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The development of targeted phage metagenomics

Primary Supervisor: Dr Ed Galyov, Department of Respiratory Sciences

Secondary supervisor: Prof Martha Clokie, Andrew Millard

PhD project title: The development of targeted phage metagenomic

University of Registration: University of Leicester

Project outline:

The challenge: To understand the diversity of phages that can target specific bacteria and to establish their host range.

BACKGROUND: Bacteriophages are the most abundant biological entities on the planet with 1031 in the biosphere. They drive the evolution of their bacterial hosts by mediating horizontal gene transfer, facilitating the spread of antimicrobial resistance and virulence genes. Despite being abundant, we have only begun to understand their diversity through culture-independent methods (eg viral metagenomics). Although viral metagenomics has expanded our understanding of phage diversity, critically it does not link the phage with host that it can infect. Research into the range of hosts a phage can infect is traditionally based on the use of plaque assays that allows the visualisation of plaques, should the infection results in a productive infection. However, there are many issues with this method. First, if the phage is temperate in nature, it will not produce plaques. Additionally, the phage may be able to attach and inject its DNA (also transferred genes) but not be able to actively replicate due to host anti-phage systems. Linking phage diversity to hosts and identifying the range of host phage can transfer genetic material between is essential to understand the role of phages in host evolution. Furthermore, with growing use of phage therapy and use of phage as delivery vectors identifying the full range of hosts will be essential for the success of such approaches.

This work will build on the preliminary data we have for the production of mini-cells of E. coli and their subsequent infection with phages. Mini-cells are "mini" bacterial cells which are formed as a result of asymmetric bacterial division. They contain no chromosomal DNA but crucially contain membrane receptors for phage attachment. Here, we hypothesise that using mini-cells, it is possible to select host-specific phages from complex phage mixtures present in the environment, and combined with metagenomics, identify all phages capable of infection of that particular host. Thus, such a targeted phage metagenomic method will enable to assess “host-specific” metagenomes directly linking phages to host.

PROPOSED WORK:

Objective 1: Expand the production of mini-cells beyond E. coli, with the first target being Vibrio natriegens

Objective 2: Test and optimise with a defined cocktail of phages, with known host ranges, the limits of detection of mini-cells combined with metagenomics to identify host specific interactions

Objective 3. Apply the use of mini-cells to produce host specific metagenomes from animals slurries (E. coli) and aquaculture (Vibrio)

Objective 4. Apply the use of mini-cells to identify the extent different phages are capable of mediated horizontal gene transfer

POTENTIAL IMPACT: Unlike traditional viral metagenomics, the use of mini-cells offers the crucial ability to directly link phages with the hosts they can infect. Furthermore, it provides a high-throughput way to identify the extent of phages can mediated the transfer of genes.

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Microbial Food Safety. Understanding the Rules of Life: Microbiology.

Techniques that will be undertaken during the project:

  • Engineering of bacteria to produce mini-cells
  • Isolation, purification and analysis of mini-cells
  • Long and short read viral metagenomics (MinION & Illumina)
  • Phage isolation
  • Bioinformatic analysis of viral metagenomes

Contact: Dr Ed Galyov, University of Leicester