Maintenance of Escherichia coli cell envelope integrity under stress

细菌的细胞膜是一个复杂的多层结构，对细胞的生存至关重要。在一大群革兰氏阴性细菌中，细胞膜由两层膜组成，中间夹着一层坚硬的细胞壁。包膜是一种选择性可渗透的屏障，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%的细胞蛋白质嵌入到包膜中，在那里它们执行关键的和不同的功能，包括构建包膜和维持其完整性。其中，脂蛋白是一类重要的和丰富的蛋白质，含有将它们固定在细胞膜上的特征脂肪链。脂肪酸的加成发生在膜本身，这需要脂蛋白修饰与膜动态平衡的紧密协调。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被膜受到环境或突变压力时，细菌控制脂蛋白修饰的方法。这项工作很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的环境中都扮演着重要的病理和非病理角色。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，从而为纽约市立大学(CCNY)的广泛本科生和研究生提供多学科和多层面的培训。与这项建议的研究目标紧密结合的探究式实验室模块将被纳入本科微生物学课程，提供接触和留住不同群体的学生，其中包括女性、第一代大学生和/或那些在生物科学中历史上代表不足的群体。在革兰氏阴性细菌中，脂蛋白的翻译后修饰与来自膜磷脂的脂肪酰链发生在内膜。这些成熟的脂蛋白中有一些保留在内膜，而大多数定位于外膜。它们的过程和功能还没有完全被理解。此外，细胞是否以及如何调节脂蛋白修饰，特别是当膜内平衡在包膜压力下改变时，尚不清楚。本项目旨在通过研究特定的分子机制来解决这一问题，这些机制有助于在环境或突变应激导致的细胞膜性质发生物理化学变化时保持脂蛋白成熟的保真度。具体的实验将探索一组保守的细菌基因之间的合成关系，如果没有这些基因，脂蛋白成熟就会出现缺陷。这一组突变导致脂蛋白定位改变，内膜与细胞壁形成异常连接，细胞质收缩，最终导致细胞死亡。拟议的实验是基于初步发现，支持存在基本的分子安全机制，以维持这一过程的适宜性，以应对膜脂组成/平衡的变化。总体而言，预计该项目将为压力下细菌细胞包膜完整性的分子机制提供基本的见解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.