Research Department, New England Biolabs, Ipswich, Massachusetts, United States of America.
PLoS Genet. 2022 Apr 4;18(4):e1009943. doi: 10.1371/journal.pgen.1009943. eCollection 2022 Apr.
Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX (BacteRiophage EXclusion) systems. These RM-like activities employ host protection by DNA modification, but immediate replication arrest occurs without evident of nuclease action on unmodified phage DNA. Here we show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a variant BREX system. A laboratory strain disabled for both the restriction and methylation activity of StySA nevertheless has wild type sequence in pglX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (μ) in their C-terminal domains. We further investigate this system in situ, replacing the mutated pglZμ and brxCμ genes with the WT counterpart. PglZ-WT supports methylation in the presence of either BrxCμ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. These results suggests that while BrxC, PglZ and PglX are principal components of the BREX modification activity, BrxL is required for restriction only. Furthermore, we show that a partial disruption of brxL disrupts transcription globally.
理解塑造原核生物水平基因转移的机制是生物学中的一个关键问题。DNA 进入的主要限制是由依赖于宿主特定部位 DNA 修饰模式的限制-修饰（RM）过程施加的。在经典的 RM 中，内切核酸酶的 DNA 切割紧随未受保护的进入 DNA 位点的检测之后。最近的研究发现了 BREX（细菌噬菌体排斥）系统。这些类似 RM 的活性通过 DNA 修饰来保护宿主，但在没有明显核酶作用于未修饰噬菌体 DNA 的情况下，立即发生复制停滞。在这里，我们表明沙门氏菌 Typhimurium 的历史 stySA RM 基因座是一种变体 BREX 系统。尽管实验室菌株的 StySA 限制和甲基化活性都被失活，但 pglX，即修饰基因同源物，仍然具有野生型序列。相反，侧翼基因 pglZ 和 brxC 在其 C 末端结构域中各携带多个突变（μ）。我们进一步在原位研究了这个系统，用 WT 对应物替换突变的 pglZμ 和 brxCμ 基因。在 BrxCμ 或 BrxC-WT 的存在下，PglZ-WT 支持甲基化，但在 BrxC 缺失/插入等位基因Δ brxC::cat 的存在下则不行。限制需要 BrxC-WT 和 PglZ-WT，这表明 BrxC 的 C 末端专门参与限制活性。这些结果表明，虽然 BrxC、PglZ 和 PglX 是 BREX 修饰活性的主要成分，但 BrxL 仅对限制是必需的。此外，我们还表明 brxL 的部分破坏会导致转录全局中断。
