Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA, 91125, USA.
Howard Hughes Medical Institute, David Geffen School of Medicine, Departments of Biological Chemistry and Physiology, University of California Los Angeles, 615 Charles E Young Drive South, Los Angeles, CA, 90095, USA.
Nat Commun. 2020 Oct 8;11(1):5080. doi: 10.1038/s41467-020-18866-y.
Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.
自然转化是细菌从环境中摄取遗传物质并通过同源重组将其整合到基因组中的过程。它代表了水平基因转移的一种模式，有助于抗生素耐药性等特征的传播。在霍乱弧菌中，一种类型 IVa 菌毛（T4aP）被认为通过从细胞表面延伸、结合外源性 DNA 并缩回将该 DNA 通过外膜分泌蛋白 PilQ 进行线程化来促进自然转化。在这里，我们使用从天然霍乱弧菌细胞中纯化的 VcPilQ 的功能标记等位基因来确定在 Amphipol 中 VcPilQ 分泌蛋白的 cryoEM 结构，分辨率约为 2.7 Å。我们使用生物信息学比较了 Proteobacteria 中的 T4aP 分泌蛋白与 VcPilQ 的结构域架构和基因邻域。该结构突出了 T4aP 分泌蛋白与 II 型和 III 型分泌系统分泌蛋白在结构上的差异。基于我们的 cryoEM 结构，我们设计了一系列突变体来可逆调节 VcPilQ 门控动力学。这些实验支持将 VcPilQ 视为潜在可药用靶标的想法，并深入了解 DNA 可能穿越的通道，以通过自然转化促进抗生素耐药性的水平基因转移。
