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Berlin, Germany

Lang I.,University of Gottingen | Lang I.,Cyano Biofuels GmbH | Hodac L.,University of Gottingen | Friedl T.,University of Gottingen | Feussner I.,University of Gottingen
BMC Plant Biology

Background: Among the various biochemical markers, fatty acids or lipid profiles represent a chemically relatively inert class of compounds that is easy to isolate from biological material. Fatty acid (FA) profiles are considered as chemotaxonomic markers to define groups of various taxonomic ranks in flowering plants, trees and other embryophytes.Results: The fatty acid profiles of 2076 microalgal strains from the culture collection of algae of Göttingen University (SAG) were determined in the stationary phase. Overall 76 different fatty acids and 10 other lipophilic substances were identified and quantified. The obtained FA profiles were added into a database providing information about fatty acid composition. Using this database we tested whether FA profiles are suitable as chemotaxonomic markers. FA distribution patterns were found to reflect phylogenetic relationships at the level of phyla and classes. In contrast, at lower taxonomic levels, e.g. between closely related species and even among multiple isolates of the same species, FA contents may be rather variable.Conclusion: FA distribution patterns are suitable chemotaxonomic markers to define taxa of higher rank in algae. However, due to their extensive variation at the species level it is difficult to make predictions about the FA profile in a novel isolate. © 2011 Lang et al; licensee BioMed Central Ltd. Source

Lang I.,Cyano Biofuels GmbH | Lang I.,Mount Allison University | Kaczmarska I.,Mount Allison University
Diatom Research

Many major advances in macro-organismal biology, population genetics and evolutionary biology may be attributed to the development of rapid and inexpensive molecular methodology. Here, a simple protocol is presented for a polymerase chain reaction (PCR) of alcohol-preserved single diatom cells that facilitate studies requiring a large number of individuals, inclusive of archived and uncultivable specimens. Our approach integrates cell isolation and PCR amplification of two molecular markers, as well as post-PCR identification by scanning electron microscopy, thereby bridging the gap between classical and molecular diatom biology. The amplification of two genes was performed in a two-step protocol. By applying multiple sets of PCR primers to a single diatom cell, an internal fragment of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (RuBisCO, rbcL) was successfully amplified and sequenced, as well as the internal transcribed spacer (ITS) region in 20% of the same, preserved cells derived from natural phytoplankton. Multiple-gene recovery from a single-cell source requires further optimization, likely specific for the taxa of interest. Single marker recovery (from first-step amplification to sequencing) from natural, fixed single-cell samples was more successful with overall success rate of ∼31% for ITS and 67% for rbcL, respectively. Admittedly it is 50-70% lower than rates for a culture-pelleted DNA source (68% for ITS and 100% for rbcL) using current standard methods of sequence recovery in diatom studies, but our method is much less labor- and cost-intensive. This approach was applied to the diatom Ditylum brightwellii to assess the quality of recovered sequences. The phred quality scores of our sequences derived from single-cell material were similar to phred scores of our own, published sequences obtained from cultures grown in our laboratory. © 2011 The International Society for Diatom Research. Source

Tillich U.M.,Wildau University of Applied Sciences | Tillich U.M.,Humboldt University of Berlin | Lehmann S.,Wildau University of Applied Sciences | Schulze K.,Wildau University of Applied Sciences | And 2 more authors.

Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria Synechocystis sp. PCC6803. Based on these results an optimal dosage of 10-50 J/m2 for UV and either 0.1 or 1 v% for MMS was determined. A Synechocystis wildtype culture was then mutagenized and selected for increased temperature tolerance in vivo. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate in vivo screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for in vivo, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods. © 2012 Tillich et al. Source

Gaudry A.,University of Strasbourg | Lorber B.,University of Strasbourg | Neuenfeldt A.,University of Strasbourg | Neuenfeldt A.,Cyano Biofuels GmbH | And 3 more authors.
Protein Engineering, Design and Selection

Mitochondrial aminoacyl-tRNA synthetases are key enzymes in translation. They are encoded by the nuclear genome, synthesized as precursors in the cytosol and imported. Most are matured by cleavage of their N-terminal targeting sequence. The poor expression of mature proteins in prokaryotic systems, along with their low solubility and stability after purification are major obstacles for biophysical and crystallographic studies. The purpose of the present work was to analyze the influence of additives on a slightly soluble aspartyl-tRNA synthetase and of the N-terminal sequence of the protein on its expression and solubility. On the one hand, the solubility of the enzyme was augmented to some extent in the presence of a chemical analog of the intermediary product aspartyl-adenylate, 5′-O-[N-(L aspartyl) sulfamoyl] adenosine. On the other hand, expression was enhanced by extending the N-terminus by seven natural amino acids from the predicted targeting sequence. The re-designed enzyme was active, monodisperse, more soluble and yielded crystals that are suitable for structure determination. This result underlines the importance of the N-terminal residue sequence for solubility. It suggests that additional criteria should be taken into account for the prediction of cleavage sites in mitochondrial targeting sequences. © 2012 The Author. Source

Neuenfeldt A.,University of Strasbourg | Neuenfeldt A.,Cyano Biofuels GmbH | Lorber B.,University of Strasbourg | Ennifar E.,University of Strasbourg | And 4 more authors.
Nucleic Acids Research

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and noncognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclearencoded proteins to cooperate with degenerated organelle RNAs. © 2013 The Author(s). Source

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