Function of PhaX in PHB and polyphosphate metabolism in Ralstonia eutropha

Ralstonia eutropha是一种聚（3-羟基丁酸）（PHB）和聚磷酸盐（PP）积累的趋化石自养β -变形菌。通过3个独立的2杂交筛选试验，鉴定了A_2274产物（假定的具有邻近pitA基因[磷酸盐转运蛋白]的磷酸盐转运调节剂）与PHB颗粒相关蛋白，即phasins PhaP2， PhaP3和PHB解聚合酶PhaZa1相互作用。Phasins是存在于所有phb积累细菌中的小的两亲性蛋白。通过分离的PHB颗粒的蛋白质组分析，获得了A_2274与PHB颗粒关联的独立证据。缺失A_2274导致PP形成增加3倍，PHB的再利用（“动员”）受损。分离的PHB颗粒中存在A_2274蛋白，其与3种PHB颗粒相关蛋白的体内相互作用以及缺失突变体的表型表明PHB代谢与PP之间存在联系；因此，A_2274被指定为phaX。本研究的目的是确定PhaX在磷酸盐和PHB代谢中的功能，并了解PHB和PP代谢之间联系的本质。为此，将研究缺失突变体（phaX、pitA、ppk1、ppk2、ppx、phoB、phaZa1、phaC +选择组合）对PP和PHB代谢特定特征的影响，包括蛋白质组学分析。

Ralstonia eutropha is a poly(3-hydroxybutyrate) (PHB) and polyphosphate (PP) accumulating chemolithoautotrophic beta-proteobacterium. The A_2274 product (putative phosphate transport regulator with adjacent pitA gene [phosphate transporter]) was identified in 3 independent 2-hybrid screening assays to interact with PHB granule associated proteins, namely with phasins PhaP2, PhaP3 and with PHB depolymerase PhaZa1. Phasins are small amphiphilic proteins present in all PHB-accumulating bacteria. Independent evidence for the association of A_2274 with PHB granules was obtained by proteome analysis of isolated PHB granules. Deletion of A_2274 resulted in 3-fold increased formation of PP and in impaired reutilization ("mobilization") of PHB. The finding that A_2274 protein is present in isolated PHB granules, its in vivo interaction with 3 PHB granule associated proteins and the phenotype of the deletion mutant suggest the presence of a link between metabolism of PHB and PP; therefore, A_2274 was designated as phaX. The aim of this study is to identify the function of PhaX in phosphate and PHB metabolism and to understand the nature of the link between PHB and PP metabolism. For this, the effect of deletion mutants (phaX, pitA, ppk1, ppk2, ppx, phoB, phaZa1, phaC + selected combinations) on specific features of PP and PHB metabolism including proteome analysis will be investigated.