Hänisch Jan, Wältermann Marc, Robenek Horst, Steinbüchel Alexander
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Corrensstraße 3, D-48149 Münster, Germany.
Department of Cell Biology and Ultrastructural Research, Leibniz Institute for Arteriosclerosis Research, University of Münster, Germany.
Microbiology (Reading). 2006 Nov;152(Pt 11):3271-3280. doi: 10.1099/mic.0.28969-0.
In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli beta-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc(2)155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia-Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1-eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1-eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1-LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of beta-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.
在真养产碱杆菌中，H16 相蛋白 PhaP1 是与聚羟基脂肪酸酯（PHA）包涵体表面结合的主要相蛋白。在本研究中，利用大肠杆菌-分枝杆菌/红球菌穿梭质粒 pJAM2 的耻垢分枝杆菌乙酰胺酶（ace）启动子，将 PhaP1 与增强型绿色荧光蛋白（eGFP）以及与大肠杆菌β-半乳糖苷酶（LacZ）的 C 端融合蛋白分别在积累三酰甘油（TAG）的放线菌——食油红球菌 PD630 和耻垢分枝杆菌 mc(2)155 中表达。PhaP1 和 PhaP1 融合蛋白在重组菌株中稳定表达。对食油红球菌细胞组分的蛋白质免疫印迹分析表明，PhaP1 和 PhaP1-eGFP 融合蛋白与 TAG 包涵体相关，而在可溶性组分和膜组分中未检测到相蛋白或相蛋白融合蛋白。额外的电子显微镜/免疫细胞化学研究表明，PhaP1 主要位于细胞内 TAG 包涵体的表面；此外，一些 PhaP1 也出现在质膜上。对 PhaP1-eGFP 融合蛋白在体内和分离的 TAG 包涵体上的亚细胞分布进行荧光显微镜研究表明，该融合蛋白在 TAG 包涵体形成的各个阶段均与 TAG 包涵体结合，并且在一定程度上与细胞质膜结合。PhaP1-LacZ 融合蛋白也与 TAG 包涵体结合，并且可以与食油红球菌粗提物中的包涵体一起分离，从而证明了β-半乳糖苷酶活性固定在包涵体上。据信，这是第一份证明 PhaP1 除了能与 PHA 包涵体结合外，还能与脂质包涵体结合的报告。此外，还证明了 PhaP1 的这种非特异性可用于将具有酶活性的融合蛋白锚定到细菌 TAG 包涵体的基质上。
