Kroth, Peter G.
2022, Kroth, Peter G., Matsuda, Yusuke
Diatoms are unicellular algae that perform photosynthesis, a process that comprises both the electron transport processes in the thylakoid membranes and the reactions involved in CO2 fixation. The latter process is the starting point for carbohydrate biosynthesis and numerous reactions and pathways in different cellular locations, including the generation, modification, conversion, subsequent storage, and degradation of carbohydrates. While there is vast knowledge of these processes available of land plants and green algae, much less is known of algae like diatoms that are derived from secondary endosymbiosis. Comparative studies on the localization and regulation of photosynthetic pathways in recent years revealed in principle a similarity of photosynthesis in land plants and diatoms, but also a number of peculiar differences, which may be due both to the general phylogenetic distance between these groups and the evolution of diatoms by secondary endosymbiosis, resulting in a different genetic background. This chapter describes the current knowledge of CO2 acquisition and fixation processes in diatoms on the molecular, cellular, and physiological levels in diatoms. Additional focus is laid on photorespiration, as well as carbohydrate pathways, carbohydrate degradation, and carbohydrate storage.
A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum
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.
Valorization of Unconventional Lipids from Microalgae or Tall Oil via a Selective Dual Catalysis One-Pot Approach
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%.
Blue-Light-Induced Unfolding of the Jalpha Helix Allows for the Dimerization of Aureochrome-LOV from the Diatom Phaeodactylum tricornutum
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.
Cell Biology of Organelles
2022, Maier, Uwe G., Moog, Daniel, Flori, Serena, Jouneau, Pierre-Henri, Falconet, Denis, Heimerl, Thomas, Kroth, Peter G., Finazzi, Giovanni
Diatoms are phototrophic, unicellular, and eukaryotic organisms. They originate from secondary endosymbiosis, a specific evolutionary process. Accordingly, their cells and organelles have a typical organisation, as revealed by ultrastructural investigations. Diatoms possess specific compartments and structures, including a silica shell surrounding the diatom cell, the so-called silica deposition vesicles (SDVs), as well as complex plastids that are surrounded by four membranes. Here we provide an overview of diatom organelles, and recapitulate recent information obtained from 3D imaging of whole diatom cells, focusing on the subcellular topology of the model diatom Phaeodactylum tricornutum. This chapter will not discuss issues of the cell wall and the SDVs, which are covered in Chaps. “Structure and Morphogenesis of the Frustule” and “Biomolecules Involved in Frustule Biogenesis and Function”.
N-Acyl Homoserine Lactone Derived Tetramic Acids Impair Photosynthesis in Phaeodactylum tricornutum
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.
Controlled supply of CO2 to batch cultures of the diatom Phaeodactylum 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.
Sensing and Signalling in Diatom Responses to Abiotic Cues
2022, Jaubert, Marianne, Duchêne, Carole, Kroth, Peter G., Rogato, Alessandra, Bouly, Jean-Pierre, Falciatore, Angela
Diatoms are prominent microalgae that proliferate in a wide range of aquatic environments. Still, fundamental questions regarding their biology, such as how diatoms sense and respond to environmental variations, remain largely unanswered. In recent years, advances in the molecular and cell biology of diatoms and the increasing availability of genomic data have made it possible to explore sensing and signalling pathways in these algae. Pivotal studies of photosensory perception have highlighted the great capacity of diatoms to accurately detect environmental variations by sensing differential light signals and adjust their physiology accordingly. The characterization of photoreceptors and light-dependent processes described in this review, such as plastid signalling and diel regulation, is unveiling sensing systems which are unique to these algae, reflecting their complex evolutionary history and adaptation to aquatic life. Here, we also describe putative sensing components involved in the responses to nutrient, osmotic changes, and fluid motions. Continued elucidation of the molecular systems processing endogenous and environmental cues and their interactions with other biotic and abiotic stress signalling pathways is expected to greatly increase our understanding of the mechanisms controlling the abundance and distribution of the highly diverse diatom communities in marine ecosystems.
Isolation of Plastid Fractions from the Diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum
2018, Schober, Alexander, Flori, Serena, Finazzi, Giovanni, Kroth, Peter G., Río Bártulos, Carolina
The so-called "complex" plastids from diatoms possessing four envelope membranes are a typical feature of algae that arose from secondary endosymbiosis. Studying isolated plastids from these algae may allow answering a number of fundamental questions regarding diatom photosynthesis and plastid functionality. Due to their complex architecture and their integration into the cellular endoplasmic reticulum (ER) system, their isolation though is still challenging. In this work, we report a reliable isolation technique that is applicable for the two model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. The resulting plastid-enriched fractions are of homogenous quality, almost free from cellular contaminants, and feature structurally intact thylakoids that are capable to perform oxygenic photosynthesis, though in most cases they seem to lack most of the stromal components as well as plastid envelopes.
Capsules of the diatom Achnanthidium minutissimum arise from fibrillar precursors and foster attachment of bacteria
2014, Leinweber, Katrin, Kroth, Peter G.
Achnanthidium minutissimum is a benthic diatom that may form biofilms on submerged, aquatic surfaces. Within these biofilms, A. minutissimum cells produce extracellular structures which facilitate substrate adhesion, such as stalks and capsules. Both consist of extracellular polymeric substance (EPS), but the microstructure and development stages of the capsules are so far unknown, despite a number of hypotheses about their function, inlcuding attachment and protection. We coupled scanning electron microscopy (SEM) to bright-field microscopy (BFM) and found that A. minutissimum capsules mostly possess an unstructured surface. However, capsule material that was mechanically stressed by being stretched between or around cells displayed fibrillar substructures. Fibrils were also found on the frustules of non-encapsulated cells, implicating that A. minutissimum capsules may develop from fibrillar precursors. Energy-dispersive X-ray (EDX) spectroscopy revealed that the capsule material contains little to no silicon, suggesting that the capsule does not arise from the cell wall. We furthermore show that bacteria attach preferentially to capsules, instead of non-encapsulated A. minutissimum cells, which supports the idea that capsules mediate diatom-bacteria interactions.