Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems
Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems
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DOI：
10.1038/s41586-022-05091-4
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发表时间：
2022-07-18
期刊：
影响因子：
64.8
通讯作者：
Typas, Athanasios
中科院分区：
文献类型：
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作者：
Bobonis, Jacob;Mitosch, Karin;Typas, Athanasios
反转录子是编码产生多拷贝单链DNA（1）（msDNA）的逆转录酶的原核遗传逆转录元件。尽管对msDNA的生物合成进行了数十年的研究（2），但逆转子的功能和生理作用仍然未知。在这里，我们表明，Retron-Sen 2的肠道沙门氏菌血清型鼠伤寒编码一个辅助毒素蛋白，STM 14_4640，我们改名为RcaT。RcaT被逆转录酶-msDNA抗毒素复合物中和，并在msDNA生物合成受到干扰时变得活跃。逆转录酶是与RcaT结合所必需的，msDNA是抗毒素活性所必需的。高度流行的含RcaT的retron家族构成了一种新型的含三重DNA的毒素-抗毒素系统。为了了解这种毒素-抗毒素系统的生理作用，我们开发了毒素激活-抑制偶联（TAC-TIC），这是一种高通量反向遗传学方法，可识别毒素-抗毒素系统的分子触发剂和阻断剂。通过将TAC-TIC应用于Retron-Sen 2，我们鉴定了噬菌体来源的多种触发和阻断蛋白。我们证明，噬菌体相关的触发直接修改的msDNA，从而激活RcaT和抑制细菌生长。相比之下，原噬菌体蛋白通过直接阻断RcaT来绕过逆转子。与最近的报道一致，视网膜毒素-抗毒素系统始终充当流产感染抗噬菌体防御系统（3，4）。因此，RcaT逆转子是三重DNA调节的毒素-抗毒素系统，其使用逆转录酶-msDNA复合物作为抗毒素和作为噬菌体蛋白活性的传感器。
Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA(1) (msDNA). Despite decades of research on the biosynthesis of msDNA(2), the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports(3,4). Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.