Function of PhaX in PHB and polyphosphate metabolism in Ralstonia eutropha

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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.