Structure and formation of PHB granule in bacteria

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Poly(3-hydroxybutyrate), PHB, is the most important storage compound for carbon and energy in prokaryotes. PHB granules at the early stages of formation are not randomly distributed in the cells but are – in contrast to literature – localized at the cell poles and/or at the inner side of the cytoplasmic membrane suggesting a specific interaction of PHB granule-associated proteins with unknown target proteins in the cell periphery. This assumption is supported by recent identification of cytoskeleton-like structures on the surface of isolated PHB granules by others. In this study subcellular localisation of active and inactive PHB synthase-Gfp fusion will be determined at different growth stages in Ralstonia eutropha (model organism for PHB accumulation, but small cell diameter) and Azotobacter vinelandii (PHB accumulation in cells with ø ≥2μm). A set of different fluorescent dyes with specificity for either cytoplasmic membrane, cytoplasm and PHB granules will be used to follow localization of PHB synthase-Gfp fusion and PHB granules formation in individual living cells. Proteins that specifically interact with PHB synthase and with phasin proteins (proteins of the PHB granule surface) will be identified by two hybrid analysis. Function of identified proteins will be investigated by disruption mutant analysis.