Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria
Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria
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DOI：
10.1128/aem.03965-12
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发表时间：
2013-05-01
影响因子：
4.4
通讯作者：
Jendrossek, Dieter
中科院分区：
文献类型：
--
作者：
Pfeiffer, Daniel;Jendrossek, Dieter
聚(3-羟基丁酸酯)(PHB)颗粒是一种细胞器样的多酶聚合物复合体(碳小体)，是原核生物中广泛存在的储存化合物。用双分子荧光互补(BIFC)显微镜研究了三种PHB颗粒结合蛋白(PHB合成酶PhaC1、Phasin PhaP5和PHB/DNA结合蛋白Pham)在体内的相互作用。为此，我们建立了一个可移动的双质粒系统，用于在革兰氏阴性菌中用阿拉伯糖控制表达增强型黄色荧光蛋白(EYFP)的N端(YN)和C端(YC)部分的融合蛋白。两种表达载体均能在大肠杆菌和富营养化杆菌的接合子中稳定表达。在PHB允许的条件下，检测到PhaC1、PhaP5和Pham的同源齐聚，以及PhaC1和Pham之间以及Pham和PhaP5之间的相互作用，并与PHB颗粒共定位。在没有PHB的情况下，Pham-PhaC1复合体在中细胞/类核区附近被检测到。与野生型细胞相比，在含有Pham的富营养化杆菌中表达BIFC复合体(Pham同源低聚物或Pham-PhaC1或Pham-PhaP5复合体)导致细胞显著延长，并且BIFC信号通常位于中细胞/类核区域附近。野生型细胞提取液的Western印迹分析和PHB颗粒结合蛋白的蛋白质组分析表明，Pham和PhaP5在富营养化乳杆菌中表达，并且Pham的表达与PHB的存在与否无关。结合纯化的PhaP5-His6和Pham-His(6)的交联实验，尺寸排斥层析分析表明，PhaP5形成二聚体，Pham以低聚(十二聚体)形式存在。这一发现对亚细胞内PHB的定位和PHB颗粒在富营养化杆菌中的形成的启动进行了讨论。
Poly(3-hydroxybutyrate) (PHB) granules are organelle-like multienzyme-polymer complexes (carbonosomes) and are widespread storage compounds in prokaryotes. The interaction of three PHB granule-bound proteins (PHB synthase PhaC1, phasin PhaP5, and PHB/DNA binding protein PhaM) was studied in vivo by bimolecular fluorescence complementation (BiFC) microscopy in Ralstonia eutropha. To this end, a mobilizable 2-plasmid system for arabinose-controlled expression of protein fusions with the N-terminal (YN) and C-terminal (YC) parts of the enhanced yellow fluorescent protein (eYfp) in Gram-negative bacteria was developed. Both plasmids were stably expressed in Escherichia coli and in transconjugants of R. eutropha. Homo-oligomerization of PhaC1, PhaP5, and PhaM and interactions between PhaC1 and PhaM and between PhaM and PhaP5 were detected in R. eutropha and colocalized with PHB granules under PHB-permissive conditions. PhaM-PhaC1 complexes were detected near the midcell/nucleoid region in the absence of PHB. Expression of BiFC complexes in R. eutropha with PhaM (PhaM homo-oligomers or PhaM-PhaC1 or PhaM-PhaP5 complexes) resulted in substantial cell elongation compared to wildtype cells and in BiFC signals that were generally located near the midcell/nucleoid region. Western blot analysis of wild-type cell extracts and proteome analysis of PHB granule-bound proteins revealed that PhaM and PhaP5 are expressed in R. eutropha and that PhaM is constitutively expressed independently of the presence or absence of PHB. Size exclusion chromatography analysis in combination with cross-linking experiments of purified PhaP5-His6 and PhaM-His(6) showed that PhaP5 forms dimers and that PhaM is present in oligomeric (dodecamer) form. Implications of this finding for subcellular PHB localization and initiation of PHB granule formation in R. eutropha will be discussed.