STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE COMPLEX INVOLVED IN dsDNA PACKAGING IN CAUDOVIRICETES BACTERIOPHAGES

Bacteriophages of the class Caudoviricetes play key roles in microbial dynamics and have considerable biotechnological potential, particularly in phage therapy. A critical step in their replication cycle is the packaging of viral dsDNA into the capsid, a process driven by the terminase complex. This project aims to structurally and functionally characterize terminase complexes from representative phages displaying distinct tail morphotypes: short-tailed (Podoviridae-like; ZC03 and ΦXacm4-11), long non-contractile-tailed (Siphoviridae-like; ZC01), and long contractile-tailed (Myoviridae-like; LafX), all isolated in our laboratory. The experimental approach includes cloning and heterologous expression of terminase-encoding genes, purification of the corresponding subunits, and characterization of protein–DNA interactions using biochemical and biophysical approaches, including EMSA, ITC, and DSF. ATPase and endonuclease activities will also be investigated. Structural characterization is being performed using negative-stain transmission electron microscopy and cryo-electron microscopy (cryo-EM). To date, two expression vectors encoding ZC01 TerS and TerL have been successfully constructed and expressed. Five additional vectors carrying terminase genes from ZC03, LafX, and ΦXacm4-11 are currently in the final stages of construction, with only the ΦXacm4-11 TerL construct remaining to be generated. Initial cryo-EM data of ZC01 TerS have enabled a preliminary reconstruction of the protein complex, and single-particle analysis indicates the formation of a homo-nonameric assembly. The preliminary reconstruction reveals a ring-shaped architecture with a well-defined central core and peripheral densities. Current efforts are focused on completing the biochemical and structural characterization of the terminase complexes. Comparative analysis of terminases from distinct phage morphotypes may reveal conserved and divergent mechanisms underlying dsDNA recognition, processing, and packaging. These findings may broaden our understanding of phage DNA-packaging strategies and provide a foundation for the future engineering of phage-derived molecular machines for biotechnological applications.