The Phage Nucleus and PhuZ Spindle: Defining Features of the Subcellular Organization and Speciation of Nucleus-Forming Jumbo Phages

Abstract

Bacteriophages and their bacterial hosts are ancient organisms that have been co-evolving for billions of years. Some jumbo phages, those with a genome size larger than 200 kilobases, have recently been discovered to establish complex subcellular organization during replication. Here, we review our current understanding of jumbo phages that form a nucleus-like structure, or "Phage Nucleus," during replication. The phage nucleus is made of a proteinaceous shell that surrounds replicating phage DNA and imparts a unique subcellular organization that is temporally and spatially controlled within bacterial host cells by a phage-encoded tubulin (PhuZ)-based spindle. This subcellular architecture serves as a replication factory for jumbo Pseudomonas phages and provides a selective advantage when these replicate in some host strains. Throughout the lytic cycle, the phage nucleus compartmentalizes proteins according to function and protects the phage genome from host defense mechanisms. Early during infection, the PhuZ spindle positions the newly formed phage nucleus at midcell and, later in the infection cycle, the spindle rotates the nucleus while delivering capsids and distributing them uniformly on the nuclear surface, where they dock for DNA packaging. During the co-infection of two different nucleus-forming jumbo phages in a bacterial cell, the phage nucleus establishes Subcellular Genetic Isolation that limits the potential for viral genetic exchange by physically separating co-infection genomes, and the PhuZ spindle causes Virogenesis Incompatibility, whereby interacting components from two diverging phages negatively affect phage reproduction. Thus, the phage nucleus and PhuZ spindle are defining cell biological structures that serve roles in both the life cycle of nucleus-forming jumbo phages and phage speciation.

Keywords: Anti CRISPR mechanism; PhuZ; jumbo phage; nucleus-like compartment; phage nucleus; spindle-like structure; subcellular organization; viral speciation.

FIGURE 1

Replication machinery of nucleus-forming jumbo phages. (A) At the beginning of infection, phages attach to the bacterial cell membrane close to the pole and inject their genome into the cell. A nucleus-like structure is then formed by the shell protein assembling around and enclosing the phage genome. PhuZ filaments that emanate from the cell pole push the phage nucleus containing the replicating phage genome toward midcell while the host genome is degraded. When the nucleus arrives at midcell, it is met by an opposing PhuZ filament, resulting in its oscillation close to midcell. (B) The phage nucleus is formed by a proteinaceous shell which provides the phage genome with protection from bacterial host defense systems that target DNA because they are excluded from the shell. Phage mRNA is transported to the cell cytoplasm to initiate protein translation. Host mRNA-targeting CRISPR-Cas systems are therefore able to destroy phage mRNAs, and as a result, these phages remain sensitive to mRNA-targeting defense mechanisms (Erb et al., 2014; Chaikeeratisak et al., 2017b, 2019; Mendoza et al., 2020). (C) Midway through infection, procapsids assemble throughout the host cell close to the cell membrane and traffic along treadmilling PhuZ filaments toward the phage nucleus where they dock on its surface to initiate DNA packaging. Mature capsids detach from the phage nucleus and assemble with tails in the cell cytoplasm. Cell lysis occurs at the end of infection and mature phage particles are released from the cell.

FIGURE 2

Subcellular Genetic Isolation and Virogenesis Incompatibility contribute to viral speciation. (A) During co-infection by either identical (such as two PhiPA3) or different phages (PhiPA3 and PhiKZ), each individual phage assembles its own compartment that physically separates its genome from another, resulting in Subcellular Genetic Isolation. In both cases of co-infection, with phages that are either identical or different, the shell potentially limits genetic exchange promoting evolutionary divergence. (B) Virogenesis Incompatibility occurs when divergent components of phage replication interact negatively, causing interference with viral replication, such as PhuZ (a cytoplasmic speciation factor) and gp210 (a nuclear speciation factor). This incompatibility results in a reduction in phage fitness (Chaikeeratisak et al., 2021).