F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SS_Fs). Assembly of T4SS_Fs and associated F pili requires 10 VirB/VirD4-like Tra subunits and eight or more F-specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SS_Fs undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F-specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SS_F structures by in situ cryoET. Mutant phenotypes supported the assignment of F-specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SS_F-associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SS_F functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure-driven model in which F-specific subunits contribute to distinct steps of T4SS_F assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.

中文翻译：

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F 特异性亚基对 F 质粒编码的 IV 型分泌系统和 F 菌毛的贡献

_{F质粒通过编码的IV型分泌系统（T4SS F} s）在肠杆菌科中广泛传播。T4SS _F s 和相关的 F pili 的组装需要 10 个 VirB/VirD4 样 Tra 亚基和 8 个或更多 F 特异性亚基。最近，我们使用原位冷冻电子断层扫描 (cryoET) 提供了证据，证明 T4SS _F在被激活以产生菌毛时会发生结构转变，并且组装的菌毛沉积在细胞表面的替代基底平台上。在这里，我们从 MOBF12C 质粒 pED208 中删除了八个保守的 F 特异性基因，并量化了对质粒转移的影响，荧光显微镜下菌毛的产生，以及 T4SS _{F的详细说明}通过原位cryoET构建结构。突变表型支持将 F 特异性亚基分配到三个功能类别中：(i) TraF、TraH 和 TraW 是所有 T4SS _F相关活动所必需的，(ii) TraU、TraN 和 TrbC 是非必需的，但对不同的T4SS _F功能，和 (iii) TrbB 对 F 菌毛的生产是必需的，但对质粒转移不是必需的。系统发育远相关的 MOB12A F 质粒中的等效突变赋予了相似的表型，并且通常支持这些类别分配。我们提出了一种新的结构驱动模型，其中 F 特异性亚基有助于 T4SS _F组装或激活的不同步骤，以调节 DNA 转移和 F 菌毛动力学和沉积到替代平台上。