Maintenance of Escherichia coli cell envelope integrity under stress

Cuny City College, New York NY

Investigators

Abstract

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 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. 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.