Chitin-based organic networks: an integral part of cell wall biosilica in the diatom Thalassiosira pseudonana.

Diatom cell walls are outstanding examples of natural hybrid materials and exhibit extraordinarily interesting mechanical and optical properties. Their structure and composition continue to inspire a variety of biomimetic synthesis approaches. Diatoms are preferred model organisms in silica biomineralization studies. Their hierarchically structured cell walls contain amorphous silica as well as special biomolecules. Over the past decade, three different classes of such biomolecules have been identified: 1) the silaffins, highly post-translationally modified peptides/proteins; 2) long-chain polyamines (LCPAs); and 3) the highly acidic silacidins. The zwitterionic silaffins self-assemble into supramolecular aggregates. The same was observed for LCPAs provided a properly chosen counterion such as orthophosphate or pyrophosphate, or a negatively charged peptide such as silacidin is present. Both the aggregated silaffins as well as the long-chain polyamines induce rapid silica precipitation in vitro from silicic acid containing solutions. To identify these molecules, biosilica was dissolved in HF or NH4F. Silaffins, LCPAs, and silacidins were then found to be dissolved in the extraction solutions. The diatom species Thalassiosira pseudonana is an established model organism in this area; its genome has been completely sequenced. Recent ion-abrasion scanning electron microscopic as well as atomic force microscopic studies on cell wall formation in T. pseudonana revealed the presence of filamentous nanoand microscale structures within the growing cell wall which apparently contain central templating organic structures (“linear proteins”). Chitin (poly-N-acetyl-d-glucosamine) occurs in numerous calciumbased biominerals. It is assumed to form insoluble scaffolds or compartments, wherein chitin-associated biomolecules control calcium biomineralization events. So far, however, chitin has not been identified in biosilica formation in diatom cell walls. On the other hand, several diatom species such as Thalassiosira sp. synthesize external fibers from highly crystalline b-chitin. Interestingly, recent gene expression studies of T. pseudonana indicate a possible role of chitin in cell wall biosynthesis. Furthermore, signals characteristic for polysaccharides such as chitin were observed in solid-state NMR spectroscopic analyses of T. pseudonana cell walls. The aim of the present work is the elucidation of the possible role of chitin in T. pseudonana cell walls, in particular with respect to the presence of “internal” chitin embedded in or tightly bound to the biosilica. SEM images of T. pseudonana cell walls are shown in Figure 1. The cell walls were extracted using the established method based on treatment with sodium dodecylsulfate (SDS) and ethylenediamine tetraacetic acid (EDTA) (see the Experimental Section). Cultures were grown under identical conditions but harvested either with the use of a flow centrifuge or a filter (see Experimental Section). The filtered samples contain large amounts of the well-known external chitin fibers (Figure 1, top). This observation is confirmed by C solid-state NMR spectroscopy (Figure 2). The spectrum of the filtered sample is dominated by intense, narrow resonances at chemical shifts characteristic of crystalline b-chitin . An intense fluorescence of the material can be observed after staining with Calcofluor White, a fluorescence dye that preferentially binds to b-1,4-bound polysaccharides. This confirms the presence of high amounts of external, dye-accessible chitin (see the Supporting Information). In contrast, external chitin is removed from samples harvested with a flow centrifuge (Figure 1, middle). This is confirmed by the complete absence of fluorescence for centrifuge-harvested Calcofluor White stained cell walls (see the Supporting Information). The C solid-state NMR spectrum of centrifuge-harvested samples is a superimposition of various resonances. However, a well-resolved signal at d = 104 ppm is observed which is characteristic for the C1 position of polysaccharides like poly-N-acetyl-d-glucosamine. In monomers such as N-acetyl-d-glucosamine this signal would occur at approximately d = 93 ppm. In other words, a significant amount of polysaccharide is indeed integrated in the biosilica. The linewidth (full width at half maximum height, FWHM) of the C1 signal is approximately 240 Hz. In contrast, for the external chitin fibers the linewidth of this signal amounts to only 110 Hz (see Figure 2A). This observation indicates that the polysaccharide bound in the biosilica is more disordered than that in the crystalline external chitin fibers. [*] Prof. Dr. E. Brunner, P. Richthammer, Dr. H. Ehrlich, Dr. S. Paasch, S. Ueberlein, Prof. Dr. K. -H. v. P e FR Chemie und Lebensmittelchemie, TU Dresden 01069 Dresden (Germany) Fax: (+ 49)351-4633-7188 E-mail: eike.brunner@tu-dresden.de