arising from piezophilic and non-piezophilic organisms.Using an integrated approach combining enzymology, biophysics and X-ray crystallography, we reveal significantly different behaviors with respect to high pressure. Our analysis show that these differences involved the dynamic component of the enzyme. These results suggest that pressure could be a discriminating parameter susceptible to induce an adaptative response.This thesis work allows to set the foundations of a protein-properties comparative method with respect to high pressure to reveal piezophilic adaptation in other protein systems.

Long non-protein coding RNAs (lncRNAs) represent a diverse and enigmatic classification of RNA. With roles associated with development and stress responses, these non-coding gene regulators are essential, and yet remain understudied in plants. Thus far, of just over 430 experimentally validated lncRNAs, only 13 are derived from plant systems and many of which do not meet the classic criteria of the RNA class. Without a solid definition of what makes a lncRNA, and few empirically validated transcripts, methods currently available for prediction fall short. To address this deficiency in lncRNA research, we constructed and applied a machine learning-based lncRNA prediction protocol that does not impose predefined rules, and utilises only experimentally confirmed lncRNAs in its training datasets. Through model evaluation, we found that our novel lncRNA prediction tool had an estimated accuracy of over 96%. In a study that predicted lncRNAs from transcriptomes of evolutionary diverse plant species, we determined that molecular features of lncRNAs display different phylogenetic signal patterns compared to protein-coding genes. Additionally, our analyses suggested that stress-resistant species express fewer lncRNAs than more stress sensitive species. To expand on these results, we used the prediction tool in concert with a transcriptomic study of two natural accessions of the drought tolerant species Eutrema salsugineum. Previously reported to show little physiological differences in a first drought, but differ significantly in a second, we instead demonstrated that the two ecotypes displayed vastly different transcriptomic responses, including the expression of lncRNAs, to a first and second drought treatment. In conclusion, the prediction tool can be applied to studies to further our knowledge of lncRNA evolution and as an additional tool in classic transcriptomic studies. The suggested importance of lncRNAs in drought resistance, and evidence of expression in two natural E. salsugineum accessions, merits further studies on the molecular and evolutionary mechanisms of these putatively regulatory transcripts. / Thesis / Doctor of Philosophy (PhD)

A cultivation-independent approach, sequence analysis of 18S rRNA genes PCR-amplified from environmental DNA, was used to explore the diversity and distribution of eukaryotic microbes inhabiting algal mats in two acidic geothermal streams in Yellowstone National Park. The objectives were to: (1) clarify the identity of mat forming algae in Nymph Creek (2) survey microbial species in the Nymph Creek mat over seasonal intervals along a thermal gradient (3) compare microbial species in the Nymph Creek mat with those in Alluvium Creek mats (4) evaluate microbial species in algal mats formed on different substrates in Alluvium Creek. The results show that a novel red alga dominates high temperature regions (~50ºC) of Nymph Creek and two "Chlorella-like" algae predominate the cooler regions (<38ºC). The predominant algae in Alluvium Creek were distinctly different from those in Nymph Creek. Several stramenophiles and fungi were detected in each algal mat.

No description available.

This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.

No description available.

No description available.