Translocation of DNA across Agrobacterium envelope

 描述(由申请人提供) 根癌农杆菌的VIRB/VirD4系统和大肠杆菌中密切相关的质粒连接系统是详细研究T4SS结构和功能的重要模型。该实验室的长期工作目标是用完整的分子术语描述T4SS机器生物发生途径，底物识别和跨细胞膜转移的基础，以及机器与靶细胞接触的性质。新的结构信息表明，革兰氏阴性菌的T4SS由一个大的外膜相关复合体(OMC；也被称为核心复合体)和一个同样大的内膜复合体(IMC)组成，两个亚结构之间有一个狭窄的柄连接。这种结构对底物分泌途径和介导结合菌毛组装的机制提出了新的问题。我们假设，T4SS既可以作为底物通道，也可以作为菌毛生物发生系统，细胞内和细胞外信号的组合调节这些替代结果。我们提出了两个特定的目标：1)确定底物-受体对接的性质和通道ATPase在底物处理和跨内膜递送中的作用；2)确定菌毛的生物发生途径和调控底物转移和菌毛成核和跨外膜延伸的调节检查点。对于目标1，最近的发现推动了我们探索易位信号在底物-受体结合中的作用的精细研究，以及测试两个ATPase在易位前对伴侣释放和底物展开的贡献的实验。对于目标2，最近的结构进展和生化发现支持对特定模型的测试，即在IMC处的菌毛成核和通过OMC的中央腔室延伸。细胞内信号的组合，包括底物结合和ATP能量，被认为是诱导OMC的构象变化，以激活底物转移，以牺牲菌毛的产生。这些研究与正在进行的合作努力相结合，以获得高分辨率的T4SS结构和供体-靶细胞交配连接。这项工作有望刺激新的基础研究方向，并为旨在抑制许多临床上重要病原体的抗生素耐药性传播和疾病进展的应用研究提供关键的知识基础。

 DESCRIPTION (provided by applicant) Despite their broad medical importance as dominant mechanisms underlying the rapid dissemination of antibiotic resistance and infection by many bacterial pathogens, there is a fundamental lack of mechanistic understanding how type IV secretion systems (T4SSs) translocate macromolecular substrates to bacterial or eukaryotic target cells. The VirB/VirD4 system of Agrobacterium tumefaciens and closely-related plasmid conjugation systems in Escherichia coli serve as important models for detailed structure-function studies of T4SSs. The long-term goal of work in this laboratory is to describe in complete molecular terms the T4SS machine biogenesis pathway, the basis for substrate recognition and transfer across the cell envelope, and the nature of the machine - target cell contact. New structural information has shown that the T4SSs of Gram- negative bacteria are composed of a large outer membrane-associated complex (OMC; also designated as the core complex) and an equally large inner membrane complex (IMC), with a narrow stalk connecting the two substructures. This structure raises new questions about the substrate secretion pathway and the mechanisms mediating assembly of conjugative pili. We hypothesize that T4SSs function either as substrate channels or as pilus biogenesis systems and that a combination of intra- and extracellular signals regulate these alternative outcomes. We propose two specific aims: 1) Define the nature of substrate-receptor docking and the contributions of channel ATPases to substrate processing and delivery across the inner membrane and ii) Define the pilus biogenesis pathway and the regulatory checkpoints governing substrate transfer and pilus nucleation and extension across the outer membrane. For Aim 1, recent findings drive our refined studies exploring the roles of translocation signals in the substrate - receptor binding, as well as experiments testing the contributions of two ATPases to chaperone release and substrate unfolding prior to translocation. For Aim 2, recent structural advances and biochemical findings support tests of specific models for pilus nucleation at the IMC and extension through the central chamber of the OMC. A combination of intracellular signals, including substrate binding and ATP energy, is postulated to induce conformational changes in the OMC for activation of substrate transfer at the expense of pilus production. These studies interface with ongoing collaborative efforts to obtain high-resolution T4SS structures and donor-target cell mating junctions. The work is innovative, in concept by exploiting a highly efficient conjugation machine that we have now shown to translocate effector proteins, and in approach through incorporation of state-of-the art in vivo assays to define dynamic steps in substrate translocation and pilus assembly. The work is expected to stimulate new basic research directions and provide a critical knowledge base for applied studies aimed at suppressing antibiotic resistance spread and disease progression by many clinically significant pathogens.