Interfakultäres Institut für Mikrobiologie und Infektionsmedizin (IMIT), Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Germany.
Microbiology (Reading). 2013 Aug;159(Pt 8):1575-1585. doi: 10.1099/mic.0.068049-0. Epub 2013 Jul 7.
Toxin-antitoxin (TA) systems are small genetic elements of prokaryotes associated with persister cell formation, phage defence, stress regulation and programmed cell arrest. In this study, we characterized two paralogues of the ribosome-dependent RNase YefM-YoeB TA system from the Gram-positive organism Staphylococcus equorum SE3. 5' Rapid amplification of cDNA ends confirmed transcriptional activity in the exponential growth phase and revealed an extended 5' untranslated region upstream of the yefM-seq1 gene. Inducible expression of the putative yoeB-seq1/2 toxins led to growth defects of Escherichia coli, which were counteracted by simultaneous induction of the cognate yefM-seq1/2 antitoxin candidates in a strictly pairwise manner. Bacterial two-hybrid assays revealed interaction between YoeB-seq1 and YefM-seq1 but not YoeB-seq1 and YefM-seq2, also indicating two independent systems. In vivo primer extensions demonstrated specific RNA cleavage adjacent to the start codons by YoeB-seq proteins, and YoeB-seq2 activity could be neutralized by the corresponding antitoxin YefM-seq2. Together, these results indicate that the two yefM-yoeB-seq1/2 paralogues from S. equorum encode functional TA systems.
毒素-抗毒素（TA）系统是原核生物的小型遗传元件，与持久细胞形成、噬菌体防御、应激调节和程序性细胞阻滞有关。在这项研究中，我们从革兰氏阳性菌马肠球菌 SE3 中鉴定了核糖体依赖性 RNase YefM-YoeB TA 系统的两个同源物。5'快速扩增 cDNA 末端证实了指数生长期的转录活性，并在上游 yefM-seq1 基因发现了一个扩展的 5'非翻译区。推定的 yoeB-seq1/2 毒素的诱导表达导致大肠杆菌生长缺陷，而在严格的成对方式下同时诱导同源 yefM-seq1/2 解毒剂候选物则可以拮抗这种缺陷。细菌双杂交试验表明 YoeB-seq1 和 YefM-seq1 之间存在相互作用，但 YoeB-seq1 和 YefM-seq2 之间不存在相互作用，这也表明存在两个独立的系统。体内引物延伸实验证明 YoeB-seq 蛋白在起始密码子附近具有特异性的 RNA 切割活性，并且相应的抗毒素 YefM-seq2 可以中和 YoeB-seq2 的活性。综上所述，这些结果表明马肠球菌的两个 yefM-yoeB-seq1/2 同源物编码功能性 TA 系统。
