Fellowship extension request for F32GM130003 "The Molecular Mechanisms of Spore Germination"

项目摘要 为了响应饥饿，芽孢杆菌目和梭菌目的细菌物种分化成孢子。 这些抗压力的细胞类型在代谢上是不活跃的，可以保持休眠多年，但很快 在感觉到适当的营养条件已经恢复时发芽并恢复生长。许多这些 物种是人类的病原体，在休眠状态下对抗生素具有高度耐药性， 否则进行灭菌处理，如加热和紫外线照射。为了更好地了解如何预防致病性 孢子形成者进入这种高度持久的状态，几乎每一步的分子机制 在孢子形成途径中的作用已经被表征。然而，同样重要的发芽过程 仍然不太清楚。更完整的发芽分子表征将有助于 开发可以防止退出休眠或引发过早发芽的处理方法， 易受抗菌治疗的影响 大多数内孢子形成细菌遵循类似的萌发程序，涉及一组保守的因子。 第一步涉及环境监测的一个大家庭的假定发芽受体。芽孢杆菌中 在枯草芽孢杆菌中，原型受体由gerA操纵子的产物- GerAA、GerAB和 GerAC。GerA蛋白是响应L-丙氨酸的孢子萌发所必需的。它们如何运作， 它们是否作为营养受体还没有确定。感受器在感受到发芽物质后 通过未知的机制起作用，从细胞中释放大量储存的小分子吡啶二羧酸（DPA）。 孢子核水取代DPA，导致部分孢子水合。一种保守的膜复合物， DPA释放所需的，但如何激活尚不清楚。最后，保守的细胞壁水解酶包装 被激活并降解一层厚的称为孢子皮层的特化肽聚糖。 去除这一保护层允许进一步的核心水合作用，代谢活动的开始，以及 恢复增长。这些酶是如何被激活的，它们的底物特异性仍然很差 明白该建议旨在定义发芽过程中所有三个步骤的分子基础 从最后一步到第一步的时间路径。具体目标是： 目的1：确定皮质降解酶CwlJ的激活机制，并确定其作用机制。 底物特异性 目的2：探讨萌发检测及信号转导机制。

PROJECT SUMMARY In response to starvation, bacterial species of the orders Bacillales and Clostridiales differentiate into spores. These stress-resistant cell types are metabolically inactive and can remain dormant for years but rapidly germinate and resume growth upon sensing that proper nutrient conditions have returned. Many of these species are human pathogens, and in their dormant state are highly resistant to antibiotics and can withstand otherwise sterilizing treatments like heat and UV irradiation. To better understand how to prevent pathogenic spore-formers from entering this highly durable state, the molecular mechanisms underlying virtually every step in the sporulation pathway have been characterized. However, the equally important process of germination remains less well understood. A more complete molecular characterization of germination will facilitate the development of treatments that can prevent exit from dormancy or trigger premature germination, leaving cells vulnerable to antibacterial therapies. Most endospore-forming bacteria follow a similar germination program that involves a conserved set of factors. The first step involves environmental monitoring by a large family of putative germinant receptors. In Bacillus subtilis, the prototypical receptor is composed of the products of the gerA operon – GerAA, GerAB, and GerAC. The GerA proteins are required for spore germination in response to L-alanine. How they function and whether or not they act as a nutrient receptor has not been established. Upon sensing germinants, receptors act by an unknown mechanism to release large stores of the small molecule dipicolinic acid (DPA) from the spore core. Water replaces the DPA resulting in partial spore hydration. A conserved membrane complex is required for DPA release but how it is activated is unknown. Finally, conserved cell wall hydrolases packaged in the spore are activated and degrade a thick layer of specialized peptidoglycan known as the spore cortex. Removal of this protective layer allows further core hydration, the onset of metabolic activity, and the resumption of growth. How these enzymes are activated and their substrate specificities remain poorly understood. This proposal seeks to define the molecular underpinning of all three steps in the germination pathway, working from the last temporal step to the first. The specific aims are: Aim 1: Determine the mechanism that activates the cortex-degrading enzyme CwlJ and define its substrate specificity. Aim 2: Investigate the mechanism of germinant detection and signal transduction.