A Comprehensive approach to bacterial osmotolerance

项目摘要 通过土壤和淡水传播的肠道细菌和大多数机会致病菌显示出特殊的 对各种环境的适应性。它们的适应性潜力的一部分是在剧烈的渗透压下生存的能力 变化在外部介质突然稀释时，例如在雨中，细菌逃避机械破裂 通过接合作为渗透剂释放阀的张力激活通道。低阈值MSCS和高- 阈值MscL是介导E.大肠杆菌， 在其结构和门控机制方面进行了广泛的研究。然而，尽管生物物理学取得了进展， 尽管对这些单独的机械敏感通道进行了研究，但对实际的释放过程知之甚少， 发生在细胞内突然的渗透压降低时。几乎没有关于肿胀程度和速度的数据， 渗透剂释放的动力学，通过特定通道逃逸的分子， 瞬时渗透性停止，最后，所有这些参数是如何与渗透适应性。我们目前 对该机制的分析有力地表明了两个关键方面：（i）通道必须快速释放渗透剂 足以超过渗透水流入并抑制细胞肿胀;另一方面（ii）大量的渗透剂 耗散必须通过低阈值沟道的失活来牢固地终止以促进恢复。的 拟议的项目旨在建立一个自洽的动力学/物理模型的救援过程的基础上， 对E. 大肠杆菌，细胞被膜力学，以及通道门控的空间和热力学性质。(1)我们将 确定在渗透过程中通过特定通道离开细胞的主要渗透剂的身份和可用性 现代代谢组学的研究。我们将研究主要渗透性和非渗透性渗透剂的影响 在MscS和MscL门控和可视化渗透和相互作用，可能会影响状态分布在MD 仿真。(2)为了解决有关MSCS开放机制的几个遗留问题， 失活后，我们将确定具有稳定的静息态和开放态的突变体的晶体结构。的 然后将在模拟中重建状态之间的过渡路径。(3)我们将采用 停流技术，以记录活培养物中光散射的动力学，并评估 细胞被膜与水和渗透剂的交换率;我们将把交换率与渗透细胞活力联系起来。使用 我们将使用射流和视频显微镜来确定细胞壁的弹性和膜的量 可拉伸肽聚糖内的储存器。并行电生理分析将提供通道 用于门控和灭活的密度和参数。两非一致行动的详细画面- 冗余通道的过程中渗透渗透率的反应和一组'重要的'参数将提供 一个定量模型的基础，该模型将预测一个特定的渗透压的大小和速度， 降档将是可容忍的或致命的。

Project Summary Enteric bacteria and most opportunistic pathogens transmitted through soil and fresh water show exceptional adaptability to a range of environments. Part of their adaptive potential is the ability to survive drastic osmolarity changes. Upon a sudden dilution of external medium, such as in the rain, bacteria evade mechanical rupture by engaging tension-activated channels that act as osmolyte release valves. The low-threshold MscS and high- threshold MscL, the two channel species that mediate the bulk of osmolyte exchange in E. coli, have been extensively studied in terms of their structure and gating mechanisms. Yet, despite the progress in biophysical studies of these individual mechanosensitive channels, little is known about the actual release process that takes place in the cell upon abrupt osmotic downshift. There is almost no data on the extent and rate of swelling, the kinetics of osmolyte release, the molecules that escape through specific channels, when and how the transient permeability ceases, and finally, how all these parameters are linked to osmotic fitness. Our current analysis of the mechanism strongly suggests two key aspects: (i) the channels must release osmolytes fast enough to outpace the osmotic water influx and curb cell swelling; on the other hand (ii) the massive osmolyte dissipation must be firmly terminated by inactivation of the low-threshold channel to facilitate recovery. The proposed project aims at a self-consistent kinetic/physical model of the rescuing process based on a comprehensive phenomenological description of osmotically-induced solute exchange in live cultures of E. coli, cell envelope mechanics, and on spatial and thermodynamic properties of channel gating. (1) We will determine identities and availabilities of major osmolytes leaving cells through specific channels during osmotic shock using modern metabolomics. We will study the effects of major permeable and impermeable osmolytes on MscS and MscL gating and visualize permeation and interactions which may affect state distributions in MD simulations. (2) To address several remaining questions about the mechanism of MscS opening and inactivation, we will determine crystal structures of mutants with stabilized resting and open states. The transition pathways between the states will then be reconstructed in simulations. (3) We will employ the stopped-flow technique to record the kinetics of light scattering in live cultures and assess permeabilities of the cell envelope to water and osmolytes; we will correlate the exchange rates with osmotic cell viability. Using fluidics and videomicroscopy we will to determine the elasticity of the cell wall and the amount of membrane reservoir inside the stretchable peptidoglycan. Parallel electrophysiological analysis will provide channel densities and parameters for gating and inactivation. The detailed picture of the concerted action of two non- redundant channels in the course of osmotic permeability response and a set of ‘vital’ parameters will provide grounds for a quantitative model which would predict whether a particular magnitude and speed of osmotic downshift will be tolerable or lethal.