Molecular factors whereby giant phage ΦKZ modulates host protein synthesis

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Phages with large genomes and complex lifestyles represent exciting opportunities to discover new molecular factors and principles of host manipulation during infection. The vast majority of the proteins encoded in the genome sequences of these phages have no matches in the current sequence databases. For example, the 280-kB genome of ΦKZ, a giant bacteriophage attacking pseudomonads, possesses 369 annotated open reading frames but only a small fraction of the encoded proteins are of known function. On the one hand, this promises a huge discovery space for what will likely be novel proteins with specialized functions in host manipulation. On the other hand, the sheer number of these uncharacterized proteins demands new approaches to accelerate functional analysis. In preliminary work for this proposal, we have used RNA-centric high-throughput techniques to map with high-resolution the transcriptomes of phage ΦKZ and infected Pseudomonas aeruginosa cells and to predict candidate phage proteins with new functions in usurping the host bacterium. For example, gradient fractionation of complexes by size and density (Grad-seq), allowed us to generate a catalogue of potential protein and RNA complexes in the early phase of ΦKZ infection of P. aeruginosa cells. This revealed a striking accumulation of phage transcripts in ribosomal fractions thereby indicating an efficient transfer of viral transcripts to the translation machinery. More importantly, we observed numerous phage proteins to occur in ribosomal fractions, raising the possibility that ΦKZ actively manipulates the protein synthesis machinery of its host to its own benefit. Thus, we hypothesize that ΦKZ expresses proteins that modify ribosomes in infected Pseudomonas cells to prioritize the synthesis of its own proteins upon infection. Therefore, in the initial phase of the SPP2330, we will identify phage protein complexes and characterise their impact on mRNA translation in host bacteria, with the long-term goal to better understand if and how host protein synthesis is controlled during phage infection. The identification of phage proteins that target host protein synthesis will provide important biological insight into the phage-host interface, has potential for exploitation in antimicrobial strategies, and may yield new tools for synthetic biology.