Function of PhaX in PHB and polyphosphate metabolism in Ralstonia eutropha

富营养罗氏杆菌是一种聚3-羟基丁酸酯(PHB)和聚磷酸盐(PP)的富含化学自养的β-蛋白细菌。A_2274产物(与PitA基因相邻的磷酸转运蛋白)在3种独立的双杂交筛选中被鉴定为与PHB颗粒相关蛋白相互作用，即与Phasins PhaP2、PhaP3和PHB解聚酶PhaZa1相互作用。Phasins是一种小的两亲性蛋白质，存在于所有积累PHB的细菌中。通过对分离的PHB颗粒进行蛋白质组学分析，获得了A_2274与PHB颗粒相关的独立证据。A_2274缺失导致PP形成增加3倍，并损害PHB的再利用(“动员”)。分离的PHB颗粒中存在A_2274蛋白，它与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的代谢与PP之间存在联系，因此将A_2274命名为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代谢之间的联系的本质。为此，将研究缺失突变体(phax、pITA、ppk1、ppk2、ppx、Phob、phaZa1、phac精选组合)对PP和PHB代谢特性的影响，包括蛋白质组分析。

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.