Pfeiffer Daniel, Jendrossek Dieter
Institute of Microbiology, University Stuttgart, Stuttgart, Germany.
Appl Environ Microbiol. 2013 May;79(9):2989-99. doi: 10.1128/AEM.03965-12. Epub 2013 Feb 22.
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 wild-type 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-His6 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.
聚（3-羟基丁酸酯）（PHB）颗粒是细胞器样的多酶聚合物复合物（碳体），是原核生物中广泛存在的储存化合物。通过双分子荧光互补（BiFC）显微镜在真养产碱杆菌中对三种与PHB颗粒结合的蛋白质（PHB合酶PhaC1、phasins蛋白PhaP5和PHB/DNA结合蛋白PhaM）之间的相互作用进行了体内研究。为此，开发了一种可移动的双质粒系统，用于在革兰氏阴性细菌中以阿拉伯糖控制表达与增强型黄色荧光蛋白（eYfp）的N端（YN）和C端（YC）部分融合的蛋白质。这两种质粒在大肠杆菌和真养产碱杆菌的转接合子中均能稳定表达。在真养产碱杆菌中检测到了PhaC1、PhaP5和PhaM的同源寡聚化以及PhaC1与PhaM之间、PhaM与PhaP5之间的相互作用，并且在允许PHB存在的条件下与PHB颗粒共定位。在没有PHB的情况下，在细胞中部/类核区域附近检测到了PhaM-PhaC1复合物。与野生型细胞相比，在真养产碱杆菌中表达带有PhaM的BiFC复合物（PhaM同源寡聚体或PhaM-PhaC1或PhaM-PhaP5复合物）会导致细胞显著伸长，并且BiFC信号通常位于细胞中部/类核区域附近。对野生型细胞提取物的蛋白质印迹分析以及对与PHB颗粒结合的蛋白质的蛋白质组分析表明，PhaM和PhaP5在真养产碱杆菌中表达，并且PhaM的表达是组成型的，与PHB的存在与否无关。结合纯化的PhaP5-His6和PhaM-His6的交联实验进行的尺寸排阻色谱分析表明，PhaP5形成二聚体，而PhaM以寡聚体（十二聚体）形式存在。将讨论这一发现对真养产碱杆菌中亚细胞PHB定位和PHB颗粒形成起始的影响。
