Readshaw Jennifer J, Doyle Lindsey A, Puiu Maria, Kelly Abigail, Nelson Andrew, Kaiser Alex J, McGuire Sydney F, Peralta Acosta Julieta, Smith Darren L, Stoddard Barry L, Kaiser Brett K, Blower Tim R
Department of Biosciences, Durham University, Stockton Road, DurhamDH1 3LE, UK.
Division of Basic Sciences, Fred Hutchinson Cancer Center, 1100 Fairview Ave. N. Seattle WA 98019, United States.
Nucleic Acids Res. 2025 Jun 20;53(12). doi: 10.1093/nar/gkaf540.
BREX (Bacteriophage Exclusion) systems, identified through shared identity with Pgl (Phage Growth Limitation) systems, are a widespread, highly diverse group of phage defence systems found throughout bacteria and archaea. The varied BREX Types harbour multiple protein subunits (between four and eight) and all encode a conserved putative phosphatase, PglZ, and an equally conserved, putative ATPase, BrxC. Almost all BREX systems also contain a site-specific methyltransferase, PglX. Despite having determined the structure and fundamental biophysical and biochemical behaviours of several BREX factors (including the PglX methyltransferase, the BrxL effector, the BrxA DNA-binding protein, and a commonly-associated transcriptional regulator, BrxR), the mechanism by which BREX impedes phage replication remains largely undetermined. In this study, we identified a stable BREX sub-complex of PglZ:BrxB, generated and validated a structural model of that protein complex, and assessed the biochemical activity of PglZ from BREX, revealing it to be a metal-dependent nuclease. PglZ can cleave cyclic oligonucleotides, linear oligonucleotides, plasmid DNA and both non-modified and modified linear phage genomes. PglZ nuclease activity has no obvious role in BREX-dependent methylation, but does contribute to BREX phage defence. BrxB binding does not impact PglZ nuclease activity. These data contribute to our growing understanding of BREX phage defence.
BREX（噬菌体排斥）系统是通过与Pgl（噬菌体生长限制）系统的共同特征鉴定出来的，它是在细菌和古细菌中广泛存在且高度多样的一组噬菌体防御系统。不同类型的BREX包含多个蛋白质亚基（4至8个），并且都编码一种保守的假定磷酸酶PglZ和一个同样保守的假定ATP酶BrxC。几乎所有的BREX系统还包含一个位点特异性甲基转移酶PglX。尽管已经确定了几种BREX因子（包括PglX甲基转移酶、BrxL效应器、BrxA DNA结合蛋白以及一种常见的相关转录调节因子BrxR）的结构和基本生物物理及生化行为，但BREX阻碍噬菌体复制的机制在很大程度上仍未确定。在本研究中，我们鉴定出了PglZ:BrxB稳定的BREX亚复合物，生成并验证了该蛋白质复合物的结构模型，并评估了BREX中PglZ的生化活性，发现它是一种金属依赖性核酸酶。PglZ可以切割环状寡核苷酸、线性寡核苷酸、质粒DNA以及未修饰和修饰的线性噬菌体基因组。PglZ核酸酶活性在BREX依赖性甲基化中没有明显作用，但对BREX噬菌体防御有贡献。BrxB的结合不影响PglZ核酸酶活性。这些数据有助于我们对BREX噬菌体防御的进一步了解。
