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

Abstract

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.

Structure of the bacteriophage PhiKZ non-virion RNA polymerase

“…It also expresses several other systems that the host lacks entirely. During infection, ΦKZ forms a ‘nucleus’ within the host cytoplasm that protects its genome from host endonucleases, and which it traffics with a tubulin cytoskeleton ( 6 , 7 ). This implies intriguing evolutionary parallels to their eukaryotic equivalents, and suggests a possible viral origin for the eukaryotic nucleus and cytoskeleton.…”

Phage phiKZ—The First of Giants

“…Recently, it was found that that phage phiKZ encodes a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection [47,48] and in this way segregates its DNA from the immunity mechanisms of its bacterial host (two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI) [49]. Phage-encoded tubulin-like protein is supposed to play a role in positioning this structure in the cell [45,50] The DNA polymerase was finally found in phiKZ-like phages genomes by way of very thorough bioinformatical analysis and appeared to represent an unexpectedly highly divergent newly discovered branch of the B-family DNA polymerases [51][52][53].…”

Emerging Aspects of Jumbo Bacteriophages

“…Giant phiKZ-like bacteriophages belong to myoviruses, which include Pseudomonas aeruginosa phiKZ, EL, OBP, and Pseudomonas chlororaphis 201phi2-1 that are found to have circular genomes. 44,45 These genomes are packaged inside the capsid through a head-full packaging mechanism ensuring that the entire interior space of the viral protein head is filled with DNA in the course of the DNA packaging. After genome sequencing of the giant phages, it is concluded that their genomes encode structural proteins of capsid and tail, RNA polymerases, 26,46 chaperonins, 45,47 and inner body proteins.…”

“…44,45 These genomes are packaged inside the capsid through a head-full packaging mechanism ensuring that the entire interior space of the viral protein head is filled with DNA in the course of the DNA packaging. After genome sequencing of the giant phages, it is concluded that their genomes encode structural proteins of capsid and tail, RNA polymerases, 26,46 chaperonins, 45,47 and inner body proteins. 48 Virus inner body possessed an internal proteinaceous structure, specific for jumbo phages.…”