Summary Bacteriophage genomes are densely packed with coding sequences and frequently encode genes of unknown function. Unbiased phage functional genomics approaches are therefore needed, particularly for large lytic phages. Here, we harness the mariner transposase to develop phage transposon mutagenesis and sequencing (TnSeq), enabling pooled sequencing to identify both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage ΦKZ (280,334 bp; 371 predicted genes), we show that ~110 genes are fitness-conferring via phage TnSeq, identifying many known and previously unknown essential genes. Moreover, this phage carries ~261 non-essential genes, including some capsid and tail proteins, many of which are important for fitness across different clinical isolates or conditions. Phage TnSeq was also extended to a base-modified phage. Together, phage TnSeq is a scalable technology that can identify essential phage genes, generate knockouts in all non-essential genes, and sensitively assign the quantitative fitness contributions of every gene in parallel.
A. Chan, Wearn-Xin Yee, Deepto Mozumdar et al.· Cell· 0 citations
Jumbo bacteriophages in the ϕKZ-like family have shown great potential as a therapeutic to treat infections from the human pathogen Pseudomonas aeruginosa, known for its high level of antibiotic resistance. ϕKZ-like phages form a proteinaceous “phage nucleus” upon infection that compartmentalizes and protects phage DNA replication and transcription mechanisms from host defenses. Transcription occurs within the phage nucleus, requiring RNA polymerase and other transcriptional components to be imported into the compartment. Selective import of phage-encoded proteins into this nucleus is critical for successful infection, yet the mechanisms governing this specificity remain incompletely understood.
In this work I investigated how the ϕKZ-encoded non-virion RNA polymerase (nvRNAP) is selectively imported. Previously, a series of genetic selections identified the genes imp1 as the major import factor and imp7 as a nvRNAP-specific import factor. Therefore, we hypothesized that Imp1 or 7 may directly interact with nvRNAP. We then purified and mixed Imp7 with nvRNAP, in both its active and inactive forms (with and without sigma factor, respectively).
We discovered that Imp7 does not directly bind to nvRNAP in vitro. Preliminary data also suggests Imp1 may bind to the catalytically inactive nvRNAP and not the active nvRNAP in the presence of Imp7.
These findings suggest multiple potential models for nvRNAP import, including the independent and separate import of catalytically inactive nvRNAP and sigma factor, forming the catalytically active nvRNAP only when both are successfully imported. Our results highlight a complex import system for the phage nvRNAP and lay the groundwork for further mechanistic dissection of protein trafficking across the phage nuclear barrier. This knowledge could uncover new principles of protein trafficking across novel biological barriers and inform the development of phage-based therapeutics against antibiotic-resistant pathogens.
NIH
Microbial, Parasitic, and Fungal Immunology (MPF)
Iris Zheng, Joseph Bondy-Denomy, Daphne F. Chen et al.· Journal of Immunology· 0 citations
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