Assembly of Chitin-based Meshworks and their Role in Silica Morphogenesis

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In this subproject we will investigate the role of the chitin-based meshwork, an insoluble organic matrix found in the diatom Thalassiosira pseudonana, in biosilica formation. For this purpose we will extract and identify the proteins/peptides associated with the chitin-based meshwork. The genes of these proteins/peptides will be cloned and homologously expressed with tags to investigate whether they are associated with the biosilica in vivo. Cloning of the six predicted chitin synthase genes and their homologous expression as GFP-fusion or peptide-fusion proteins will allow for the localization of the chitin synthases in the diatom cells, to identify the enzyme(s) involved in biosynthesis of the chitin-based meshwork. Transcription analysis of chitinase genes by qPCR in synchronized cells, in combination with biochemical identification of extracellular and intracellular chitinases is expected to identify candidate chitinases (out of the 22 predicted chitinases of T. pseudonana) that may be involved in remodeling of the chitin-based meshwork in vivo. By expression of microbial chitinases in T. pseudonana we will attempt to inhibit or reduce the formation of the chitin-based meshwork, and analyze the effect on cellular growth rate and morphology of the silica. In complementary studies at later stages of the project we will attempt to generate and analyze chitin synthase knock-down mutants. The interaction of the chitin-based meshwork with silacidins (highly phosphorylated peptides), and the role of silacidins in biosilica formation will be further investigated by studying the effect of hypo-phosphorylated silacidin variants on silica formation in vivo and in vivo. Additionally, it will be attempted to generate silacidin knock-down mutants to analyze the role of silacidins on the survival, growth and silica morphology of T. pseudonana in vivo.