Biogenesis of Polyhydroxyalkanoate Inclusion Bodies in Bacillus Megaterium

聚羟基烷酸（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的影响，令人惊讶的是，人们对它们的结构和生物发生却知之甚少。本项目将鉴定与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，并利用巨型芽孢杆菌确定精确定位所需的精确氨基酸序列。在巨芽孢杆菌中鉴定出2个位点的6个pha基因。其中三个基因（phaA， B和C）的功能可以通过与已知基因的同源性来预测，而其余三个基因（phaP， Q和R）的功能是未知的。利用绿色荧光蛋白（GFP）的翻译融合物检测phaQ和phaR的细胞内定位。将确定phaP和phaC（以及phaQ和phaR，如果合适的话）定位到包涵体所需的氨基酸序列的身份。将对phaP、Q和R缺失突变体的表型进行表征，并通过分析各种缺失突变体中每个pha蛋白的亚细胞定位来测试这些蛋白相互依赖以准确定位。这些实验的数据应该表明PHA、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.