Colicin-mediated transport of DNA through the iron transporter FepA
Colicin-mediated transport of DNA through the iron transporter FepA
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大肠菌素介导的 DNA 通过铁转运蛋白 FepA 的转运
DOI：
10.1101/2021.05.11.443673
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Cohen-Khait R
中科院分区：
文献类型：
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作者：
Cohen-Khait R
大肠杆菌素是大肠杆菌用来消除竞争菌株的蛋白质抗生素。大肠杆菌素经常利用外膜 (OM) 营养转运蛋白来穿透选择性渗透的细菌细胞膜。在这里，通过应用活细胞荧光成像，我们能够监测成孔毒素大肠菌素 B (ColB) 进入大肠杆菌并将其定位在周质内。我们进一步证明，与 ColB 偶联的单链 DNA 也可以被转运到周质，这强调了大肠菌素的输入途径可以被用来将大的货物分子携带到细菌中。此外，我们通过应用光活化交联、质谱和结构建模的组合来表征 ColB 与其 OM 受体 FepA 关联的分子机制。我们证明复杂的形成与大肠杆菌素的大规模构象变化是一致的。此后，ColB 通过 FepA 的主动转运涉及大肠菌素取代通常封闭该铁转运蛋白的插头结构域的 N 端一半。 重要性几十年来过度使用现成抗生素已经产生了全球性的抗生素耐药性问题，因此迫切需要新型抗生素解决方案。细菌素是由细菌产生的基于蛋白质的抗生素，用于消除密切相关的竞争菌株。细菌素毒素已进化为绕过复杂的细胞包膜以杀死细菌细胞。在这里，我们揭示了一种众所周知但知之甚少的细菌素（大肠菌素 B）的细胞渗透机制，它对大肠杆菌具有活性。此外，我们证明了大肠菌素 B 输入途径可用于将缀合的 DNA 货物输送到细菌细胞中。我们的工作使人们更好地了解细菌素作为潜在的替代抗生素的作用方式，并强调如何在革兰氏阴性细菌的基因组操作中利用它们。
Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter.IMPORTANCEDecades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.