Kroth, Peter G.
Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum
2019-09-13, Buck, Jochen Mario, Sherman, Jonathan, Río Bártulos, Carolina, Serif, Manuel, Halder, Marc, Henkel, Jan, Falciatore, Angela, Lavaud, Johann, Kroth, Peter G., Lepetit, Bernard
Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms.
Discovery of a kleptoplastic 'dinotom' dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids
2019-07-19, Yamada, Norico, Bolton, John J., Trobajo, Rosa, Mann, David G., Dąbek, Przemysław, Witkowski, Andrzej, Onuma, Ryo, Horiguchi, Takeo, Kroth, Peter G.
A monophyletic group of dinoflagellates, called 'dinotoms', are known to possess evolutionarily intermediate plastids derived from diatoms. The diatoms maintain their nuclei, mitochondria, and the endoplasmic reticulum in addition with their plastids, while it has been observed that the host dinoflagellates retain the diatoms permanently by controlling diatom karyokinesis. Previously, we showed that dinotoms have repeatedly replaced their diatoms. Here, we show the process of replacements is at two different evolutionary stages in two closely related dinotoms, Durinskia capensis and D. kwazulunatalensis. We clarify that D. capensis is a kleptoplastic protist keeping its diatoms temporarily, only for two months. On the other hand, D. kwazulunatalensis is able to keep several diatoms permanently and exhibits unique dynamics to maintain the diatom nuclei: the nuclei change their morphologies into a complex string-shape alongside the plastids during interphase and these string-shaped nuclei then condense into multiple round nuclei when the host divides. These dynamics have been observed in other dinotoms that possess permanent diatoms, while they have never been observed in any other eukaryotes. We suggest that the establishment of this unique mechanism might be a critical step for dinotoms to be able to convert kleptoplastids into permanent plastids.
Genome editing in diatoms : achievements and goals
2018-10, Kroth, Peter G., Bones, Atle M., Daboussi, Fayza, Ferrante, Maria I., Jaubert, Marianne, Kolot, Misha, Nymark, Marianne, Río Bártulos, Carolina, Serif, Manuel, Falciatore, Angela
Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.
Production of Chemicals from Microalgae Lipids : Status and Perspectives
2018-01, Hess, Sandra K., Lepetit, Bernard, Kroth, Peter G., Mecking, Stefan
The engineering of algae strains, cultivation and further processing steps in microalgae production are considered in view of the utilization of microalgae lipids for chemicals. Insights from biofuel production trials on the one hand and existing processes for very high-value pharmaceuticals on the other hand are instructive to this end. A recent example of the production of a surfactant from chemical intermediates gained from algae oil and further opportunities are discussed.
Influence of the algal microbiome on biofouling during industrial cultivation of Nannochloropsis sp. in closed photobioreactors
2019-09, Giraldo, Javier B., Stock, Willem, Dow, Lachlan, Roef, Luc, Willems, Anne, Mangelinckx, Sven, Kroth, Peter G., Vyverman, Wim, Michiels, Mark
Industrial cultivation of microalgae is becoming increasingly important, yet the process is still hampered by many factors, including contamination and biofouling of the algal reactors. We characterized a subset of microorganisms occurring in the broth and different biofilm stages of industrial scale photobioreactors applied for the cultivation of Nannochloropsis sp. A total of 69 bacterial strains were isolated, belonging to at least 24 different species. In addition, a green microalga was isolated and identified as Chlamydomonas hedleyi. The effect of C. hedleyi and 24 of the bacterial isolates on the productivity of Nannochloropsis was evaluated through growth and biofilm assays. C. hedleyi was shown to reduce growth and induce biofilm formation in Nannochloropsis. These effects were however indirect as they could be attributed to the bacteria associated to C. hedleyi and not C. hedleyi itself. Although most bacterial strains reported no effect, several were able to induce biofilm formation.
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.
The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum
2018-08, Ewe, Daniela, Tachibana, Masaaki, Kikutani, Sae, Gruber, Ansgar, Río Bártulos, Carolina, Konert, Grzegorz, Kaplan, Aaron, Matsuda, Yusuke, Kroth, Peter G.
Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.
Organelle Studies and Proteome Analyses on Mitochondria and Plastids Fractions from the Diatom Thalassiosira pseudonana
2019-08-01, Schober, Alexander, Río Bártulos, Carolina, Bischoff, Annsophie, Lepetit, Bernard, Gruber, Ansgar, Kroth, Peter G.
Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration, and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from western blots and mass spectrometry. LC-ESI-MS/MS studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.
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.
Reduced vacuolar β-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum
2018-05-01, Huang, Weichao, Haferkamp, Ilka, Lepetit, Bernard, Molchanova, Mariia, Hou, Shengwei, Jeblick, Wolfgang, Río Bártulos, Carolina, Kroth, Peter G.
The β-1,3-glucan chrysolaminarin is the main storage polysaccharide of diatoms. In contrast to plants and green algae, diatoms and most other algal groups do not accumulate storage polysaccharides in their plastids. The diatom Phaeodactylum tricornutum possesses only a single gene encoding a putative β-1,3-glucan synthase (PtBGS). Here, we characterize this enzyme by expressing GFP fusion proteins in P. tricornutum and by creating and investigating corresponding gene silencing mutants. We demonstrate that PtBGS is a vacuolar protein located in the tonoplast. Metabolite analyses of two mutant strains with reduced amounts of PtBGS reveal a reduction in their chrysolaminarin content and an increase of soluble sugars and lipids. This indicates that carbohydrates are shunted into alternative pathways when chrysolaminarin production is impaired. The mutant strains show reduced growth and lower photosynthetic capacities, while possessing higher photoprotective abilities than WT cells. Interestingly, a strong reduction in PtBGS expression also results in aberrations of the usually very regular thylakoid membrane patterns, including increased thylakoid thickness, reduced numbers of thylakoids per plastid, and increased numbers of lamellae per thylakoid stack. Our data demonstrate the complex intertwinement of carbohydrate storage in the vacuoles with carbohydrate metabolism, photosynthetic homeostasis, and plastid morphology.