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.

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Structure and function of virion RNA polymerase of a crAss-like phage.

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.

Maturation of Pseudo-Nucleus Compartment in P. aeruginosa, Infected with Giant phiKZ Phage.

The giant phiKZ phage infection induces the appearance of a pseudo-nucleus inside the bacterial cytoplasm. Here, we used RT-PCR, fluorescent in situ hybridization (FISH), electron tomography, and analytical electron microscopy to study the morphology of this unique nucleus-like shell and to demonstrate the distribution of phiKZ and bacterial DNA in infected Pseudomonas aeruginosa cells. The maturation of the pseudo-nucleus was traced in short intervals for 40 min after infection and revealed the continuous spatial separation of the phage and host DNA. Immediately after ejection, phage DNA was located inside the newly-identified round compartments; at a later infection stage, it was replicated inside the pseudo-nucleus; in the mature pseudo-nucleus, a saturated internal network of filaments was observed. This network consisted of DNA bundles in complex with DNA-binding proteins. On the other hand, the bacterial nucleoid underwent significant rearrangements during phage infection, yet the host DNA did not completely degrade until at least 40 min after phage application. Energy dispersive x-ray spectroscopy (EDX) analysis revealed that, during the infection, the sulfur content in the bacterial cytoplasm increased, which suggests an increase of methionine-rich DNA-binding protein synthesis, whose role is to protect the bacterial DNA from stress caused by infection.