Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
Department of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089.
Proc Natl Acad Sci U S A. 2018 Apr 3;115(14):E3246-E3255. doi: 10.1073/pnas.1718810115. Epub 2018 Mar 19.
Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of OM extensions, using electron cryotomography (ECT). We developed a robust method for fluorescence light microscopy imaging of OM extension growth on electron microscopy grids and used correlative light and electron microscopy to identify and image the same structures by ECT. Our results reveal that OM extensions are dynamic chains of interconnected outer membrane vesicles (OMVs) with variable dimensions, curvature, and extent of tubulation. Junction densities that potentially stabilize OMV chains are seen between neighboring vesicles in cryotomograms. By comparing wild type and a cytochrome gene deletion mutant, our ECT results provide the likely positions and packing of periplasmic and outer membrane proteins consistent with cytochromes. Based on the observed cytochrome packing density, we propose a plausible ET path along the OM extensions involving a combination of direct hopping and cytochrome diffusion. A mean-field calculation, informed by the observed ECT cytochrome density, supports this proposal by revealing ET rates on par with a fully packed cytochrome network.
细菌纳米线作为一种将细菌电子传递链与远离细胞的固相电子受体连接起来的细胞外电子转移 (EET) 途径,近来引起了人们的关注。最近的研究表明,MR-1 产生的外膜 (OM) 和周质延伸包含 EET 成分,并暗示它们可能作为细菌纳米线发挥作用。然而,其在天然条件下的细胞色素电子载体的精细结构和分布仍不清楚,这使得难以评估 OM 延伸部位潜在的电子传递 (ET) 机制。在这里,我们使用电子 cryotomography (ECT) 报告了 OM 延伸的高分辨率图像。我们开发了一种在电子显微镜网格上生长 OM 延伸的荧光显微镜成像的强大方法,并使用相关的光和电子显微镜通过 ECT 来识别和成像相同的结构。我们的结果表明,OM 延伸是具有不同尺寸、曲率和管状化程度的相互连接的外膜囊泡 (OMV) 的动态链。在 cryotomograms 中可以看到相邻囊泡之间存在潜在稳定 OMV 链的连接密度。通过比较野生型和细胞色素基因缺失突变体,我们的 ECT 结果提供了与细胞色素一致的周质和外膜蛋白的可能位置和包装。基于观察到的细胞色素包装密度,我们提出了一种可能的 ET 途径,涉及直接跳跃和细胞色素扩散的组合。基于观察到的 ECT 细胞色素密度的平均场计算支持了这一建议,表明 ET 速率与完全填充的细胞色素网络相当。
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