Functional studies of BacteRiophage EXclusion (BREX) systems

Background:
Bacteriophages (phages) outnumber bacteria by ten to one, with an estimated 1030 phages causing infections at a rate of 1025 a second. This huge selection pressure drives diversity of the global microbiome, and phage-mediated horizontal gene transfer disseminates traits such as antimicrobial resistance between hosts. This pressure has also led to the development of bacterial systems that protect from phage predation. Many of these phage-resistance systems have already proved useful to biochemists: the restriction-modification, abortive infection and CRISPR-cas systems underpinned the recombinant DNA and genome editing revolutions. A novel resistance mechanism, the BacteRiophage Exclusion (BREX) system, presents an excellent opportunity to explore another weapon in the ongoing molecular warfare between bacteria and phages, to better understand phage-mediated transfer of host DNA and to further assess the ability of phage therapy to act as a viable alternative to antibiotics.
Aims:
In this cross-institution, multi-disciplinary proposal we will use a combination of molecular biology, microbiology, functional genomics, protein biochemistry, structural biology and next generation sequencing to investigate uncharacterised multi-gene BREX loci. There has been one publication on BREX to date1 (examining a Gram-positive example from Bacillus), which described putative activities for the system and showed that BREX was encoded by 6-8 ORFs found in approximately a quarter of bacterial genomes. The student will examine Gram-negative BREX systems that we have identified within Escherichia and Salmonella strains and approach multiple aspects, such as; 1) The ability of BREX to protect bacterial cultures from phages; 2) The nature of phage BREX-resistance mutations; 3) The ability of BREX to control DNA transfer by infecting phages; 4) The localisation of and interactions between products of the BREX locus and 5) The identification of potential candidates for protein crystallography trials and structural elucidation of these uncharacterised proteins. This work will highlight new aspects of bacterial and phage co-evolution, and potentially provide new methods for the control of both horizontal gene transfer and deleterious phage infections in biotechnological settings (such as industrial fermentations), and contribute to the development of novel molecular biological tools.
Methodology and Research Training:
The proposed work will provide an excellent training opportunity in a range of cross-disciplinary methodologies:
1.Molecular Microbiology: The student will learn molecular biological and microbiological techniques, focussing on the growth of micro-organisms, phage-infection assays and selection of BREX-resistant phages, together with utilising the necessary recombinant DNA technologies (Blower)
2.Protein biochemistry: The student will use protein purification techniques to produce BREX proteins for downstream biochemical activity assays, that will include Western blotting and
co-immunoprecipitation (Blower)
3.Structural biology: The student will learn to prepare crystals for X-ray diffraction experiments, data collection (Diamond Light Source), data processing and structural modelling (Blower)
4.Functional Genomics: The student will isolate wild-type and BREX-resistant phages and identify mutations within the resistant phage genomes with next generation sequencing, to identify the gene products required for BREX activation and susceptibility (Hinton)