Structure and formation of PHB granule in bacteria

聚3-羟基丁酸酯（Poly（3-hydroxybutyrate），简称PHB）是原核生物中最重要的碳和能量储存化合物。在形成的早期阶段的PHB颗粒不是随机分布在细胞中，而是-与文献相反-定位在细胞极和/或细胞质膜的内侧，这表明在细胞外周中的PHB颗粒相关蛋白与未知靶蛋白的特异性相互作用。这一假设是支持最近鉴定的cytokerion样结构的表面上分离的聚羟基丁酸颗粒由他人。在这项研究中，将在真养罗尔斯通氏菌（PHB积累的模式生物，但细胞直径小）和棕色固氮菌（细胞中的PHB积累≥2μm）的不同生长阶段确定活性和非活性PHB酶-GFP融合的亚细胞定位。将使用一组对细胞质膜、细胞质和PHB颗粒具有特异性的不同荧光染料来跟踪单个活细胞中的PHB内切酶-Gfp融合和PHB颗粒形成的定位。通过双杂交分析鉴定与PHB合酶和与相蛋白（PHB颗粒表面的蛋白）特异性相互作用的蛋白质。将通过破坏突变体分析研究鉴定的蛋白质的功能。

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.