Unpacking the terminase: Revealing the distinctions between Gene Transfer Agent and viral DNA translocation motors

Horizontal Gene Transfer (HGT) is a fundamental and powerful process for the exchange of genes between bacteria. HGT drives bacterial evolution, adaptation and spread into new ecological niches and is the primary means for rapid distribution of characteristics such as antibiotic resistance and pathogenicity. Viruses that infect bacteria are known as bacteriophages, or phages, and are one of the most influential mechanisms of HGT. Gene Transfer Agents (GTAs) are small virus-like particles that are related to bacteriophages. Whereas viruses are selfish elements that use the host cell’s resources to replicate themselves and to infect new hosts, GTAs do not copy their own genome and do not promote their own survival at the host’s expense. Instead GTAs package the entire genome of their bacterial host in bitesize pieces and distribute these to recipient cells. If the species that produces GTAs contains genes for enhanced pathogenesis or antibiotic resistance, this indiscriminate gene transfer becomes of great concern. Research into the activity of GTAs in the environment revealed that antibiotic resistance genes could be spread at extremely high frequencies and thus GTA-mediated spread of antibiotic resistance and virulence genes in pathogens has huge potential clinical and economic consequences. The overarching goal of this research project is to characterize the structure and function of the GTA DNA recognition machinery. In bacteriophages, the protein that is responsible for specific recognition of the phage genome is called the small terminase. The small terminase also regulates the enzymatic activities of the large terminase protein, which cuts the target DNA and rapidly feeds it into the viral head until the whole genome is packaged. We were the first group to identify a GTA small terminase and predicted similar TerS in diverse GTAs. During previous BBSRC funding, we optimized purification of cognate TerS/L pairs and produced compelling structural and biochemical evidence for a DNA packaging initiation complex. Here, we will build on this data 1) to examine in detail the similarities and differences between GTA and phage terminase proteins, 2) to produce high resolution structures of DNA packaging complexes and intermediates, 3) to develop biotechnological outputs to exploit the unique biology of GTAs. We envisage that our results will allow efficient detection of novel GTAs, which will enhance our understanding of their environmental impact and provide new tools for biotechnology. GTAs are already used as a genome editing method in Rhodobacter, and detection of a broader suite of GTAs will allow development of similar tools for otherwise intractable hosts. GTAs are far simpler in organization than most phages and, therefore, there is scope to establish GTAs as tractable model systems to answer longstanding fundamental questions in the field of viral DNA recognition and packaging with associated applications for synthetic biology. In addition, for dsDNA viruses, recognition of their own DNA is integral to their ability to successfully propagate and infect new hosts. Our data will inform novel approaches to prevent DNA packaging via development of antivirals or pharmaceuticals that slow bacterial evolution and onset of antibiotic resistance.