2019-05, Madhuri, Shvaita, Río Bártulos, Carolina, Serif, Manuel, Lepetit, Bernard, Kroth, Peter G.

The recent availability of genome editing tools like TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) for the diatom Phaeodactylum tricornutum has dramatically increased the options to explore diatom biology via reverse genetics. In order to verify that an observed phenotype indeed is directly related to a specific gene knockout and not due to a secondary effect, complementation of the inactivated gene with the wildtype gene and restoration of the wild type phenotype is an essential tool in molecular biology. So far, no strategy for a complementation method has been published for P. tricornutum. Here we demonstrate, as a proof-of principle, the complementation of P. tricornutum AUREO1a knockout strains previously created by TALEN technology. These strains are deficient in the PtAureo1a gene, which is encoding a blue-light dependent transcription factor. pPTbsr, a modified pPha-T1 vector with an antibiotic resistance cassette against Blasticidin served as a complementation vector. In order to avoid the modification of the complementing gene via the potentially still active TALEN nucleases, we have modified the TALEN binding sites of the complementing PtAureo1a gene using synonymous codons. The altered PtAureo1a gene along with its native promoter and terminator was transformed by particle gun bombardment into PtAUREO1a TALEN knockout strains of P. tricornutum. The integration and the expression of PtAUREO1a was confirmed by PCR and western blotting. Due to random integration within the genome, the expression level of the complemented gene may be variable in different lines. Physiological parameters indicated the successful rescue of the wild type phenotype in several lines that showed a similar PtAUREO1a protein content as wild type cells. Our method provides a rapid and efficient tool to complement knockout lines generated by genome editing approaches in P. tricornutum.

2017, Gruber, Ansgar, Kroth, Peter G., Yu, Guilan

For the growth of photosynthetic organisms, supply of CO2 is essential. Experimental work on the uptake and utilisation of inorganic carbon, requires that CO2 concentrations can be adjusted and kept stable. Here we tested the suitability of a culture method that allows supply of CO2 to a cell suspension, without the need of a continuous external gas supply for experimental work with the diatom Phaeodactylum tricornutum. This approach utilizes buffers with different ratios of HCO3-/CO32- in one chamber of a two-tier vessel, releasing different amounts of CO2 to the gas phase of the vessel, which is shared with the cell culture in the other chamber of the vessel. We cultured P. tricornutum under three different CO2 concentrations, while monitoring cell density, CO2 concentration in the gas phase, and pH within the cultures. We found the method very useful for work with P. tricornutum and found that the method also allows the creation of CO2 deplete conditions. This culturing system, while not as precise as a chemostat culture with supply of gas mixtures containing CO2, is simple to use and offers the possibility to adjust CO2 growth conditions.

2013, Rottberger, Julia, Gruber, Ansgar, Boenigk, Jens, Kroth, Peter G.

Chrysophyte algae show wide variation in their nutritional modes, which can be especially advantageous in oligotrophic conditions. However, the capacities and strategies of the algae to adapt to changing conditions may vary due to different nutritional requirements of individual species. In this context, comparative analyses evaluating the physiological range of mixotrophic chrysophytes are important to predict possible changes in phytoplankton composition. We compared 4 freshwater chrysophytes—Poteriospumella lacustris, Poterioochromonas malhamensis, Dinobryon divergens and Mallomonas annulata—under different growth conditions. The diatom Phaeodactylum tricornutum served as a photoautotrophic reference strain. We demonstrate active growth of P. lacustris and the mixotrophic P. malhamensis under chemoheterotrophic conditions. Neither of the chrysophytes were growing photoautotrophically; however, P. malhamensis showed some photosynthetic activity and survived longer when kept in the light. M. annulata, P. tricornutum and the mixotrophic D. divergens showed light-limited growth when kept in inorganic media. Even though D. divergens consumed living bacteria, it did not grow in the dark in the presence of the bacteria only. We detected large differences in the general nutritional preferences between the 2 mixotrophs in relation to the nutritional features of all investigated strains.

2011, Vugrinec, Sascha, Gruber, Ansgar, Kroth, Peter G.

Plastids of diatoms are surrounded by four membranes. The outermost membrane is continuous with the endoplasmic reticulum and therefore is termed chloroplast ER (CER) membrane. The complex ultra structure of diatom plastids naturally requires more transport steps to import nucleus encoded proteins into the plastid compared to higher plant plastids which possess only two envelope membranes.
Several hypothetic models for the import of preproteins into the complex plastids of diatoms are discussed. Common to all these models is the postulation of a first cotranslational transport step into the chloroplast endoplasmic reticulum lumen via the Sec61 translocon. Furthermore, all models postulate transport via a translocator in the innermost membrane similar to the Tic complex (translocon of the inner chloroplasts envelope) of higher plant plastids. The models differ, however, with respect to their explanation of transport out of the CERlumen and into the interenvelope space: either translocators, vesicles crossing the periplastidic space or putative membrane channels connecting CERlumen and the interenvelope space have been proposed. To investigate the presence of such a hypothetic connection between the CERlumen and the interenvelope space, we expressed different preproteins in the diatom Phaeodactylum tricornutum that were fused to selfassembling fragments of GFP (GFP110 and GFP11). Complementary fragments were fused to marker proteins of the CERlumen and the interenvelope space, respectively. Our data indicate that the GFP110 and GFP11 fusion proteins are located in two separate compartments which are not connected to each other.

2019-02-15, Stock, Frederike, Syrpas, Michail, Graff van Creveld, Shiri, Backx, Simon, Blommaert, Lander, Dow, Lachlan, Stock, Willem, Lepetit, Bernard, Kroth, Peter G., Mangelinckx, Sven

Marine bacteria contribute substantially to nutrient cycling in the oceans and can engage in close interactions with microalgae. Many microalgae harbor characteristic satellite bacteria, many of which participate in N-acyl homoserine lactone (AHL) mediated quorum sensing. In the diffusion-controlled phycosphere, AHLs can reach high local concentrations, with some of them transforming into tetramic acids, compounds with a broad bioactivity. We tested a representative AHL, N-(3-oxododecanoyl) homoserine lactone, and its tetramic acid rearrangement product on the diatom Phaeodactylum tricornutum. While cell growth and photosynthetic efficiency of photosystem II were barely affected by the AHL, exposure to its tetramic acid rearrangement product had a negative effect on photosynthetic efficiency and led to growth inhibition and cell death in the long term, with a minimum inhibitory concentration between 20 and 50 μΜ. These results strengthen the view that AHLs may play an important role in shaping the outcome of microalgae-bacteria interactions.

2013-05-07, Herman, Elena, Sachse, Matthias, Kroth, Peter G., Kottke, Tilman

Aureochromes have recently been shown to act as blue-light-regulated transcription factors in the stramenopile alga Vaucheria frigida. They comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module with flavin mononucleotide (FMN) as a chromophore and a basic region leucine zipper (bZIP) domain as an effector. Aureochromes are the only members of a large LOV protein family, where the effector domain is located N-terminal to the sensor domain. This domain inversion positions the linking Jα helix of other LOV proteins to the terminus, raising the question of the role of Jα in aureochrome signaling. In phototropins, signaling proceeds from LOV2 via dissociation and unwinding of the Jα helix to the C-terminal kinase effector domain. In contrast, other LOV proteins have been demonstrated to activate the effector without the unfolding of Jα. We investigated the LOV domain of aureochrome1a from the diatom Phaeodactylum tricornutum both with and without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence that the Jα helix unfolds upon illumination. This unfolding is prerequisite for light-induced dimerization of LOV. Under illumination, full conversion to the dimer was observed by size exclusion chromatography. In the absence of the helix, a monomer was detected in the dark and in the light. As a further effect, the recovery of the dark state is 6-fold slower in LOV-Jα than LOV. We therefore postulate that the Jα helix plays an important role in aureochrome signaling.

2012-06-08, Kikutani, Sae, Tanaka, Rie, Yamazaki, Yukiko, Hara, Satoshi, Hisabori, Toru, Kroth, Peter G., Matsuda, Yusuke

Thioredoxins (Trxs) are important regulators of photosynthetic fixation of CO(2) and nitrogen in plant chloroplasts. To date, they have been considered to play a minor role in controlling the Calvin cycle in marine diatoms, aquatic primary producers, although diatoms possess a set of plastidic Trxs. In this study we examined the influences of the redox state and the involvement of Trxs in the enzymatic activities of pyrenoidal carbonic anhydrases, PtCA1 and PtCA2, in the marine diatom Phaeodactylum tricornutum. The recombinant mature PtCA1 and -2 (mPtCA1 and -2) were completely inactivated following oxidation by 50 μm CuCl(2), whereas DTT activated CAs in a concentration-dependent manner. The maximum activity of mPtCAs in the presence of 6 mm reduced DTT increased significantly by addition of 10 μm Trxs from Arabidopsis thaliana (AtTrx-f2 and -m2) and 5 μm Trxs from P. tricornutum (PtTrxF and -M). Analyses of mPtCA activation by Trxs in the presence of DTT revealed that the maximum mPtCA1 activity was enhanced ∼3-fold in the presence of Trx, whereas mPtCA2 was only weakly activated by Trxs, and that PtTrxs activate PtCAs more efficiently compared with AtTrxs. Site-directed mutagenesis of potential disulfide-forming cysteines in mPtCA1 and mPtCA2 resulted in a lack of oxidative inactivation of both mPtCAs. These results reveal the first direct evidence of a target of plastidic Trxs in diatoms, indicating that Trxs may participate in the redox control of inorganic carbon flow in the pyrenoid, a focal point of the CO(2)-concentrating mechanism.

2017-09-27, Hess, Sandra K., Schunck, Natalie S., Goldbach, Verena, Ewe, Daniela, Kroth, Peter G., Mecking, Stefan

A dual catalysis approach enables selective functionalization of unconventional feedstocks composed of complex fatty acid mixtures with highly unsaturated portions like eicosapentaenoate (20:5) along with monounsaturated compounds. The degree of unsaturation is unified by selective heterogeneous hydrogenation on Pd/γ-Al₂O₃, complemented by effective activation to a homogeneous carbonylation catalyst [(dtbpx)PdH(L)]⁺ by addition of diprotonated diphosphine (dtbpxH₂)(OTf)₂. By this one-pot approach, neat 20:5 as a model substrate is hydrogenated to up to 80% to the monounsaturated analogue (20:1), this is functionalized to the desired C₂₁ α,ω-diester building block with a linear selectivity of over 90%. This catalytic approach is demonstrated to be suitable for crude microalgae oil from Phaeodactylum tricornutum genetically engineered for this purpose, as well as tall oil, an abundant waste material. Both substrates were fully converted with an overall selectivity to the linear α,ω-diester of up to 75%.

2013, Rottberger, Julia, Gruber, Ansgar, Kroth, Peter G.

Mixotrophy is a combination of autotrophic and heterotrophic nutrition. The regulation of the nutritional mode in mixotrophic algae is not yet fully understood. Changes in the light regime are likely to affect cell size of autotrophic, mixotrophic and heterotrophic chrysophytes to a varying extend. Here we demonstrate that an automatic particle analyser (Coulter counter) is a helpful tool not only for cell counts, but also for the detection of changes in mean cell size, total cell bio-volume or size distribution of chrysophyte cultures. The method is time saving and therefore allows large comparative studies, however, the method tends to underestimate the absolute size compared to microscopic measurements. The limitations of automated particle analysis opposed to traditional microscopical measurements are discussed exemplarily for Dinobryon divergens.

2011-04, Bruckner, Christian G., Rehm, Charlotte, Grossart, Hans-Peter, Kroth, Peter G.

Phototrophic epilithic biofilms harbour a distinct assemblage of heterotrophic bacteria, cyanobacteria and photoautotrophic algae. Secretion of extracellular polymeric substances (EPS) by these organisms and the physicochemical properties of the EPS are important factors for the development of the biofilms. We have isolated representative diatom and bacteria strains from epilithic biofilms of Lake Constance. By pairwise co-cultivating these strains we found that diatom growth and EPS secretion by diatoms may depend on the presence of individual bacteria. Similar results were obtained after addition of spent bacterial medium to diatom cultures, suggesting that soluble substances from bacteria have an impact on diatom physiology. While searching for putative bacterial signal substances, we found that concentrations of various dissolved free amino acids (DFAA) within the diatom cultures changed drastically during co-cultivation with bacteria. Further, the secretion of extracellular carbohydrates and proteins can be influenced by bacteria or their extracellular substances. We have performed mass spectrometric peptide mapping to identify proteins which are secreted when co-cultivating the diatom Phaeodactylum tricornutum Bohlin and Escherichia coli. The identified proteins are possibly involved in signalling, extracellular carbohydrate modification and uptake, protein and amino acid modification, and cell/cell aggregation of diatom and bacteria strains. Our data indicate that diatom–bacteria biofilms might be regulated by a complex network of chemical factors involving EPS, amino acid monomers and other substances. Thus interactions with bacteria can be considered as one of the main factors driving biofilm formation by benthic diatoms.