Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCE The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial β and β′-like RNAP subunits.

The infection of Pseudomonas aeruginosa by the giant bacteriophage phiKZ is resistant to host RNA polymerase (RNAP) inhibitor rifampicin. phiKZ encodes two sets of polypeptides that are distantly related to fragments of the two largest subunits of cellular multisubunit RNAPs. Polypeptides of one set are encoded by middle phage genes and are found in the phiKZ virions. Polypeptides of the second set are encoded by early phage genes and are absent from virions. Here, we report isolation of a five-subunit RNAP from phiKZ-infected cells. Four subunits of this enzyme are cellular RNAP subunits homologs of the non-virion set; the fifth subunit is a protein of unknown function. In vitro, this complex initiates transcription from late phiKZ promoters in rifampicin-resistant manner. Thus, this enzyme is a non-virion phiKZ RNAP responsible for transcription of late phage genes. The phiKZ RNAP lacks identifiable assembly and promoter specificity subunits/factors characteristic for eukaryal, archaeal and bacterial RNAPs and thus provides a unique model for comparative analysis of the mechanism, regulation and evolution of this important class of enzymes.

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.

Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a ‘nucleus’, erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial β/β′ subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.