Abstract
BackgroundCystoviruses 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.ResultsUsing 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/cm3 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.ConclusionsOur 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.
Highlights
Introduction of heterologousP9‐green fluorescent protein (GFP) fusion protein P9‐GFP is susceptible to protease degradation Previously, the phi6 major envelope protein P9 was used as an expression partner to enhance heterologous membrane protein production in E. coli [26]
Bacterial strains and plasmids Escherichia coli JM109 [30] was used as a cloning strain and E. coli BL21(DE3) [31] as an expression strain
P9 induces vesicle formation in the absence of P12 Detection of spherical structures in E. coli thin sections The phi6 major envelope protein P9 and the non-structural protein P12 are the minimum requirements for viral envelope formation in the Pseudomonas host [23]
Summary
P9‐GFP fusion protein P9‐GFP is susceptible to protease degradation Previously, the phi major envelope protein P9 was used as an expression partner to enhance heterologous membrane protein production in E. coli [26]. In this system, GFP was used as an indicator for proper folding of the expressed membrane proteins [26, 38]. The observed pattern of the light scattering zone was similar to that observed for the lysate of E. coli BL21(DE3) control cells, except for an intense green zone that appeared at the bottom of the tube (Fig. 3a) This suggested that the expressed GFP was not associated with lipids (Fig. 3a). The lipid-protein envelope around the nucleocapsid [6] consists of phospholipids derived from the host cytoplasmic membrane (CM) [15] and the following five viral membrane proteins: the major envelope protein P9, fusogenic protein P6, spike protein P3, putative holin protein P10, and minor membrane protein P13 [13, 16,17,18]
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