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条件致病菌显示出例外 对一系列环境的适应性。它们的部分适应潜力是在极端渗透压下生存的能力。 改变。一旦外部介质突然稀释，如在雨中，细菌就能避开机械破裂。 通过接合作为渗透分子释放阀的张力激活通道。低门槛MSCs和高门槛MSCs 阈值MSCL，这两种介导大肠杆菌中大部分渗透分子交换的通道物种，已经被 对它们的结构和门控机制进行了广泛的研究。然而，尽管生物物理学取得了进展 对这些单独的机械敏感通道的研究，对实际的释放过程知之甚少 在细胞中发生在突然的渗透降档。几乎没有关于肿胀程度和速度的数据， 渗透分子释放的动力学，即通过特定通道逃逸的分子，何时以及如何 暂态渗透性停止了，最后，所有这些参数如何与渗透适合性联系在一起。我们目前的情况 对这种机制的分析强烈地提出了两个关键方面：(I)通道必须快速释放渗透物质 足以超过渗透水流入并抑制细胞肿胀；另一方面(Ii)巨大的渗透分子 必须通过停用低阈值通道来牢固地终止耗散，以促进恢复。这个 拟议的项目旨在建立一个自洽的救援过程的动力学/物理模型 渗透诱导活体培养中溶质交换的唯象描述 Coli，细胞包膜力学，以及通道门的空间和热力学特性。(1)我们会 确定在渗透过程中通过特定渠道离开细胞的主要渗透物质的特性和可用性 使用现代代谢组学进行电击。我们将研究主要渗透性和非渗透性渗透分子的影响。 在MSCs和MSCL上进行门控并可视化可能影响MD状态分布的渗透和相互作用 模拟。(2)解决关于MSCs开放机制的几个遗留问题 失活后，我们将确定稳定休眠状态和开放状态的突变体的晶体结构。这个 然后，将在模拟中重建状态之间的转换路径。(3)我们会采用 停流技术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压有关；我们将把交换率与渗透压细胞的活力联系起来。vbl.使用 流体学和视频显微镜，我们将确定细胞壁的弹性和膜的数量 可伸缩的肽聚糖内部的储存物。并行电生理分析将提供渠道 用于选通和失活的密度和参数。两个非政府组织协同行动的详细画面 渗透通透性反应过程中的冗余通道和一组关键参数将提供 一个定量模型的基础，该模型将预测特定的渗透量和速度 减速将是可以容忍的，或者是致命的。

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.