Molecular structure of the bacterial chemotaxis apparatus

我们正在开展一项持续和系统的方法来解决预测细菌趋化性行为的挑战。在过去一年的研究报告中，我们已经证明，在完整的革兰氏阴性细菌中，直接可视化和确定趋化机制和细胞骨架成分的分子成分结构是可能的。我们使用冷冻电子断层扫描对完整大肠杆菌细胞中化学受体阵列的直接可视化和空间组织进行了研究，结果表明，在野生型细胞中，三元复合物排列成一个扩展晶格，可能是有序的，也可能不是有序的，在同一种群的细胞中，其大小和特定位置存在显著差异。在缺乏CheA和CheW的情况下，化学感受器不会形成可观察到的簇，而是弥漫性地定位于细胞极。在不成比例的高受体水平，膜内陷包含无功能，轴向相互作用的受体组装形成。然而，功能性的化学受体阵列可以通过增加CheA和CheW的细胞水平来重建。这些结果表明，大肠杆菌的趋化性需要化学受体阵列的存在，而这些阵列的形成需要信号分子CheA和CheW的脚手架相互作用。（详见Zhang， Khursigara等人（2007）和Zhang， Weis等人（2007））。在相关研究中，我们使用低温电子断层扫描来确定野生型新月形茎杆菌细胞的极性化学受体阵列的结构。我们证明了这种革兰氏阴性细菌中的化学受体是由受体二聚体组成的三聚体，在细胞质膜中形成部分有序的六边形信号复合物排列。这种在有序/无序界面上的新型受体组织提示了受体和效应器如何被包装在信号组装中，以动态响应细菌趋化性的激活和适应步骤。（详见Khursigara， Wu和Subramaniam(2008)）。为了继续努力确定整个细菌细胞天然环境中单个受体的分子结构，我们使用冷冻电子断层扫描结合3D平均来确定大肠杆菌细胞中化学受体组装的原位结构，这些细胞已经被改造成过量产生丝氨酸化学受体Tsr。我们已经证明，化学受体被组织为受体二聚体的三聚体，并显示出两种不同的构象，主要不同于HAMP结构域在每个三聚体中的排列。配体结合和甲基化改变了化学受体在两种构象之间的分布，丝氨酸结合有利于扩大构象，而化学受体甲基化有利于紧凑构象。因此，在三聚体单元中，化学受体HAMP结构域的不同位置可能代表了与趋化性信号相关的化学受体结构变化的重要方面。基于这些结果，我们提出紧凑和扩展的构象分别代表了化学受体三聚体的激酶开启和激酶关闭状态。我们可以确定完整细胞中分子复合物的结构，这一事实彻底改变了我们对趋化装置和细胞骨架结构的动态变化进行有意义计算的方法，并比较了如何通过遗传改变改变细胞的生理机能，从而可以用来探测和理解潜在复杂机制的行为。（详见Khursigara， Wu， Zhang等人（2008））。我们正在开展一项持续和系统的方法来解决预测细菌趋化性行为的挑战。在过去一年的研究报告中，我们已经证明，在完整的革兰氏阴性细菌中，直接可视化和确定趋化机制和细胞骨架成分的分子成分结构是可能的。我们使用冷冻电子断层扫描对完整大肠杆菌细胞中化学受体阵列的直接可视化和空间组织进行了研究，结果表明，在野生型细胞中，三元复合物排列成一个扩展晶格，可能是有序的，也可能不是有序的，在同一种群的细胞中，其大小和特定位置存在显著差异。在缺乏CheA和CheW的情况下，化学感受器不会形成可观察到的簇，而是弥漫性地定位于细胞极。在不成比例的高受体水平，膜内陷包含无功能，轴向相互作用的受体组装形成。然而，功能性的化学受体阵列可以通过增加CheA和CheW的细胞水平来重建。这些结果表明，大肠杆菌的趋化性需要化学受体阵列的存在，而这些阵列的形成需要信号分子CheA和CheW的脚手架相互作用。（详见Zhang， Khursigara等人（2007）和Zhang， Weis等人（2007））。在相关研究中，我们使用低温电子断层扫描来确定野生型新月形茎杆菌细胞的极性化学受体阵列的结构。我们证明了这种革兰氏阴性细菌中的化学受体是由受体二聚体组成的三聚体，在细胞质膜中形成部分有序的六边形信号复合物排列。这种在有序/无序界面上的新型受体组织提示了受体和效应器如何被包装在信号组装中，以动态响应细菌趋化性的激活和适应步骤。（详见Khursigara， Wu和Subramaniam(2008)）。为了继续努力确定整个细菌细胞天然环境中单个受体的分子结构，我们使用冷冻电子断层扫描结合3D平均来确定大肠杆菌细胞中化学受体组装的原位结构，这些细胞已经被改造成过量产生丝氨酸化学受体Tsr。我们已经证明，化学受体被组织为受体二聚体的三聚体，并显示出两种不同的构象，主要不同于HAMP结构域在每个三聚体中的排列。配体结合和甲基化改变了化学受体在两种构象之间的分布，丝氨酸结合有利于扩大构象，而化学受体甲基化有利于紧凑构象。因此，在三聚体单元中，化学受体HAMP结构域的不同位置可能代表了与趋化性信号相关的化学受体结构变化的重要方面。基于这些结果，我们提出紧凑和扩展的构象分别代表了化学受体三聚体的激酶开启和激酶关闭状态。我们可以确定完整细胞中分子复合物的结构，这一事实彻底改变了我们对趋化装置和细胞骨架结构的动态变化进行有意义的计算的方法，并比较了通过遗传改变如何改变细胞的生理机能。

We are carrying out a sustained and systematic approach to address the challenge of predicting bacterial chemotaxis behavior. In studies reported over the course of the last year, we have demonstrated that it is possible to directly visualize and determine structures of molecular components of the chemotaxis machinery and cytoskletal components in intact gram-negative bacteria. Our work on direct visualization and spatial organization of chemoreceptor arrays in intact E. coli cells using cryo-electron tomography shows that in wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing non-functional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be re-established by increasing cellular levels of CheA and CheW. These results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays, and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW. (see Zhang, Khursigara et al (2007) and Zhang, Weis et al (2007) for more details). In related studies we have used cryo-electron tomography to determine the architecture of polar chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrated that chemoreceptors in this Gram-negative bacterium are organized as trimers of receptor dimers, forming partially ordered, hexagonally-packed arrays of signaling complexes in the cytoplasmic membrane. This novel receptor organization at the order/disorder interface suggests how receptors and effectors can be packed in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis. (see Khursigara, Wu and Subramaniam (2008) for more details). In continuing efforts to define the molecular structures of the individual receptors in the native environment of whole bacterial cells, we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We have demonstrated that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the expanded conformation, and chemoreceptor methylation favoring the compact conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the kinase on and kinase off states of chemoreceptor trimers, respectively. The fact that we can determine structures of molecular complexes in intact cells revolutionizes our approach to carrying out meaningful calculations of the dynamic changes in the chemotaxis apparatus and cytoskeletal architecture, and to compare how changing the physiology of the cells by genetic alterations can be used to probe and understand the behavior of the underlying complex machinery. (see Khursigara, Wu, Zhang et al (2008) for more details).We are carrying out a sustained and systematic approach to address the challenge of predicting bacterial chemotaxis behavior. In studies reported over the course of the last year, we have demonstrated that it is possible to directly visualize and determine structures of molecular components of the chemotaxis machinery and cytoskletal components in intact gram-negative bacteria. Our work on direct visualization and spatial organization of chemoreceptor arrays in intact E. coli cells using cryo-electron tomography shows that in wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing non-functional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be re-established by increasing cellular levels of CheA and CheW. These results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays, and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW. (see Zhang, Khursigara et al (2007) and Zhang, Weis et al (2007) for more details). In related studies we have used cryo-electron tomography to determine the architecture of polar chemoreceptor arrays in wild-type Caulobacter crescentus cells. We demonstrated that chemoreceptors in this Gram-negative bacterium are organized as trimers of receptor dimers, forming partially ordered, hexagonally-packed arrays of signaling complexes in the cytoplasmic membrane. This novel receptor organization at the order/disorder interface suggests how receptors and effectors can be packed in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis. (see Khursigara, Wu and Subramaniam (2008) for more details). In continuing efforts to define the molecular structures of the individual receptors in the native environment of whole bacterial cells, we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We have demonstrated that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the expanded conformation, and chemoreceptor methylation favoring the compact conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the kinase on and kinase off states of chemoreceptor trimers, respectively. The fact that we can determine structures of molecular complexes in intact cells revolutionizes our approach to carrying out meaningful calculations of the dynamic changes in the chemotaxis apparatus and cytoskeletal architecture, and to compare how changing the physiology of the cells by genetic alterations can be [summary truncated at 7800 characters]