Characterization of the Bacterial BREX Phage Restriction System

摘要 细菌和噬菌体之间的军备竞赛促使细菌进化出广泛的防御系统， 其中许多问题仍有待定性。一种基本不具特征的噬菌体限制系统，称为“Brex” (‘噬菌体排斥’)，最初描述于35年前，在细菌和古生菌中广泛分布 基因组。Brex介导的噬菌体限制机制可能很复杂，因为有大量 由Brex系统编码的基因(4-8个基因，取决于亚家族)和酶活性范围。 对1型和2型Brex系统(总共有6个亚家族)的研究表明，PglX DNA甲基转移酶通过改变细菌基因组来建立自我/非自我辨别。然而， Brex介导的限制似乎没有通过降解噬菌体基因组来发挥作用，这令人惊讶 鉴于类似的甲基转移酶在限制修饰(R-M)中的作用已被很好地描述 系统。1型Brex系统在吸附之后和DNA复制之前抑制噬菌体，但更多细节 关于噬菌体限制的机制尚不清楚。长期的目标是理解这种机制。 通过Brex限制噬菌体，包括阐明每个Brex组分的功能作用。 我们对Brex介导的噬菌体限制的理解是有限的，因为大多数个体的功能 Brex蛋白尚不清楚，也不清楚它们是如何被调控的。为了解决这些问题，我们 正在使用生化和结构研究相结合的方法来表征不动杆菌1型Brex系统 利用以大肠杆菌为基础的噬菌体感染系统进行生物活性分析。第一个目标将解决 假设Brx0，一种不动杆菌特异性因子(即在其他Brex Type 1系统中不存在)， 转录调控其他Brex ORF。第二个目标将解决BrxL是AAA的假设 专门针对底物蛋白质进行降解的小室蛋白水解酶。揭开两者的角色 蛋白质将提供对Brex介导的噬菌体限制机制的关键洞察，不仅是在 不动杆菌，但作为一般策略，在其他细菌分支。 对细菌防御系统有一个基本的了解可能会在许多方面影响人类健康 方法，包括为治疗抗药性细菌的策略做出贡献，以及了解 微生物组关系到健康和疾病。

SUMMARY The arms race between bacteria and phage has driven bacteria to evolve a wide range of defense systems, many of which remain to be characterized. A largely uncharacterized phage restriction system called ‘BREX’ (‘Bacteriophage Exclusion’), initially described 35 years ago, is widely distributed in bacterial and archaeal genomes. The mechanism of BREX-mediated phage restriction is likely complex given the large number of genes (4-8 genes, depending on the subfamily) and range of enzymatic activities encoded by BREX systems. Studies of type 1 and type 2 BREX systems (there are 6 total subfamilies) have demonstrated that the PglX DNA methyltransferase establishes self / non-self discrimination by modifying the bacterial genome. However, BREX-mediated restriction does not appear to function by degrading the phage genome, which is a surprise given the well-characterized role for similar methyltransferase enzymes in restriction-modification (R-M) systems. Type 1 BREX systems inhibit phage after adsorption and prior to DNA replication, but further details about the mechanism of phage restriction are not known. The long-term goal is to understand the mechanism by which BREX restricts phage, including elucidating the functional role of each BREX component. Our understanding of BREX-mediated phage restriction is limited because the function of most individual BREX proteins is not understood, nor is it understood how they are regulated. To address these questions, we are characterizing the Acinetobacter type 1 BREX system using biochemical and structural studies combined with biological activity assays using an E. coli-based phage infection system. The first aim will address the hypothesis that Brx0, an Acinetobacter-specific factor (i.e. not present in other BREX Type 1 systems), transcriptionally regulates other BREX ORFs. The second aim will address the hypothesis that BrxL is a AAA+ chambered protease that specifically targets substrate proteins for degradation. Uncovering the roles of both proteins will provide key insight into the mechanism of BREX-mediated phage restriction, not only in Acinetobacter but as a general strategy in other bacterial clades. Gaining a fundamental understanding of bacterial defense systems could impact human health in numerous ways, including contributing to strategies to treat antibiotic-resistant bacteria and understanding how the microbiome relates to health and disease.