Characterizing Import of RNA Polymerase into a Phage Nucleus 2310316

Abstract Introduction 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. Methods 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). Results 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. Conclusion 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. Funding Source NIH Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)