Genetic Mainpulation of Silica Morphogenesis in the Diatom Thalassiosira pseudonana
Zusammenfassung der Projektergebnisse
The species specific nano- and micropatterned silica (SiO2) cell walls of diatoms, a group of unicellular eukaryotic algae, are fascinating examples for the biological phenomenon of biomineralization. Silica formation in diatoms involves longchain polyamines and phosphoproteins (silaffins and silacidins), which spontaneously form dynamic supramolecular assemblies that accelerate the deposition of silica in vitro. However, in vitro synthesis of diatom-like silica structures has not yet been accomplished, indicating that the molecular program underlying biosilica morphogenesis in diatoms is still incomplete understood. To enable the investigation of diatom biosilica morphogenesis in vivo, the development of genetic methods for “gene silencing” and “gene replacement by homologous recombination (HR)” in the model diatom Thalassiosira pseudonana was attempted. To establish RNA-antisense mediated gene silencing in T. pseudonana, two mutant strains which constitutively express the reporter enzyme betaglucuronidase (GusA) were transformed with anti-gusA antisense constructs of different designs. Expression of the antisense constructs was inducible which allows for measuring GusA activity when expression of antisense constructs is turned “on” and turned “off”. Reduction of GuA activity triggered by each construct was less than 35 % and the level of down-regulation in both GusA parent strains was not consistent. Therefore it remains unclear how effective gene silencing can be trigger in T. pseudonana. The challenge for gene replacement in diatoms is that random chromosomal integration of DNA introduced by transformation is an order of magnitude more frequent than homologous integration. To select for integration into actively transcribed genes we tested the promoterless antibiotic resistance marker nat for homologous replacement of the tpSil3 gene (codes for the cell wall protein silaffin3). Transformation of T. pseudonana with the tpSil3-replacement vector consisting of the promoterless nat gene flanked by tpSil3-homologous sequence and a GFP expression cassette, yielded antibiotic resistant, non-fluorescent transformants. tpSil3-replacement mutants were expected to show this phenotype. Eighteen transformants were analyzed for replacement of tpSil3 but none of them had the nat gene inserted into tpSil3. To identify new members of the biosilica forming machinery, a novel sequence homology-independent bioinformatics screen for silaffin-like proteins the genome database of T. pseudonana was pursued. This screen retrieved 86 silaffin-like proteins of mostly unknown function. In vivo localization of six silaffin-like proteins (denoted cingulins) revealed them to be integral constituents of an insoluble organic matrix that exhibits the nanopattern and steric architecture of T. pseudonana girdle band silica. In vitro, the novel insoluble matrix precipitated silica in a controlled manner along preferred sites thereby producing non-porous silica structures reminiscent of girdle band silica. Insoluble organic matrices with silica-specific nanoand micropattern and silica forming activity were also discovered in other diatoms and therefore may present a new mechanistic principle for silica morphogenesis in diatoms.
