Structural and Functional Diversity of alpha-Proteobacterial Acidocalcisomes/polyphosphate

在拟议的项目中，我们将确定细菌酸钙体（多磷酸盐夹杂物中的液泡质子焦磷酸酶[V-H+-PPase，HppA]驱动的酸性膜封闭区室）和非膜封闭的多磷酸盐颗粒的非酸钙体形成细菌之间的结构和功能差异。我们假设hppA基因的存在表明形成酸钙体的能力（所有基因组测序的α-变形菌都具有hppA基因，并且已经显示在根癌农杆菌和红杜鹃中形成酸钙体）。我们将从A.根癌农杆菌（具有膜）和新月柄杆菌（假定具有膜封闭的酸钙体）。我们将确定酸钙体的元素组成，确定酸钙体相关蛋白，并通过体外和体内方法确定酸钙体膜的脂质组成。这些知识将使我们能够比较细菌酸钙体与真核单细胞酸钙体（e. G.克氏锥虫）和来自在聚磷酸盐颗粒周围缺乏脂质膜的细菌的聚P颗粒。我们将通过结合生长和生命成像实验以及野生型和产生的突变菌株的表型分析来研究细菌酸钙体的功能，在所述突变菌株中，在A. tumefaciens或C. crescentus。该项目将大大增加我们对细菌细胞器的了解。

In the proposed project we will determine the structural and functional differences between bacterial acidocalcisomes (polyphosphate inclusions in a vacuolar proton pyro-phosphatase [V-H+-PPase, HppA]-driven acidic membrane-enclosed compartment) and non-membrane-enclosed polyphosphate granules of non-acidocalcisome-forming bacteria. We postulate that the presence of an hppA gene is indicative for the ability to form acidocalcisomes (all genome-sequenced alpha-proteobacteria have a hppA gene and the formation of acidocalcisomes in Agrobacterium tumefaciens and Rhodospirillum rubrum has been shown). We will isolate acidocalcisomes from A. tumefaciens (with membranes) and from Caulobacter crescentus (postulated to have membrane-enclosed acidocalcisomes). We will determine the elemental composition of acidocalcisomes, identify the acidocalcisome-associated proteins and determine the lipid-composition of the acidocalcisome membrane by in vitro and in vivo methods. This knowledge will enable us to compare bacterial acidocalcisomes with acidocalcisomes from eukaryotic unicellular counterparts (e. g. Trypanosomas cruzi) and with polyP granules from bacteria lacking a lipid membrane around polyphosphate granules. We will investigate the function of bacterial acidocalcisomes by a combination of growth and life imaging experiments and phenotype analysis of the wild type and generated mutant strains in which the key gene necessary for the formation of the acidic acidocalcisome compartment (V-H+-PPase, hppA) and/or the key gene of polyP synthesis (polyP kinase gene, ppk) are deleted in A. tumefaciens or C. crescentus. This project will considerably increase our knowledge on bacterial organelles.