Genomic transfer via membrane vesicle: a strategy of giant phage phiKZ for early infection.

During infection, the giant phiKZ phage forms a specialized structure at the center of the host cell called the phage nucleus. This structure is crucial for safeguarding viral DNA against bacterial nucleases and for segregating the transcriptional activities of late genes. Here, we describe a morphological entity, the early phage infection (EPI) vesicle, which appears to be responsible for earlier gene segregation at the beginning of the infection process. Using cryo-electron microscopy, electron tomography (ET), and fluorescence microscopy with membrane-specific dyes, we demonstrated that the EPI vesicle is enclosed in a lipid bilayer originating, apparently, from the inner membrane of the bacterial cell. Our investigations further disclose that the phiKZ EPI vesicle contains both viral DNA and viral RNA polymerase (vRNAP). We have observed that the EPI vesicle migrates from the cell pole to the center of the bacterial cell together with ChmA, the primary protein of the phage nucleus. The phage DNA is transported into the phage nucleus after phage maturation, but the EPI vesicle remains outside. We hypothesized that the EPI vesicle acts as a membrane transport agent, efficiently delivering phage DNA to the phage nucleus while protecting it from the nucleases of the bacterium. IMPORTANCE: Our study shed light on the processes of phage phiKZ early infection stage, expanding our understanding of possible strategies for the development of phage infection. We show that phiKZ virion content during injection is packed inside special membrane structures called early phage infection (EPI) membrane vesicles originating from the bacterial inner cell membrane. We demonstrated the EPI vesicle fulfilled the role of the safety transport unit for the phage genome to the phage nucleus, where the phage DNA would be replicated and protected from bacterial immune systems.

Assembly of phiKZ bacteriophage Inner Body during infection.

The giant myovirus phiKZ is characterised by an Inner Body (IB) structure within its capsid, crucial for orderly DNA packaging. The IB is composed of six phiKZ-specific proteins. Notably, four of these IB proteins are co-injected with DNA into the host cell, where they potentially play a role in attacking the bacterial cell. The dynamics of IB assembling within the phiKZ capsid during infection remain poorly understood. In this study, we used fluorescent microscopy to track the localisation of IB proteins fused to fluorescent proteins within the cell throughout the infection process. Our findings reveal that the proteins Gp97 and Gp162 are incorporated into new virion heads during phage head maturation. In contrast, proteins Gp90, Gp93, and Gp95 are likely integrated into the virion shortly before the DNA packaging.