Kroth, Peter G.
Genetic transformation methods for diatom Nitzschia captiva : New tools to better understand dinotom endosymbiosis
2023-05, Sprecher, Brittany N., Buck, Jochen Mario, Ropella, L. Loraine, Ramsperger, Annette, Kroth, Peter G., Yamada, Norico
Endosymbiosis is a widespread and ecologically significant phenomenon in the marine environment. How these endosymbiotic partners evolve into an organism with a new organelle is still mostly unknown and requires investigation into modern symbioses. Dinotoms, dinoflagellates with evolutionarily intermediate diatom plastids, are considered excellent models for studying organellogenesis as they remain at three successive but distinct stages. Efforts to understand the host dinoflagellate-endosymbiotic diatom relationship has been limited by the lack of genetic transformation methods for either member of the symbiosis. To address this absence, we modified existing diatom biolistic and conjugation transformation methods and cryopreservation protocols for the diatom Nitzschia captiva, an essential prey for the kleptoplastic dinotom Durinskia capensis. Through the use of Phaeodactylum tricornutum, Cylindrotheca fusiformis, and native Nitzschia captiva diatom designed plasmids, we successfully express and target EGFP to the cytosol, mitochondria, and plastids of N. captiva, and visualize these organelles inside D. capensis in vivo, allowing specific labeling and tracking of organelles and proteins after ingestion. Furthermore, we attempt to utilize CRISPR/Cas9 to target the introduced EGFP gene but find no evidence of successful gene editing.
Characterization of a trimeric light-harvesting complex in the diatom Phaeodactylum tricornutum built of FcpA and FcpE proteins
2010, Joshi-Deo, Jidnyasa, Schmidt, Matthias, Gruber, Ansgar, Weisheit, Wolfram, Mittag, Maria, Kroth, Peter G., Büchel, Claudia
Fucoxanthin chlorophyll proteins (Fcps), the light-harvesting antennas of heterokont algae, are encoded by a multigene family and are highly similar with respect to their molecular masses as well as to their pigmentation, making it difficult to purify single Fcps. In this study, a hexa-histidine tag was genetically added to the C-terminus of the FcpA protein of the pennate diatom Phaeodactylum tricornutum. A transgenic strain expressing the recombinant His-tagged FcpA protein in addition to the endogenous wild type Fcps was created. This strategy allowed, for the first time, the purification of a specific, stable trimeric Fcp complex. In addition, a pool of various trimeric Fcps was also purified from the wild-type cells using sucrose density gradient ultracentrifugation and gel filtration. In both the His-tagged and the wild-type Fcps, excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated using circular dichroism spectroscopy. Mass spectrometric analyses of the trimeric His-tagged complex indicated that it is composed of FcpA and FcpE polypeptides. It is confirmed here that a trimer is the basic organizational unit of Fcps in P. tricornutum. From circular dichroism spectra, it is proposed that the organization of the pigments on the polypeptide backbone of Fcps is a conserved feature in the case of chlorophyll a/c containing algae.