Maintenance of Escherichia coli cell envelope integrity under stress

细菌的细胞包膜是一个复杂的多层结构，对细胞的生存至关重要。在革兰氏阴性细菌的一大群中，细胞包膜由两层膜组成，中间夹着坚硬的细胞壁。包膜是一种选择性渗透屏障，允许从外部吸收重要的营养物质，从内部排出有毒废物，是抵御环境压力的第一道防线。近30%的细胞蛋白质嵌入在包膜中，它们在包膜的构建和维持其完整性方面发挥着重要而多样的功能。在这些蛋白质中，脂蛋白是一类重要而丰富的蛋白质，它具有将它们固定在细胞膜上的特征脂肪酸链。脂肪酸的添加发生在膜本身，这就需要脂蛋白修饰与膜稳态的紧密协调。然而，人们对调节这种耦合的机制知之甚少。该项目描述了当细胞包膜受到环境或突变压力时，细菌控制脂蛋白修饰的方法。这项工作可能适用于其他革兰氏阴性细菌，这些细菌在地球上几乎所有环境中都起着重要的病理和非病理作用。为了实现其目标，该项目将结合遗传学、生物化学和细胞生物学方法，从而为纽约城市学院（CCNY）的本科生和研究生提供多学科和多维度的培训。与本提案的研究目标密切相关的探究式实验室模块将被纳入本科微生物学课程，提供并留住多样化的学生群体，包括女性、第一代大学生和/或那些属于历史上代表性不足的生物科学群体的学生。在革兰氏阴性菌中，由膜磷脂衍生的脂肪酰基链对脂蛋白的翻译后修饰发生在内膜。这些成熟的脂蛋白有些保留在内膜上，而大多数位于外膜上。它们的加工过程和功能尚不完全清楚。此外，细胞是否以及如何调节脂蛋白修饰，特别是当膜稳态在包膜胁迫下改变时，尚不清楚。该项目旨在通过研究特定的分子机制来解决这个问题，这些机制有助于维持脂蛋白成熟的保真度，以响应由环境或突变应激引起的膜特性的物理化学改变。具体的实验将探讨一组保守的细菌基因之间的合成关系，缺乏脂蛋白成熟是缺陷的。这组突变导致脂蛋白定位改变，细胞膜与细胞壁形成异常连接，细胞质收缩，最终导致细胞死亡。提出的实验是基于支持在响应膜脂组成/平衡改变时维持该过程适应性的基本分子安全机制的初步发现。总的来说，该项目有望为细菌在压力下包膜完整性的分子机制提供基本的见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The bacterial cell envelope is a complex multi-layered structure that is essential for cell viability. In a large group of bacteria, the Gram-negatives, the cell envelope is composed of two membranes with a rigid cell wall sandwiched between. The envelope is a selectively permeable barrier allowing the uptake of important nutrients from the outside, expulsion of toxic wastes from the inside, and is a first line of defense against environmental stress. Nearly 30% of the cell's proteins are embedded in the envelope where they perform critical and diverse functions including in construction of the envelope and maintenance of its integrity. Among these, lipoproteins are an important and abundant class of proteins that bear characteristic fatty acid chains which anchor them to the cell's membranes. The fatty acid addition takes place on the membrane itself, and this necessitates the tight coordination of lipoprotein modification with membrane homeostasis. However, little is known about the mechanisms that regulate this coupling. This project characterizes the means by which bacteria control lipoprotein modification when the cell envelope is subject to environmental or mutational stresses. The work will likely be applicable to other Gram-negative bacteria which play important pathological and non-pathological roles in virtually all environments on the planet. To achieve its goals, this project will use a combination of genetics, biochemical and cell biological methodologies thereby providing multidisciplinary and multidimensional training for a broad range of undergraduate and graduate students at the City College of New York (CCNY). An inquiry-based laboratory module that is closely aligned with the research goals of this proposal will be incorporated into an undergraduate microbiology course providing access to and retaining a diverse group of students that include women, first-generation college students, and/or those belonging to historically underrepresented groups, in the biological sciences.In Gram-negative bacteria, the post-translation modification of lipoproteins with fatty acyl chains derived from membrane phospholipids occurs at the inner membrane. Some of these mature lipoproteins are retained at the inner membrane while most are localized to the outer membrane. Their processing and functions are not fully understood. Further, if and how cells regulate lipoprotein modification, particularly when membrane homeostasis is altered under envelope stress, is unclear. This project aims to address this question by investigating specific molecular mechanisms that serve to maintain the fidelity of lipoprotein maturation in response to physicochemical alterations in membrane properties resulting from environmental or mutational stress. Specific experiments will probe the synthetic relationship between a set of conserved bacterial genes in whose absence lipoprotein maturation is defective. Mutations within this set leads to altered lipoprotein localization, formation of aberrant links of the inner membrane with the cell wall, cytoplasmic shrinkage, culminating in cell death. The proposed experiments are based on initial findings that support the presence of an essential molecular safety mechanism to maintain the fitness of this process in response to alterations in membrane lipid composition/balance. Overall, it is expected that this project will provide fundamental insights into the molecular mechanisms of bacterial cell envelope integrity under stress.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.