基因的丢失、复制与水平转移是微生物基因组进化的重要动力，也是微生物遗传学关注的热点。细菌常常通过同源重组或随机方式丢失某些基因，以适应环境的改变。课题组近期报道了一种新的基因丢失现象，即铜绿假单胞菌（PA）可通过丢失大片段DNA而抵抗噬菌体感染，这种丢失具有概率高、片段大、位点非特异三大特点，其发生机制不清。通过转座子突变文库筛选，发现PA的核酸酶mutL突变后即不能发生大片段丢失。推测MutL在介导PA基因组大片段丢失从而耐受噬菌体中发挥重要作用。本项目拟通过基因敲除、回补等实验证实MutL在PA基因组大片段丢失中的作用，再通过克隆表达制备MutL重组蛋白，建立体外DNA剪切模型探索MutL切割双链DNA的能力及特异性识别位点，阐明MutL导致PA大片段DNA丢失的分子机制。该研究不仅有望阐明MutL的新功能，而且有助于揭示一种细菌耐受噬菌体的新机制，丰富我们对微生物遗传变异的认识。

Most bacterial genomes are continuously changing over time with respect to gene content and size, and several processes including deletion, duplication, and lateral gene transfer contribute to this genome evolution. The molecular mechanism of gene deletion includes homologous recombination and random deletion. Recently, our group reported a new deletion phenomenon: Pseudomonas aeruginosa phage resistance mutants deleted a large genomic fragment, which includes a gene involved in the biosynthesis of phage receptor. However, this deletion event exhibits three clear features: large genomic fragment deletions (200~300kb), a high frequency (10-6), and no site-specificity. To find out the deletion mechanism, we screened a P. aeruginosa transposon mutant library, and found that the fragment deletion is depended on mutL. Thus, this project aims to use genetic approaches to prove that MutL possess double stand DNA cutting function, which resulted in the genomic fragment deletion. The goal of this grant application is to provide some solid genetic and molecular evidence to a novel pathway leading to phage resistance by mutL-dependent chromosomal DNA deletion. The results of these studies will also provide new insight into bacterial genome deletion mechanisms.