Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei
Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei
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DOI：
10.1128/aem.07114-11
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发表时间：
2012-03-01
影响因子：
4.4
通讯作者：
Xiang, Hua
中科院分区：
文献类型：
--
作者：
Cai, Shuangfeng;Cai, Lei;Xiang, Hua
聚羟基链烷酸酯（PHA）颗粒相关蛋白（PGAPS）是PHA合成和颗粒形成的重要组成部分，但目前对盐古菌PGAPS的研究甚少。本研究对地中海盐古菌PGAPs进行了鉴定和功能分析。这些PGAP可视化与二维凝胶电泳（2-DE）和鉴定基质辅助激光解吸电离-串联飞行时间质谱（MALDI-TOF/TOF MS）。颗粒上最丰富的蛋白质被鉴定为一种假设的蛋白质，命名为PhaP。全基因组分析显示，phaP基因位于先前鉴定的phaEC基因的上游。通过基因敲除/互补和发酵分析的综合方法，我们证明了PhaP参与PHA积累。与野生型菌株相比，Delta phaP突变体在PHA生物合成和细胞生长方面都有缺陷。此外，透射电子显微镜结果表明，在Delta phaP突变体细胞中PHA颗粒的数量显著降低，并且在大多数Delta phaP细胞中仅观察到单个大颗粒。这些结果表明，H. mediacheiPhaP是PHA颗粒上的主要结构蛋白（phasin），参与PHA积累和颗粒形成。此外，BLASTp和系统发育的结果表明，这种类型的PhaP是唯一的盐古菌中保守的，这意味着它是一个代表盐古菌型PHA相。
The polyhydroxyalkanoate (PHA) granule-associated proteins (PGAPs) are important for PHA synthesis and granule formation, but currently little is known about the haloarchaeal PGAPs. This study focused on the identification and functional analysis of the PGAPs in the haloarchaeon Haloferax mediterranei. These PGAPs were visualized with two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). The most abundant protein on the granules was identified as a hypothetical protein, designated PhaP. A genome-wide analysis revealed that the phaP gene is located upstream of the previously identified phaEC genes. Through an integrative approach of gene knockout/complementation and fermentation analyses, we demonstrated that this PhaP is involved in PHA accumulation. The Delta phaP mutant was defective in both PHA biosynthesis and cell growth compared to the wild-type strain. Additionally, transmission electron microscopy results indicated that the number of PHA granules in the Delta phaP mutant cells was significantly lower, and in most of the Delta phaP cells only a single large granule was observed. These results demonstrated that the H. mediterranei PhaP was the predominant structure protein (phasin) on the PHA granules involved in PHA accumulation and granule formation. In addition, BLASTp and phylogenetic results indicate that this type of PhaP is exclusively conserved in haloarchaea, implying that it is a representative of the haloarchaeal type PHA phasin.