Biogenesis of Polyhydroxyalkanoate Inclusion Bodies in Bacillus Megaterium

储存聚合物聚羟基链烷酸（PHA）在大多数细菌和古细菌的包涵体中积累。 鉴于这些包涵体的广泛存在及其对细胞代谢和生理学的影响，令人惊讶的是，人们对它们的结构和生物发生知之甚少。 该项目将鉴定与 PHA 包涵体共纯化的小蛋白（pha 蛋白）的功能。 重点是在整个生长过程中定位这些蛋白质，并使用巨大芽孢杆菌确定精确定位所需的精确氨基酸序列。 已在巨大芽孢杆菌中鉴定出两个基因座的六个 pha 基因。 其中三个基因（phaA、B 和 C）的功能可以根据与已知基因的同源性进行预测，而其余三个基因（phaP、Q 和 R）的功能未知。 将使用绿色荧光蛋白 (GFP) 的翻译融合来检查 phaQ 和 phaR 的细胞内定位。 将确定 phaP 和 phaC（以及 phaQ 和 phaR，如果合适）定位到包涵体所需的氨基酸序列的同一性。 将表征 phaP、Q 和 R 缺失突变体的表型，并通过分析各种缺失突变体中每种 pha 蛋白的亚细胞定位来测试这些蛋白相互之间的相互依赖性以实现准确定位。 这些实验的数据应表明 phaP、Q 和 R 参与 PHA 包涵体组装的程度，以及它们在该过程中如何相互作用。

The storage polymer polyhydroxyalkanoic acid (PHA) accumulates in inclusion bodies in most bacteria and archaea. Given the widespread occurrence and influence on the metabolism and physiology of the cell of these inclusion bodies, it is surprising that so little is known about their structure and biogenesis. This project will identify the functions of small proteins (pha proteins) that co-purify with PHA inclusion bodies. The focus is on localizing these proteins throughout growth, and on determining the precise amino acid sequences required for accurate localization to take place, using Bacillus megaterium. Six pha genes at two loci have been identified in B. megaterium. The functions of three of these genes (phaA, B and C) can be predicted based on homology to known genes, while those of the remaining three genes (phaP, Q and R) are unknown. Intracellular localization of phaQ and phaR will be examined using translational fusions to the green fluorescent protein (GFP). The identity of the amino acid sequences required for localization of phaP and phaC (and phaQ and phaR, if appropriate) to the inclusion bodies will be determined. The phenotypes of phaP, Q and R deletion mutants will be characterized, and the interdependence of these proteins on each other for accurate localization will be tested by analyzing subcellular localization of each of the pha proteins in various deletion mutants. The data from these experiments should indicate the extent to which phaP, Q and R are involved in the assembly of PHA inclusion bodies, and how they interact with each other in that process.