Department of Medical Microbiology & Immunology, College of Medicine and Life Sciences, University of Toledo, 3100 Transverse Drive, Toledo, OH 43614, USA.
BMC Evol Biol. 2013 Oct 2;13:218. doi: 10.1186/1471-2148-13-218.
Restriction-modification (RM) systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease (REase) is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase (MTase) is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused.
The gene for CR.NsoJS138I fusion protein (nsoJS138ICR, from the bacterium Niabella soli) was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene.
Fusions between C proteins and REases have not previously been characterized, though other fusions have (such as between REases and MTases). These results reinforce the evidence for impressive modularity among RM system proteins, and raise important questions about the implications of the C-REase fusions on expression kinetics of these RM systems.
限制-修饰（RM）系统似乎在调节细菌和古菌之间的基因流动方面发挥着关键作用。由于限制内切酶（REase）对未甲基化的新宿主细胞具有潜在的致命性，因此调节以确保保护性 DNA 甲基转移酶（MTase）的预表达对于 RM 基因的传播至关重要。对于 IIP 型 RM 系统来说尤其如此，其中 REase 和 MTase 是独立的、独立活性的蛋白质。大量的 II 型 RM 系统由一种称为 C 蛋白的激活物-抑制剂控制。在这些系统中，C 控制其自身基因的启动子，以及下游缺乏自身启动子的 REase 基因的启动子。因此，MTase 在 RM 基因进入新细胞后立即表达，而 REase 的表达则延迟到 C 蛋白积累足够为止。为了研究这种调节机制的变化和进化，我们搜索了与研究充分的 C 蛋白依赖性 PvuII RM 系统密切相关的 RM 系统。出乎意料的是，在发现的系统中，有几个系统中的 C 蛋白和 REase 基因融合在一起。
克隆了来自 Niabella soli 细菌的 CR.NsoJS138I 融合蛋白（nsoJS138ICR）的基因，并生产和部分纯化了融合蛋白。Western blot 分析没有证据表明，在所测试的条件下，除全长融合蛋白外，没有其他产物生成。该蛋白在体外具有 REase 活性，并且从序列相似性来看，其特异性与 PvuII REase 相同，尽管最佳反应条件不同。此外，正如 nsoJS138ICR 基因启动子区域的 lacZ 报告融合物的体内激活所揭示的那样，融合蛋白作为 C 蛋白发挥作用。
以前没有对 C 蛋白和 REase 之间的融合进行过特征描述，尽管其他融合已经存在（例如 REase 和 MTase 之间的融合）。这些结果强化了 RM 系统蛋白之间存在令人印象深刻的模块化的证据，并提出了关于 C-REase 融合对这些 RM 系统表达动力学的影响的重要问题。
