Molecular structure of the bacterial chemotaxis apparatus

我们一直在开展持续和系统的方法来应对预测细菌趋化行为的挑战。在过去几年的研究中，我们已经证明，在完整的革兰氏阴性细菌中，可以直接可视化和确定趋化机制的分子成分和细胞质成分的结构。我们利用冷冻电子断层扫描技术对完整的大肠杆菌细胞中的化学感受器阵列进行直接可视化和空间组织的工作表明，在野生型细胞中，三元复合体以扩展的晶格形式排列，同一种群中细胞之间的大小和特定位置存在显著差异。在将这些研究扩展到新月弯孢杆菌的过程中，我们证明了这种革兰氏阴性细菌中的化学受体是以受体二聚体的三聚体的形式组织起来的，在细胞质膜上形成了部分有序的、六角形排列的信号复合体。这种位于有序/无序界面的新的受体组织表明，受体和效应器如何被包装在信号组件中，以在细菌趋化的激活和适应步骤中做出动态反应。我们还使用冷冻电子断层扫描与3D平均相结合的方法来确定大肠杆菌细胞中化学感受器组件的原位结构。这些研究是在完整的细菌细胞中确定完整膜蛋白结构的第一个报告。我们证明了化学受体被组织成受体二聚体的三聚体，并显示出两种不同的构象，主要是在每个三聚体中HAMP结构域的排列上不同。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展构象，化学感受器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体的结构，这一事实彻底改变了我们的方法，即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，并比较如何通过遗传改变改变细胞的生理，以探索和理解潜在复杂机械的行为。在最近的研究中，我们进一步扩展了分析，使用冷冻电子断层扫描来描述和比较三种不同革兰氏阴性细菌中化学感受器阵列的空间分布、定位和结构。我们发现，虽然每个生物都有一个看似共同的阵列结构，但大肠杆菌阵列是分散和扩展的，这与在Caulbacter和Bdellovibrio细胞中观察到的紧凑阵列形成了鲜明对比。化学感受器阵列的大小也更一致，与Caulbacter和Bdellovibrio细胞中的单极鞭毛密切相关，而它们的大小差异很大，与大肠杆菌细胞中存在的多鞭毛没有明显的空间相关性。层析平均结果表明，在六边形对称性的应用下，所有三种生物都具有相似的单位间距和三聚体距离。然而，对单个阵列中受体分布的分析表明，不同物种之间最近邻的亲和力有很大差异，并对生长介质的变化做出反应。虽然不同生物体中化学受体阵列的大小和定位有很大的差异，但当细胞在最低限度的生长培养基中生长时，信号复合体在大肠杆菌化学受体阵列中的堆积密度比在富营养液中生长的细胞更高。我们表明，这些堆积密度的变化可以在伊辛型模型的背景下进行参数化，以获得细胞对细胞外配体浓度变化的协作性和敏感性的理论预测。我们使用荧光共振能量转移(FRET)显微镜在与断层扫描实验相同的条件下测量CHEA的激活，对预测进行了实验验证。预测的反应与实验测量的反应之间的良好相关性表明，通过冷冻电子断层扫描确定的化学受体阵列的分子组织的知识可以转化为有意义的计算模型，用于预测细菌趋化反应的定量方面。

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.