The Evolutionary Ecology of CRISPR-Cas Adaptive Immunity

The bacterium Pseudomonas aeruginosa is the main bio-pesticide used to control insect crop pests and is widely used in agriculture in the UK. The bacterium is produced in large-scale fermentations, but this method often suffers from contaminations with viruses that feed on the bacteria (bacteriophages; phages), resulting in large economical losses. An estimated 30% of the bacterial fermentation batches suffer from phage infections and there is therefore a pressing need to develop phage-resistant P. Aeruginosa strains to replace the current phage-sensitive variants. Here we will manipulate P. Aeruginosa phage-resistance through its CRISPR-Cas systems. CRISPR-Cas systems are bacterial adaptive immune systems and unlike alternative phage-resistance strategies of bacteria, CRISPR-Cas can potentially provide cost-free resistance to unlimited numbers of phage species. In addition to increasing production of bacteria, the resultant P. Aeruginosa genotypes are likely to have greater biocontrol properties, because their in vivo growth will not be compromised by phages. However, a main hurdle for using CRISPR-Cas to generate phage-resistant bacteria is that bacteria frequently evolve phage resistance through other mechanisms, and such resistant mutants are expected to have reduced virulence in hosts as well as being outcompeted by phage sensitive bacteria.

We have recently shown how we can predict and manipulate the environmental conditions that promote the evolution of different resistance mechanisms, and we have identified conditions where the bacterium Pseudomonas aeruginosa exclusively utilizes CRISPR-Cas-mediated resistance. Here, we aim to investigate (i) the conditions where P. Aeruginosa CRISPR-Cas-mediated phage resistance evolves; (ii) the level of protection offered by CRISPR-Cas-mediated phage immunity; and (iii) any in vitro costs associated with this resistance. Once we have pre-evolved phage resistant P. Aeruginosa, we will (iv) investigate their biocontrol efficacy in insect hosts.