Molecular structure of the bacterial chemotaxis apparatus

我们一直在进行持续和系统的方法来解决预测细菌趋化性行为的挑战。在过去几年的研究中，我们已经证明，可以直接可视化和确定完整革兰氏阴性细菌中趋化机制和细胞骨架成分的分子组成部分的结构。我们的工作直接可视化和空间组织的化学感受器阵列在完整的E。大肠杆菌细胞使用冷冻电子断层扫描显示，在野生型细胞中，三元复合物排列为扩展的晶格，在同一群体中的细胞之间的大小和特定位置有显着变化。将这些研究扩展到C. crescentus，我们证明了在这种革兰氏阴性细菌中的化学感受器被组织为受体二聚体的三聚体，在细胞质膜中形成部分有序的、六边形包装的信号复合物阵列。这种新的受体组织在秩序/无序界面表明受体和效应器可以包装在信号组件动态响应细菌趋化性的激活和适应步骤。 我们还使用冷冻电子断层扫描结合三维平均，以确定在大肠杆菌细胞中的化学感受器组件的原位结构。这些研究代表了完整细菌细胞中完整膜蛋白的结构测定的第一份报告。我们证明，化学感受器组织为三聚体的受体二聚体，并显示两个不同的构象，主要不同的HAMP结构域内的每个三聚体的安排。配体结合和甲基化改变了两种构象之间的化学受体的分布，丝氨酸结合有利于扩展构象，化学受体甲基化有利于紧凑构象。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性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，并比较了如何通过遗传改变来改变细胞的生理学，以探索和理解潜在的复杂机制的行为。 在最近的研究中，我们进一步扩展了分析，以描述和比较的空间分布，定位和架构的化学感受器阵列在三个不同的革兰氏阴性细菌使用冷冻电子断层扫描。我们表明，虽然每个生物体共享一个看似共同的阵列架构，E。大肠杆菌阵列是分散的和扩展的，在柄杆菌和蛭弧菌细胞中观察到的紧凑的阵列。化学感受器阵列在大小上也更一致，并且与柄杆菌属和蛭弧菌属细胞中的单极鞭毛密切相关，而它们在大小上变化很大，并且与E. coli细胞。 层析平均结果表明，与应用程序的六边形对称性，所有三种生物体具有相似的单位间距和三聚体的二聚体距离。然而，在个别阵列中的受体分布的分析揭示了从一个物种到另一个物种的最近邻接近度的实质性变化，并响应于生长介质的变化。虽然在不同生物体中化学感受器阵列的尺寸和定位存在广泛的变化，但与丰富培养基相比，当细胞在基本生长培养基中生长时，大肠杆菌化学感受器阵列内的信号传导复合物的堆积密度更高。我们发现，这些变化的密度的包装可以参数化的伊辛型模型的背景下，以获得理论预测的协同性和灵敏度的细胞外配体浓度的变化的细胞反应。我们测试的预测实验使用荧光共振能量转移（FRET）显微镜来测量CheA激活条件相同的断层实验。预测和实验测量的响应之间的良好的相关性表明，由冷冻电子断层扫描确定的化学感受器阵列的分子组织的知识可以转化为有意义的计算模型，用于预测细菌趋化性响应的定量方面。

We have been 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 few years, 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, with significant variations in the size and specific location among cells in the same population. In an extension of these studies to C. crescentus, 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. We also used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells. These studies represent the first report of structure determination of an integral membrane protein in intact bacterial cells. We 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 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. In more recent studies, we have further extended the analysis to describe and compare the spatial distribution, localization and architecture of chemoreceptor arrays in three different Gram-negative bacteria using cryo-electron tomography. We show that although each organism shares a seemingly common arrayed architecture, E. coli arrays are disperse and extended, in contrast to the compact arrays observed in Caulobacter and Bdellovibrio cells. Chemoreceptor arrays are also more consistent in size and are closely associated with the single polar flagella in Caulobacter and Bdellovibrio cells, while they vary greatly in size and demonstrate no discernable spatial correlation to the multiple flagella present in E. coli cells. Tomographic averaging results demonstrate that with the application of hexagonal symmetry, all three organisms have a similar unit spacing and trimer-of-dimer distance. However, analysis of receptor distribution in individual arrays reveals substantial variations in nearest neighbor proximities from one species to another, and in response to changes in growth medium. While there are broad variations in size and localization of the chemoreceptor arrays in the different organisms, the packing density of signaling complexes within E.coli chemoreceptor arrays is higher when cells are grown in minimal growth medium as compared to rich medium. We show that these changes in density of packing can be parametrized in the context of an Ising-type model to obtain theoretical predictions for the cooperativity and sensitivity of the cellular response to changes in extracellular ligand concentration. We tested the predictions experimentally using Fluorescence Resonance Energy Transfer (FRET) microscopy to measure CheA activation under conditions identical to those used for the tomographic experiments. The excellent correlation between the predicted and experimentally measured responses demonstrates that knowledge of the molecular organization of the chemoreceptor arrays as determined by cryo-electron tomography can be translated into meaningful computational models for predicting quantitative aspects of the bacterial chemotaxis response.