Harnessing the potential of atypical gDNA processing by domesticated viruses

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 simply phages, and are generally accepted to be the most influential mechanism of HGT. Gene Transfer Agents (GTAs) are small viral particles that are related to bacteriophages and are able to indiscriminately transfer almost any gene between bacterial cells. 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. Viruses are usually selfish elements whose main goal is to use the resources of their host to make copies of themselves that can then move on to infect new hosts. Despite being similar to traditional viruses in many ways, GTAs do not copy their own genome and do not promote their own survival at the expense of their host. Instead GTAs package the entire genome of their bacterial host in bitesize pieces and distribute these to recipient bacteria. When the species that produces GTAs contains genes for enhanced pathogenesis or antibiotic resistance, this indiscriminate gene transfer becomes of great concern. 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 a pre-formed empty viral head until the whole genome is packaged. Although GTA large terminases are easily identified through bioinformatics owing to classical ATP hydrolysis sequence motifs, no GTA small terminase has ever been identified.

Our preliminary data provide the first evidence that a GTA possesses a small terminase and allows prediction of similar small terminases in other diverse GTAs. We will examine the biochemistry and structure of GTA small terminases, which will allow the fundamental properties of these atypical terminases to be defined with a view to increasing the efficiency of detection of novel GTAs and to provide invaluable insights into the mechanism of viral DNA recognition and packaging in general. Our results are likely to have a broad appeal to the scientific community and could answer long standing questions in Virology and Bacterial Evolution. Almost all aspects of modern medicine rely on effective antibiotics but this is being undermined by the alarming spread of antibiotic resistance. Understanding the methods used by microbes to rapidly acquire virulence genes is crucial if we are to develop new treatments or even to preserve the current antimicrobial armoury.

Bacteria are highly adaptable and can rapidly exchange genes in response to environmental pressures. Gene transfer agents (GTAs) are an understudied mechanism of genetic exchange that package the entire bacterial genome into bacteriophage-like particles, and therefore can transfer any gene between bacteria. The primary aim of true phages is to propagate themselves and to invade new hosts. During replication, the phage DNA packaging machinery recognizes signals near the end of its own genome that target it for inclusion in newly synthesized phages. In contrast to true phages, there is no evidence that GTAs recognize any packaging signals but instead DNA packaging begins at random locations. Clearly there are fundamental differences but also similarities in the way GTAs and phage select DNA to be packaged and this will be the topic of this proposal. Phage packaging is carried out by two multimeric proteins known as the small and large terminases. The large terminase has all the catalytic functions required for DNA processing, while the small terminase (TerS) regulates these activities. Crucially, TerS is responsible for imbuing DNA binding specificity. We have recently identified the first GTA TerS in Rhodobacter capsulatus, and this discovery has in turn allowed analogous proteins to be predicted for other GTAs. We have also identified potential structural differences between GTA and phage TerS proteins. Our aim is to fully characterize a cohort of GTA TerS proteins, to determine 3D structure of a GTA TerS alone and in complex with DNA and other GTA proteins, and to probe the roles of individual structural features on function. The proposal is highly novel and is likely to answer long standing questions in the GTA field but also phage biology and viral DNA packaging. The data produced will interest a wide range of academic beneficiaries and also has potential biotechnology outputs, such as production of bespoke molecular tools for gene delivery.