No description available.

We can find a lot of microorganisms living in snow including psychrophilic snow algae from the order Chlamydomonadales (Chlorophyta). They are adapted to the extreme conditions in this habitat and can cause the phenomenon of coloured snow. The species Chlamydomonas nivalis (Bauer) Wille is the most commonly associated with red snow in alpine and polar regions during summer season worldwide. In the field material, we can find red spherical cells without flagella and any morphological characteristics suitable for species determination. Until now, this species has not been isolated into laboratory culture and its life cycle is unclear. Furthermore it has been shown that red coloured snow can be caused by more species which used to be determined as Chlamydomonas nivalis. The aim of this study was to collect samples of red snow from different parts of Europe, to describe the morphological variability of Chlamydomonas nivalis-like snow algae in relation to region of origin, to try to isolate laboratory strain of this species and to describe its position and distribution by phylogenetic analysis of laboratory strains and field samples. Red snow samples were collected from 30 European localities in Slovenian Alps, Romania, Dolomites, Ötztal, Wallis and Sarntal Alps, High Tauern, Ortler massif, in Norway,...

The outer dynein arm-docking complex (ODA-DC) specifies the outer dynein arm-binding site on the flagellar axoneme. The ODA-DC of Chlamydomonas contains equimolar amounts of three proteins termed DC1, DC2, and DC3 (Takada et al., 2002). DC1 and DC2 are predicted to be coiled-coil proteins, and are encoded by ODA3 and ODA1, respectively (Koutoulis et al., 1997; Takada et al., 2002). Prior to this work, nothing was known about DC3. To fully understand the function(s) of the ODA-DC, a detailed analysis of each of its component parts is necessary. To that end, this dissertation describes the characterization of the smallest subunit, DC3. In Chapter II, I report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 D protein with four EF hands that is a member of the CTER (Calmodulin, Troponin C, Essential and Regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming due to loss of some but not all, outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly. As mentioned above, DC3 has four EF-hands: two fit the consensus pattern for calcium binding and one contains two cysteine residues within its binding loop. To determine if the consensus EF-hands are functional, I purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of DTT and Mg2+. As reported in Chapter III, the protein bound one calcium ion with an affinity (Kd) of ~1 x 10-5 M. Calcium binding was observed only in the presence of DTT and thus is redox sensitive. DC3 also bound Mg2+ at physiological concentrations, but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium-binding activity of the bacterially expressed protein. To investigate the role of the EF hands in vivo, I transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the E to Q mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains obscure. In Chapter IV, I report the initial characterization of a DC3 insertional mutant having a phenotype intermediate between that of the DC3-deletion strain and wild type. Furthermore, I suggest future experiments that may help elucidate the specific role of DC3 in outer arm assembly and ODA-DC function. Lastly, I speculate that the ODA-DC may play a role in flagellar regeneration.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20525号 / 生博第367号 / 新制||生||48(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 佐藤 文彦, 教授 河内 孝之, 教授 福澤 秀哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

No description available.

Les triacylglycérols d'algues (TAG) représentent une source prometteuse de biocarburants. Les principales étapes de la synthèse des acides gras et du métabolisme du TAG des algues ont été déduites de celles des plantes terrestres, mais on en sait peu sur les sources de carbones et d’énergie intervenant dans la synthèse de lipides de réserve. Nous avons donc étudié la synthèse des acides gras chez l’algue modèle Chlamydomonas reinhardtii en utilisant une combinaison d'approches génétiques, biochimiques et microscopiques. Plus précisément, j'ai d'abord examiné la localisation subcellulaire de gouttelettes de lipides dans des cellules d'algues exposées à une forte lumière, conditions où une plus grande quantité de pouvoir réducteur est produite. J'ai ensuite contribué à mettre en évidence que la bêta-oxydation des acides gras est un processus peroxysomal, et que pendant une carence en azote réalisée en conditions photoautotrophe, des mutants dépourvus de la malate déshydrogénase 2 peroxysomale (mdh2) accumulent 50% plus TAG que les souches parentales. Ces résultats nous ont permis de mettre en évidence l'importance du contexte redox cellulaire sur la synthèse lipidique. Cette étude a également permis de révéler l’existence d'un échange d’énergie entre le peroxysome et le chloroplaste. Enfin, en caractérisant des mutants déficients dans la dégradation des acides aminés à chaîne ramifiée (BCAA), j'ai montré que le catabolisme des BCAAs joue un double rôle dans la synthèse de TAG en fournissant des précurseurs carbonés et de l'ATP. L'ensemble de ces travaux ouvert de nouvelles pistes pour l'amélioration génétique future de souches d'algues pour la production de biocarburants. / Algal triacylglycerols (TAG) represent a promising source for biofuel. The major steps for fatty acid synthesis and TAG metabolism have been deduced based on that of land plants, but little is known about carbon and energy sources. To address this question, we investigated fatty acid synthesis in algal cells using a combination of genetic, biochemical and microscopic approaches in the model microalga Chlamydomonas reinhardtii. Specifically, I first examined subcellular localization of lipid droplets in algal cells exposed to high light, a condition favoring production of reducing power. Secondly, I contributed to put on evidence that the beta-oxidation of fatty acids is a peroxisomal process, and that during photoautotrophic nitrogen starvation, knock-out mutants of the peroxisomal malate dehydrogenase 2 (mdh2) made 50% more TAG than parental strains, highlighting the importance of cellular redox context on lipid synthesis. This study also revealed for the first time the occurrence of an energy trafficking pathway from peroxisome to chloroplast. And finally, by characterizing mutants defected in degradation of branched-chain amino acids (BCAAs), I showed that BCAA catabolism plays a dual role in TAG synthesis via providing carbon precursors and ATP. Taken together, this work highlighted the complex interplay between carbon and energy metabolism in green photosynthetic cells, and pointed future directions for genetic improvement of algal strains for biofuel productions.

Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht. / The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state.

Biotests sind für die toxikologische Bewertung von Chemikalien, Pflanzenschutzmitteln und schadstoffbelasteten Gewässern oder Böden von besonderer Bedeutung, da sie Auskünfte über die biologische Wirksamkeit eines Stoffes auf Organismen geben. Bislang gibt es für die ökotoxikologische Bewertung, insbesondere von Chemikalien und Pflanzenschutzmitteln, für die autotrophe Organismenebene neben verschiedenen Biotests mit höheren Pflanzen den DIN 28 692 Biotest "Wachstumshemmtest mit den Süßwasseralgen Scenedesmus subspicatus und Selenastrum capricornutum", der auch als OECD 201 Biotest "Algal, Growth Inhibition Test" vorliegt. Dieser aquatische Biotest wird nur mit einer Süßwasseralgenart durchgeführt und trotzdem zunehmend für die Bewertung von belasteten Böden und Sedimenten eingesetzt. Untersuchungen über aquatische Biotests, die Bodenalgen als Testorganismen nutzen, oder Boden-Biotests mit Bodenalgen gibt es nur vereinzelt. Ein Biotestsystem, das sowohl aus einem aquatischen als auch aus einem terrestrischen Biotest besteht und mehrere Bodenalgenarten als Testorganismen nutzt, existiert bisher nicht. Dieses wurde in vorliegender Arbeit entwickelt und an dem Schwermetall Cadmium als Cadmiumchlorid und dem Herbizid Arelon, Wirkstoff Isoproturon erprobt. Um Bodenalgen, die keine Resistenzen oder Toleranzen gegenüber Schadstoffen aufweisen, als Testorganismen nutzen zu können, wurden aus unbelasteten Böden Algen isoliert, Klonkulturen erstellt und die Arten bestimmt. Dies führte zu einer Sammlung mit 35 Algenarten. Aus den in die Bodenalgensammlung aufgenommenen Arten wurden Xanthonema tribonematoides, Stichococcus bacillaris, Klebsormidium flaccidum, Xanthonema montanum und Chlamydomonas noctigama für das Testsystem ausgewählt. Zusätzlich zu diesen wurde die Süßwasseralge Scenedesmus subspicatus als Referenzalge ausgewählt. Mit diesen Algen wurde der Gel-Biotest, bestehend aus einem flüssigen gelartigen Medium, das die Kontaminationspfade im Wasser nachbildet, und ein Boden-Biotest mit einem naturnahen sorptionsschwachen Boden entwickelt, der die Kontaminationspfade über Gas-, Wasser- und Festphase im Boden nachbildet. Bei der Erprobung dieses Biotestsystems mit Cadmiumchlorid und Isoproturon zeigte sich, daß Bodenalgen gegenüber Cadmiumchlorid im Gel-Biotest eine geringe bis mittlere Sensibilität aufwiesen. Im Boden-Biotest lag eine sehr geringe Sensibilität vor, wie dies auch bei anderen Bodenorganismengruppen in Biotests festgestellt wurde. Dies kann mit der Sorption der Cadmiumionen im Boden erklärt werden und dem damit geringen für die Organismen bioverfügbaren Cadmiumionenanteil. Für Isoproturon lag sowohl im Gel- als auch im Boden-Biotest eine hohe Sensibilität der Bodenalgen vor. Erstaunlich war, daß die Sensibilität in beiden Biotests nahezu identisch war, obwohl Isoproturon in sorptionsschwachen Böden zu ca. 30 % adsorbiert wird. Im Vergleich zur Sensibilität von Scenedesmus subspicatus waren die Bodenalgen bei Cadmiumchlorid bis auf zwei Ausnahmen um den Faktor 5 bis 10 unsensibler. Die Bodenalge Klebsormidium flaccidum besaß eine vergleichbare Sensibilität und Xanthonema montanum war um den Faktor 20 unsensibler. Für Isoproturon konnten keine Unterschiede in der Sensibilität zwischen Scenedesmus subspicatus und den geprüften Bodenalgen ermittelt werden, außer bei Stichococcus bacillaris, die um den Faktor 5 unempfindlicher war. Das entwickelte miniaturisierte Biotestsystem eignet sich dazu, differenzierte Aussagen über das ökotoxische Potential von Stoffen auf Bodenalgen und der Süßwasseralge Scenedesmus subspicatus zu erhalten. Durch den Einsatz von zwei unterschiedlichen Testsubstraten (Flüssigmedium und naturnaher Boden) werden der Einfluß dieser Substrate sowie die daraus resultierenden Kontaminationspfade der Teststoffe und ihre ökotoxikologische Wirkung auf Algen feststellbar und vergleichbar. Ein Normenentwurf des Biotestsystems wurde inzwischen in das "Technical Committee 190 - Soil Quality" der International Standards Organization (ISO) eingereicht. / Biotests are an important device to assess the toxicity of chemicals, pesticides, polluted water, and soils because they can provide direct information about the influence of a compound on the organism level. Besides various biotests using higher plants there is only the DIN 28 692 biotest "Growth-inhibition test using fresh water algae Scenedesmus subspicatus and Selenastrum capricornutum" (DIN 28 692) also known as the OECD 201 biotest "Algal, Growth Inhibition Test" which is currently available for an ecotoxicological assessment of chemicals such as pesticides on the autotrophic organism level. This aquatic biotest is based on a single specie of fresh water algae and is increasingly applied to evaluate polluted soils and sediments. There is almost no information on aquatic biotests which are using soil algae as test organisms instead. A more comprehensive biotest system which actually combines aquatic and terrestric biotests using several soil algae species as test organisms has not been reported, yet. Thus, a biotest system was developed and subsequently evaluated by using cadmium (cadmium chloride) as a heavy metal, and the herbicide arelon containing isoproturon as the active ingredient. Soil algae were isolated from unpolluted soil in order to obtain test organisms which are not resistant or tolerant to pollutants. The algae isolates were then cultivated, and subsequently identified. A total of 35 algae species was collected. Algae species used in the biotest system were Xanthonema tribonematoides, Stichococcus bacillaris, Klebsormidium flaccidum, Xanthonema montanum, Chlamydomonas noctigama. In addition, the fresh water specie Scenedesmus subspicatus served as a reference algae. Based on these different algae species a gel biotest using liquid gel medium was developed to investigate the contamination path via water, and also a soil biotest with a pre-treated soil of low sorption capacity was deviced to simulate the contamination path through gas, water, and solid phase. The evaluation of the biotest system using cadmium chloride and isoproturon did reveal that soil algae have had only low to medium sensitivity to cadmium chloride in the gel biotest. Algae sensitivity in the soil biotest was very low which was in accordance with data from other biotests using different soil organisms. The weak response of the algae was most likely caused by the sorption of the cadmium ions to the soil matrix what may have decreased the bioavailability of cadmium. In comparison, soil algae were very sensitive to isoproturon in both, the gel biotest and the soil biotest. Both biotests indicated almost identical sensitivities of the tested soil algae which was surprising since 30 % of the isoproturon was sorbed even in soils with a low sorption capacity. Soil algae when compared to the water algae Scenedesmus subspicatus were generally 5 to 10-fold less sensitive to cadmium chloride. Only Klebsormidium flaccidum has proved to have a similar sensitivity as Scenedesmus subspicatus had, whereas Xanthonema montanum was about 20-fold less sensitive. With isoproturon, however, no differences in sensitivity could be seen between Scenedesmus subspicatus and the tested soil algae, except Stichococcus bacillaris which was about 5-fold less sensitive. The biotest system as developed in this study has shown to be suitable for obtaining valuable information about ecotoxicological effects of chemicals on soil and water algae. Since the biotest system consists of two different test media (liquid gel and soil) it is possible to determine ecotoxicological effects on algae in both, water and soil. A first draft of the developed biotest system has been submitted to the "Technical Committee 190 - Soil Quality" of the International Standards Organization (ISO) for review.

Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as ​Escherichia coli​ and ​Pichia pastoris​ might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum​ , ​Lactococcus lactis​ , ​Pseudomonas fluorescens​ , Pseudoalteromonas haloplanktis​ , ​Ralstonia eutropha​ and ​Streptomyces lividans​ – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – ​Aspergillus fungi, the green algae ​Chlamydomonas reinhardtii​ , the yeast ​Hansenula polymorpha​ , the parasite ​Leishmania tarentolae​ , the moss ​Physcomitrella patens​ and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression systems and vector systems, providing the basis for successful expression of all kinds of complex proteins. Based on the evaluation of expression systems in this review, such toolbox can be obtained.