Geobacter species are critically involved in elemental biogeochemical cycling and environmental bioremediation processes via extracellular electron transfer (EET), but the underlying biomolecular mechanisms remain elusive due to lack of effective analytical tools to explore into complicated EET networks. Here, a simple and highly efficient cytosine base editor was developed for engineering of the slow-growing Geobacter sulfurreducens (a doubling time of 5 h with acetate as the electron donor and fumarate as the electron acceptor). A single-plasmid cytosine base editor (pYYDT-BE) was constructed in G. sulfurreducens by fusing cytosine deaminase, Cas9 nickase, and a uracil glycosylase inhibitor. This system enabled single-locus editing at 100% efficiency and showed obvious preference at the cytosines in a TC, AC, or CC context than in a GC context. Gene inactivation tests confirmed that it could effectively edit 87.7–93.4% genes of the entire genome in nine model Geobacter species. With the aid of this base editor to construct a series of G. sulfurreducens mutants, we unveiled important roles of both pili and outer membrane c-type cytochromes in long-range EET, thereby providing important evidence to clarify the long-term controversy surrounding their specific roles. Furthermore, we find that pili were also involved in the extracellular reduction of uranium and clarified the key roles of the ExtHIJKL conduit complex and outer membrane c-type cytochromes in the selenite reduction process. This work developed an effective base editor tool for the genetic modification of Geobacter species and provided new insights into the EET network, which lay a basis for a better understanding and engineering of these microbes to favor environmental applications.

中文翻译：

---

使用碱基编辑器系统对减少硫还原地杆菌细胞外污染物途径的生物分子研究

Geobacter物种通过细胞外电子转移 (EET) 关键参与元素生物地球化学循环和环境生物修复过程，但由于缺乏有效的分析工具来探索复杂的 EET 网络，其潜在的生物分子机制仍然难以捉摸。在这里，开发了一种简单且高效的胞嘧啶碱基编辑器，用于对生长缓慢的Geobacterthioreducens进行工程改造（以醋酸盐作为电子供体，富马酸盐作为电子受体的倍增时间为 5 小时）。在G.sulfurreducens中构建了一个单质粒胞嘧啶碱基编辑器 (pYYDT-BE)通过融合胞嘧啶脱氨酶、Cas9 切口酶和尿嘧啶糖基化酶抑制剂。该系统能够以 100% 的效率实现单基因座编辑，并且在 TC、AC 或 CC 环境中比在 GC 环境中显示出对胞嘧啶的明显偏好。基因灭活测试证实，它可以有效编辑 9 个模型Geobacter物种中整个基因组的 87.7-93.4% 的基因。在此基础编辑器的帮助下构建了一系列G.硫还原菌突变体，我们揭示了菌毛和外膜c的重要作用型细胞色素在远程 EET 中的作用，从而为澄清围绕其特定作用的长期争议提供了重要证据。此外，我们发现菌毛还参与了铀的细胞外还原，并阐明了 ExtHIJKL 导管复合物和外膜c型细胞色素在亚硒酸盐还原过程中的关键作用。这项工作为Geobacter物种的遗传修饰开发了一种有效的碱基编辑器工具，并为 EET 网络提供了新的见解，这为更好地理解和设计这些微生物以有利于环境应用奠定了基础。