Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, 00014, Helsinki, Finland.
Laboratory of Molecular Epidemiology and Evolutionary Genetics, St. Petersburg Pasteur Institute, Mira St. 14, St. Petersburg, 197101, Russia.
Microb Cell Fact. 2019 Feb 7;18(1):29. doi: 10.1186/s12934-019-1079-z.
Cystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli.
Using transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles.
Our results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.
囊病毒在其核衣壳周围有一层磷脂包膜。这种特征在细菌病毒（即噬菌体）中是独一无二的，而噬菌体在细菌宿主内包膜的机制在很大程度上尚不清楚。囊病毒假单胞菌噬菌体 phi6 具有包膜，包膜中含有五种病毒膜蛋白和来自其革兰氏阴性宿主细胞质膜的磷脂。phi6 的主要包膜蛋白 P9 和非结构蛋白 P12 是其病毒包膜所必需的。在假单胞菌宿主中共表达 P9 和 P12 会导致形成细胞内囊泡，这些囊泡可能是 phi6 病毒衣壳组装途径中的中间产物。本研究评估了形成 phi6 特异性囊泡的最低要求，以及将 P9 标记的异源蛋白定位到此类结构中的可能性在大肠杆菌中。
使用透射电子显微镜，我们在表达 P9 的大肠杆菌细胞的细胞质中检测到膜状结构。P9 特异性膜部分的密度较低（约 1.13 g/cm 在蔗糖中），低于细菌细胞质和外膜部分的密度。使用 P9-GFP 融合蛋白研究了异源蛋白靶向 P9 囊泡的情况。GFP 标记的 P9 囊泡的产生需要 P12，P12 可保护融合蛋白免受蛋白水解切割。分离的囊泡主要含有 P9-GFP，表明 P9 标记的融合蛋白选择性地掺入囊泡中。
我们的结果表明，phi6 的主要包膜蛋白 P9 可以在没有任何其他病毒蛋白的情况下触发大肠杆菌细胞质膜结构的形成。在细菌中，细胞内膜结构很少见，因此它们是基于细胞的囊泡生产的理想选择。将异源蛋白定位到 P9 脂质囊泡中为生产具有新型特性的囊泡结构提供了可能。这些产品在生物技术和生物医学中有潜在的用途。
