Phasin PhaF, a multifunctional protein associated with the surface of polyhydroxyalkanoate (PHA) granules that also interacts with the nucleoid, contributes significantly to PHA biogenesis in pseudomonads. As a protein present on the surface of PHA granules, PhaF participates in granule stabilization and segregation, whereas its deletion has a notable impact on overall transcriptome, PHA accumulation and cell physiology, suggesting more extensive functions besides solely being a granule structural protein. Here, we followed a systematic approach to detect potential interactions of PhaF with other components of the cell, which could pinpoint unexplored functions of PhaF in the regulation of PHA production. We determined the PhaF interactome in Pseudomonas putida KT2440 via pull‐down‐mass spectrometry (PD‐MS) experiments. PhaF complexed with PHA‐related proteins, phasin PhaI and the transcriptional regulator PhaD, interactions that were verified to be direct using in vivo two‐hybrid analysis. The determination of the PHA granule proteome showed that PhaI and three other potential PhaF interacting partners, but not PhaD, were granule‐associated proteins. Analysis of the interaction of PhaF and PhaD with the phaI promoter by EMSA suggested a new role for PhaF in interacting with PhaD and raises new questions on the regulatory system controlling pha gene expression.

中文翻译：

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Phasin相互作用组揭示了恶臭假单胞菌中PhaF与多羟基链烷酸酯转录调节蛋白PhaD的相互作用。

Phasin PhaF是一种与聚羟基链烷酸酯（PHA）颗粒表面相关的多功能蛋白质，该蛋白质也与该类核苷酸相互作用，对伪单胞菌中PHA的生物发生有重要贡献。作为PHA颗粒表面上的一种蛋白质，PhaF参与了颗粒的稳定和分离，而其缺失对整体转录组，PHA积累和细胞生理有显着影响，表明它不仅是颗粒结构蛋白，还具有更广泛的功能。在这里，我们遵循一种系统化的方法来检测PhaF与细胞其他成分的潜在相互作用，这可以指出PhaF在调节PHA产生中的未开发功能。我们确定了恶臭假单胞菌中的PhaF相互作用基因组通过下拉质谱（PD-MS）实验进行KT2440。PhaF与PHA相关蛋白，phasin PhaI和转录调节因子PhaD复合，通过体内二杂交分析证实相互作用是直接的。对PHA颗粒蛋白质组的测定表明，PhaI和其他三个潜在的PhaF相互作用伴侣是颗粒相关蛋白，但不是PhaD。EMSA对PhaF和PhaD与phaI启动子的相互作用的分析表明，PhaF在与PhaD相互作用中具有新的作用，并且对控制pha基因表达的调控系统提出了新的问题。