Exploring the role of diversity in bacteria-phage ecology and evolution.

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Bacteria in the rhizosphere can benefit agriculture by making nutrients available to plants. However, their beneficial effect is thought to be limited by viruses (phage) in soil. CRISPR-Cas adaptive immune systems of bacteria provide phage resistance but it is frequently very difficult to have bacteria evolve this type of phage resistance, and when it evolves, phage can readily escape CRISPR-Cas by point mutation. Recent studies suggest phage resistance may be improved when the adaptive immune system is integrated with an innate immune system, known as restriction-modification (RM). However, synergism between CRISPR and RM has not been clearly demonstrated yet, and because RM systems are highly diverse, it is also unclear whether such synergism would apply to all or only some RM systems. The key aim of this project is therefore to investigate the existence of synergy between different types of RM and CRISPR-Cas, and examine how this synergistic interaction affects bacteria-phage co-evolution. We already have a bacterial host that evolves high levels of CRISPR immunity in response to some, but not all phages. We will use this experimental system to first examine if levels of evolved CRISPR immunity increase when bacteria are equipped with different types of RM systems, to reveal putative synergistic RM-CRISPR interactions. We will then compare the genetic changes that accompany the evolution of CRISPR immunity in the presence and absence of RM, in order to reconstruct the mechanistic basis underlying the synergistic RM-CRISPR interactions. This will be complemented with in vitro biochemical studies that examine how purified RM components digest DNA molecules. Finally, we will investigate how an integrated RM-CRISPR immune response impacts bacteria-phage co-evolution, specifically the ability of phage to overcome immunity, compared to bacteria carrying a single immune mechanism and examine trade-offs associated with encoding multiple immune mechanisms. The student will receive training in experimental evolution, biochemistry, molecular microbiology and genetics. This interdisciplinary project integrates expertise on CRISPR-Cas (Westra, Exeter), RM (Sczcelkun, Bristol) and bacteria-phage co-evolution (Buckling, Exeter).