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The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host’s metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by the bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

Vegetative cell division in diatoms often results in a decreased cell size of one of the daughter cells, which during long-term cultivation may lead to a gradual decrease of the mean cell size of the culture. To restore the initial cell size, sexual reproduction is required, however, in many diatom cultures sexual reproduction does not occur. Such diatom cultures may lose their viability once the average size of the cells falls below a critical size. Cell size reduction therefore seriously restrains the long-term stability of many diatom cultures. In order to study the bacterial influence on the size diminution process, we observed cell morphology and size distribution of the diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea for more than two years in bacteria-free conditions (axenic cultures) and in cultures that contain bacteria (xenic cultures). We found considerable morphological aberrations of frustule microstructures in A. minutissimum and C. affiniformis when cultivated under axenic conditions compared to the xenic cultures. These variations comprise significant cell length reduction, simplification and rounding of the frustule contour and deformation of the siliceous cell walls, features that are normally found in older cultures shortly before they die off. In contrast, the xenic cultures were well preserved and showed less cell length diminution. Our results show that bacteria may have a fundamental influence on the stability of long-term cultures of diatoms.

Diatoms are unicellular algae, which due to their importance for the global primary production and their cellular and genetic complexity, became popular subjects of physiological and molecular biological research in the recent years. The increasing genomic information gathered on diatoms since the last decade promotes diverse analyses of their steady state RNA levels, which are commonly performed via quantitative real-time PCR (qPCR), a technique which excels in sensitivity and dynamic range. Up to now there are only a few studies on suitable endogenous reference genes in diatoms. Such reference genes are crucial for any relative qPCR study and must feature stable transcript levels between all the investigated experimental conditions. Therefore we expanded the data on suitable endogenous reference genes by thorough testing of ten potential genes in the model diatom Phaeodactylum tricornutum at light and time discriminate conditions. Stably expressed genes for these conditions will be of great use for any diatom study dealing with time and light dependent effects. Samples of algae grown in a 16 hours low light photoperiod and dark transitioned cells were investigated over a period of up to 33 hours. A set of three endogenous reference genes was found to be stably expressed in a light and time independent manner: the TATA box binding protein TBP, the ribosomal protein S1 RPS and the hypoxanthine-guanine phosphoribosyltransferase HPRT. Other commonly used reference genes like actin, histone H4 or 18S ribosomal ribonucleic acid did not perform well and are thus unsuited for expression analysis in light or time dependent experimental setups.

Benthic diatoms and bacteria often co-operatively build up phototrophic epilithic biofilms. Studying the properties and contributions of the individual partners requires the establishment and maintenance of axenic cultures of the involved organisms. Axenification of biofilm organisms is often challenging, because bacteria as well as diatom cells are embedded in a matrix of extracellular polymeric substances (EPS). Due to this mucilage, the cells stick together and also are less affected by antimicrobial substances. Here we describe a short and feasible protocol for culture axenification, which was successfully applied to cultures of the benthic diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea. Our protocol includes treatment of the cultures with the antibiotic imipenem and might also be useful for the purification of other cultivated diatom strains. Once axenified, diatom cultures often decay after a certain life span. Our protocol is especially useful to re-establish axenic cultures from co-cultures of diatoms with their accompanying bacteria (also referred to as xenic cultures).

Diatoms are photoautotrophic, unicellular organisms, found in many aquatic habitats. Diatom plastids most likely evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into another eukaryotic host cell and the subsequent evolutionary reduction and specialisation of this endosymbiont to a cell organelle. Diatom plastids differ from plastids of higher plants in many characteristics, e.g. they are surrounded by four (instead of two) membranes.Plastids and mitochondria contain independent genomes that trace back to the genomes of their free living ancestors, cyanobacteria and Alphaproteobacteria, respectively. The ratio of plastid and mitochondrial genome copies to nuclear genome copies was determined via quantitative real-time polymerase chain reaction (qPCR). Fusion of a plastid targeted recombinase (RecA) to the green fluorescent protein (GFP) leads to selective labelling of plastid nucleoids in the diatom Phaeodactylum tricornutum.Many essential photosynthesis enzymes are encoded in the nuclear genome, the gene products thus have to be imported into the plastids. A conserved sequence motif of unknown function ( ASAFAP -motif) within the N-terminal presequence of plastid preproteins is particularly important for the import reaction. The molecular characterisation of the conserved presequence motif allows conclusions on the targeting mechanisms involved and facilitates the prediction of plastid localised proteins on a genomic scale.The genomes of the model diatoms P. tricornutum and Thalassiosira pseudonana have been sequenced completely. By analysis of the putative intracellular localisations of enzymes based on identified presequences, indications for a C4-like photosynthesis in P. tricornutum were found, and models for carbon concentrating mechanisms and CO2 fixation in P. tricornutum and T. pseudonana have been inferred. Peculiarly, multiple isoforms of enzymes of the Calvin cycle and glycolysis are present in the investigated diatoms. The isoforms differ in their presequences and are putatively active in the plastids, the mitochondria and the cytosol.The exchange of metabolites between stroma, cytosol and other organelles is crucial for plastid function. Eight and six putative plastidic nucleotide transporters are encoded in the genomes of T. pseudonana and P. tricornutum respectively. Fusion proteins of nucleotide transporter presequences or full length fusions to GFP show that the investigated nucleotide transporters are plastid associated.

Effects of ultraviolet radiation on the formation and the germination of zoospores have been investigated in Laminaria digitata. Excised phylloid discs were exposed to controlled ultraviolet radiation of different qualities and intensities in the laboratory. Both sides of the discs were irradiated alternately. Rate of sorus formation, sorus area, zoospore release, amount of cyclobutane-pyrimidine dimers in DNA and germination rate of the released zoospores were determined. In order to determine the relevance of ultraviolet radiation for L.digitata, field measurements of irradiance were collected. The absorption characteristics of zoospore suspensions of the three Helgoland species of Laminaria were investigated. The investigations showed that for phylloid discs irradiated on both sides, ultraviolet radiation has no effect on sorus formation and sorus area. The germination rate of the zoospores was not negatively influenced by ultraviolet radiation during the formation of the sorus. This suggests an effective protection of the sporogenic tissue from ultraviolet radiation. DNA damage was shown in UVB irradiated phylloid discs. The DNA damage was higher in the non-fertile regions of the phylloid discs than in the sorus tissue. Therefore, additional UVB protection mechanisms are suspected. In the field, UVB intensities capable of damaging zoospores were found. In L. digitata, both the zoospores and exudates contribute to the UV absorption of zoospore suspensions. The proportion of the total absorption caused by exudates is higher in L. saccharina and L. hyperborea. In these species, the zoospores themselves show hardly any absorption. Possibly, a significant proportion of exudation takes place in the sorus tissue. All results indicate that L. digitata has adaptive answers to ultraviolet radiation, which counteract the detrimental effects of ultraviolet radiation. This diploma thesis was carried out at the Biologische Anstalt Helgoland. 1. Referee (Advisor): Professor Dr. P. Kroth, University of Konstanz; 2. Referee: Professor Dr. C. Wiencke, Alfred Wegener Institute for Polar and Marine Research, Bremerhaven.