PDF(Pubmed)
Abstract：
Plasmid-borne Type II restriction-modification (RM) systems mediate post-segregational killing (PSK). PSK is thought to be caused by the dilution of restriction and modification enzymes during cell division, resulting in accumulation of unmethylated DNA recognition sites and their cleavage by restriction endonucleases. PSK is the likely reason for stabilization of plasmids carrying RM systems in the absence of selection for plasmid maintenance. In this study, we developed a CRISPR interference-based method to eliminate RM-carrying plasmids and study PSK-related phenomena with minimal perturbation to the Escherichia coli host. Plasmids carrying the EcoRV, Eco29kI, and EcoRI RM systems were highly stable, and their loss resulted in SOS response and PSK. In contrast, plasmids carrying the Esp1396I system were poorly stabilized; their loss led to a temporary cessation of growth, followed by full recovery. We demonstrate that this unusual behavior is due to a limited lifetime of the Esp1396I restriction endonuclease activity, which, upon Esp1396I plasmid loss, disappears approximately after two cycles of cell division, i.e., before unmethylated sites appear in significant numbers. Our results indicate that whenever PSK induced by a loss of RM systems, and, possibly, other toxin-antitoxin systems, is considered, the lifetimes of individual system components and the growth rate of host cells shall be taken in account. Mathematical modeling shows, that unlike the situation with classical toxin-antitoxin systems, RM system-mediated PSK is possible when the lifetimes of restriction endonuclease and methyltransferase activities are similar, as long as the toxic restriction endonuclease activity persists for more than two chromosome replication cycles.IMPORTANCEIt is widely accepted that many Type II restriction-modification (RM) systems mediate post-segregational killing (PSK) if plasmids that encode them are lost. In this study, we harnessed an inducible CRISPR-Cas system to remove RM plasmids from Escherichia coli cells to study PSK while minimally perturbing cell physiology. We demonstrate that PSK depends on restriction endonuclease activity lifetime and is not observed when it is less than two replication cycles. We present a mathematical model that explains experimental data and shows that unlike the case of toxin-antitoxin-mediated PSK, the loss of an RM system induced PSK even when the RM enzymes have identical lifetimes.
摘要：
质粒携带的II型限制性修饰(RM)系统介导分离后杀伤(PSK)。PSK被认为是由细胞分裂过程中限制和修饰酶的稀释引起的，导致未甲基化的DNA识别位点的积累和限制性内切酶的切割。PSK是在没有选择质粒维持的情况下使携带RM系统的质粒稳定的可能原因。在这项研究中,我们开发了一种基于CRISPR干扰的方法来消除携带RM的质粒，并研究与PSK相关的现象，同时对大肠杆菌宿主的扰动最小。携带EcoRV的质粒,Eco29kI,EcoRIRM系统高度稳定，它们的损失导致了SOS响应和PSK。相比之下,携带Esp1396I系统的质粒稳定性差；它们的损失导致生长暂时停止，其次是完全恢复。我们证明了这种不寻常的行为是由于Esp1396I限制性内切核酸酶活性的有限寿命，which,在Esp1396I质粒丢失时，大约在两个细胞分裂周期后消失，即，在未甲基化位点出现大量之前。我们的结果表明，每当由RM系统丢失引起PSK时，and,可能,其他毒素-抗毒素系统，被认为，应考虑单个系统组件的寿命和宿主细胞的生长速率。数学建模显示，与经典的毒素-抗毒素系统不同，当限制性内切核酸酶和甲基转移酶活性的寿命相似时，RM系统介导的PSK是可能的，只要毒性限制性内切核酸酶活性持续超过两个染色体复制周期。重要性如果编码II型限制性修饰（RM）系统的质粒丢失，则许多II型限制性修饰（RM）系统可介导分离后杀死（PSK）。在这项研究中,我们利用诱导型CRISPR-Cas系统从大肠杆菌细胞中去除RM质粒，以研究PSK，同时最小程度地干扰细胞生理学。我们证明了PSK取决于限制性内切核酸酶活性寿命，并且在少于两个复制周期时观察不到。我们提出了一个解释实验数据的数学模型，并表明与毒素-抗毒素介导的PSK的情况不同，即使当RM酶具有相同的寿命时，RM系统的损失也会引起PSK。
