The Evolutionary Ecology of CRISPR-Cas Adaptive Immunity

铜绿假单胞菌是用于控制作物害虫的主要生物农药，在英国广泛用于农业。这种细菌是在大规模发酵中生产的，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，导致巨大的经济损失。估计30%的细菌发酵批次遭受噬菌体感染，因此迫切需要开发抗噬菌体的产气荚膜杆菌菌株以取代目前的噬菌体敏感变体。在这里，我们将通过其CRISPR-Cas系统来操纵嗜气单胞菌噬菌体抗性。CRISPR-Cas系统是细菌适应性免疫系统，与细菌的替代噬菌体抗性策略不同，CRISPR-Cas可以潜在地提供对无限数量的噬菌体物种的无成本抗性。除了增加细菌的产量外，所得的产气假单胞菌基因型可能具有更大的生物控制特性，因为它们的体内生长不会受到细菌的影响。然而，使用CRISPR-Cas产生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噬菌体抗性，并且这种抗性突变体预计在宿主中具有降低的毒力以及被噬菌体敏感细菌竞争。我们最近展示了如何预测和操纵促进不同抗性机制进化的环境条件，并且我们已经确定了细菌铜绿假单胞菌专门利用CRISPR-Cas介导的抗性的条件。在这里，我们的目标是研究（i）铜绿假单胞菌CRISPR-Cas介导的噬菌体抗性演变的条件;（ii）CRISPR-Cas介导的噬菌体免疫提供的保护水平;以及（iii）与这种抗性相关的任何体外成本。一旦我们有了预先进化的噬菌体抗性铜绿假单胞菌，我们将（iv）研究它们在昆虫宿主中的生物防治功效。

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.