2012, Windler, Miriam, Gruber, Ansgar, Kroth, Peter G.

Benthic diatoms and bacteria often co-operatively build up phototrophic epilithic biofilms. Studying the properties and contributions of the individual partners requires the establishment and maintenance of axenic cultures of the involved organisms. Axenification of biofilm organisms is often challenging, because bacteria as well as diatom cells are embedded in a matrix of extracellular polymeric substances (EPS). Due to this mucilage, the cells stick together and also are less affected by antimicrobial substances. Here we describe a short and feasible protocol for culture axenification, which was successfully applied to cultures of the benthic diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea. Our protocol includes treatment of the cultures with the antibiotic imipenem and might also be useful for the purification of other cultivated diatom strains. Once axenified, diatom cultures often decay after a certain life span. Our protocol is especially useful to re-establish axenic cultures from co-cultures of diatoms with their accompanying bacteria (also referred to as xenic cultures).

2009, Bruckner, Christian G., Kroth, Peter G.

In this study, we describe different combinations of physical separation and antibiotic treatment to remove associated bacteria from freshwater diatoms. Diatoms were purified either from natural epilithic biofilms or from unialgal cultures. We determined that for most strains, different purification procedures have to be combined individually. In a new approach, we show that for some diatom strains, the substitution of associated aquatic bacteria by an antibiotic-sensitive Escherichia coli strain and subsequent treatment with antibiotics may be a successful strategy to obtain axenic diatom cultures. Axenic diatom cultures are essential to study the physiology and biochemistry of individual strains as well as their responses to environmental changes without interference of accompanying bacteria.

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.

2009, Materna, Arne C., Sturm, Sabine, Kroth, Peter G., Lavaud, Johann

Diatoms play a crucial role in the biochemistry and ecology of most aquatic ecosystems, especially because of their high photosynthetic productivity. They often have to cope with a fluctuating light climate and a punctuated exposure to excess light, which can be harmful for photosynthesis. To gain insight into the regulation of photosynthesis in diatoms, we generated and studied mutants of the diatom Phaeodactylum tricornutum Bohlin carrying functionally altered versions of the plastidic psbA gene encoding the D1 protein of the PSII reaction center (PSII RC). All analyzed mutants feature an amino acid substitution in the vicinity of the QB-binding pocket of D1. We characterized the photosynthetic capacity of the mutants in comparison to wildtype cells, focusing on the way they regulate their photochemistry as a function of light intensity. The results show that the mutations resulted in constitutive changes of PSII electron transport rates. The extent of the impairment varies between mutants depending on the proximity of the mutation to the QB-binding pocket and/or to the nonheme iron within the PSII RC. The effects of the mutations described here for P. tricornutum are similar to effects in cyanobacteria and green microalgae, emphasizing the conservation of the D1 protein structure among photosynthetic organisms of different evolutionary origins.