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

Yana A Danilova,Andrey V Moiseenko,Viktoriia V Belousova,Olga S Sokolova,Maria V Yakunina,Innokentii E Vishnyakov

doi:10.3390/v12101197

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.

Highlights

Giant phiKZ-like bacteriophages of the Myoviridae family, which include: Pseudomonas aeruginosa phiKZ, EL, OBP and Pseudomonas chlororaphis 201phi2-1, possess circular genetic maps [1,2] packaged inside the capsid according to the head-full mechanism
The distribution of DNA in the cytoplasm of bacteria was estimated by Energy Loss Spectroscopy (EELS), according to our previous study [28], using the Phosphorus signal (DNA contains three phosphate groups per each nucleotide) that was detected and mapped onto the cell image (Figure 2)
It was recently shown that phiKZ-like bacteriophages are resistant to many immune mechanisms of bacteria that normally target DNA in vivo, including two subtypes of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas3, Cas9, Cas12a, and restriction enzymes, such as HsdRMS and EcoRI [15]

Summary

Introduction

Giant phiKZ-like bacteriophages of the Myoviridae family, which include: Pseudomonas aeruginosa phiKZ, EL, OBP and Pseudomonas chlororaphis 201phi, possess circular genetic maps [1,2] packaged inside the capsid according to the head-full mechanism. This means that, in the course of the DNA packaging, the entire interior space of the head is filled with DNA. Sequencing of the giant phage genome revealed that the genome encodes the structural proteins of the phage capsid and tail and RNA polymerases [3,4], chaperonins [1,5], and proteins of the inner body [6]. The function of the inner body is to support the DNA inside the capsid, Viruses 2020, 12, 1197; doi:10.3390/v12101197 www.mdpi.com/journal/viruses

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Conclusion