Agrobacterium tumefaciens VirB9, an outer-membrane-associated component of a type IV secretion system, regulates substrate selection and T-pilus biogenesis
Agrobacterium tumefaciens VirB9, an outer-membrane-associated component of a type IV secretion system, regulates substrate selection and T-pilus biogenesis
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DOI：
10.1128/jb.187.10.3486-3495.2005
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发表时间：
2005-05-01
影响因子：
3.2
通讯作者：
Christie, PJ
中科院分区：
文献类型：
--
作者：
Jakubowski, SJ;Cascales, E;Christie, PJ
根癌土壤杆菌通过IV型分泌系统（T4 SS）在细胞之间转运DNA和蛋白质底物，所述IV型分泌系统的通道亚基包括VirD 4偶联蛋白、VirB 11 ATP酶、VirB 6、VirB 8、VirB 2和VirB 9。在这项研究中，我们使用连接子插入突变来表征外膜相关的VirB 9对VirB/D4 T4 SS的组装和功能的贡献。25个二肽插入突变被分类为允许细胞间底物转移（Tra（+））、完全转移缺陷（Tra（-））或底物区分，例如，选择性地仅允许致癌转移DNA和VirE 2蛋白底物或可移动的IncQ质粒底物的转移。抑制DNA底物转移的突变不影响底物与内膜通道亚基紧密接触的形成，但阻断了与Vir 132和VirB 9通道亚基接触的形成，这表明分泌通道远端部分的组装或功能缺陷。N-和C-末端区域的几个突变破坏了VirB 9与外膜相关脂蛋白VirB 7或内膜能量传感器VirB 10的复合物形成。几种VirB9.i2-产生Tra（+）菌株未能以可检测的水平（Pil（-））产生T菌毛，并且三种这样的Tra（+）Pil（-）突变菌株在缺失virB 2后被赋予Tra（-），表明菌毛蛋白的细胞形式对于底物易位是必需的。我们的研究结果，再加上基于计算机的分析，支持一个模型，其中不同领域的VirB 9有助于基板的选择和易位，建立通道亚基接触，和T菌毛生物发生。
Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra(+)), completely transfer defective (Tra(-)), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the Virl32 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outermembrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra(+) strains failed to elaborate T pilus at detectable levels (Pil(-)), and three such Tra(+) Pil(-) mutant strains were rendered Tra(-) upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.