The Evolutionary Ecology of CRISPR-Cas Adaptive Immunity

铜绿假单胞菌是防治农作物害虫的主要生物农药，在英国农业中被广泛使用。这种细菌是在大规模发酵中产生的，但这种方法经常受到以细菌为食的病毒（噬菌体；噬菌体）的污染，造成巨大的经济损失。估计有30%的细菌发酵批次遭受噬菌体感染，因此迫切需要开发耐噬菌体的铜绿假单胞菌菌株来取代目前的噬菌体敏感变种。在这里，我们将通过其CRISPR-Cas系统操纵铜绿假单胞菌的噬菌体抗性。CRISPR-Cas系统是细菌的适应性免疫系统，与细菌的其他噬菌体抗性策略不同，CRISPR-Cas系统可以对无限数量的噬菌体物种提供免费的抗性。除了增加细菌的产量外，由此产生的铜绿假单胞菌基因型可能具有更大的生物防治特性，因为它们的体内生长不会受到噬菌体的影响。然而，使用CRISPR-Cas生成噬菌体抗性细菌的一个主要障碍是细菌经常通过其他机制进化出噬菌体抗性，并且这种抗性突变体预计会降低宿主的毒力，并被噬菌体敏感细菌所淘汰。我们最近展示了我们如何预测和操纵促进不同耐药机制进化的环境条件，我们已经确定了铜绿假单胞菌专门利用crispr - cas介导的耐药的条件。在这里，我们的目标是研究(i) P. Aeruginosa crispr - cas介导的噬菌体耐药性进化的条件；（ii） crispr - cas介导的噬菌体免疫提供的保护水平；（iii）与这种耐药性相关的任何体外成本。一旦我们预先进化出抗噬菌体的铜绿假单胞菌，我们将（iv）研究它们在昆虫宿主中的生物防治效果。

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.