Analysis of organo-silica interactions during valve formation in synchronously growing cells of the diatom Navicula pelliculosa.

Biologically formed silica is produced at ambient conditions under the control of molecular and physicochemical processes that are apparently integrated in biosilica morphogenesis, but the mechanisms are not yet fully understood. With the recent identification of small polypeptides and proteins that are encapsulated inside the biosilica and functional in silica polymerization in vitro, it is of importance to determine whether interactions between inorganic silica species and these organic compounds occur in vivo. A time-resolved analysis of valve formation in synchronously growing cells of the diatom species Navicula pelliculosa enabled us to characterize the relevant chemical bonds by attenuated total reflectance Fourier-transformed infrared (ATR-FTIR) spectroscopy. Typically, inorganic bonds of Si-O-Si (bands at 1058, 843 cm(-1)), Si-OH (3689 cm(-1)), and P=O (1239 cm(-1)) and organic bonds of proteinaceous matter (with the amide I and II bands at 1642 and 1543 cm(-1), respectively) were positively identified during one cycle of valve formation. The observed variations in FTIR band intensity and location represented specific interactions between organic and inorganic molecules during the major silicification event, during which stretching of the Si-O bonds was predominantly noticed. The experimentally obtained frequencies (nu) of the major bonds corresponded to those that were obtained by MM+ and PM3 FTIR simulations for organo-silica interactions based on biomolecules that are proposed to be involved in biosilica formation. The results indicated that hydrogen bonds originated from interactions, albeit weak, between organic phosphate or amine groups to the inorganic hydroxyl groups or oxygen atoms from the silicic acid and/or silica. The existence of covalent P-O-Si bonds and electrostatic interactions could not be excluded. These interactions clearly suggest that biomolecules actively contribute to the silica polymerization process during valve formation in N. pelliculosa, and also might act comparably in other diatoms species in which similar biomolecules have been identified.

Possible role of ubiquitin in silica biomineralization in diatoms: identification of a homologue with high silica affinity.

In diatom silicon biomineralization peptides are believed to play a role in silica precipitation and the consequent structure direction of the cell wall. Characterization of such peptides should reveal the nature of this organic-inorganic interaction, knowledge that may eventually well be used to expand the existing range of artificial silicas ("biomimicking"). Biochemical studies on Navicula pelliculosa revealed a set of proteins, which have a high affinity for a solid silica matrix; some were only eluted from the matrix when SDS-denaturation was applied. One of the proteins with an affinity for silica, about 8.5 kDa, is shown to be a homologue of ubiquitin on the basis of its N-terminal amino acid sequence; ubiquitin itself is a highly conserved 8.6 kDa protein that is involved in protein degradation. This finding is in line with a model of silica biomineralization in diatoms that implies the removal of templating polypeptides when pores in the growing cell wall develop. Western blotting with specific anti-ubiquitin antibodies confirmed cross-reactivity. Immunocytochemical localization of ubiquitin indicates that it is present along the diatom cell wall and inside pores during different stages of valve formation.