Date of Degree

6-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Anuradha Janakiraman

Committee Members

David Jeruzalmi

Itzhak Mano

Fevzi Daldal

Lars Dietrich

Subject Categories

Biology | Genetics and Genomics | Microbiology

Keywords

cell envelope, membrane integrity, bacterial genetics, e. coli

Abstract

The biological membrane is an essential, defining feature of all cells. Biological membranes comprise phospholipid bilayers as well as a complement of proteins which are unique to a given organism. These proteins play a central role in dictating the biochemical state of the cell’s internal cytoplasm by controlling selective passage of solutes in and out of the cell, transducing signals in response to extracellular stimuli, and controlling the biogenesis of the bilayer itself which is critical towards barrier function. For most bacteria, the periphery of the cell is multi-layered, including both a biological membrane as well as a peptidoglycan cell wall, collectively referred to as the cell envelope. The cell envelope of Escherichia coli, as well as most other Gram-negative bacteria, is distinguished by the presence of two biological membranes, referred to as the inner and outer membrane which form a compartment separated from the cytoplasm known as the periplasm. The cell envelope proteome of E. coli encompasses a large amount of the cell’s genetic landscape, representing more than a quarter of all genes present in the organism. The majority of cell envelope proteins reside as the inner membrane. Approximately one-third of these are of unknown function and are highly represented by small integral membrane proteins. Studies aimed at elucidating the functions of this largely unexplored class of membrane proteins will not only provide better fundamental understanding of cell envelope biology in bacteria, but also identify novel targets for antibacterial therapeutics.

This thesis concerns the characterization of one such small integral inner membrane protein, YciB, conversed across most Gram-negative bacterial species. Towards characterizing the function of this protein, a genetic screen was conducted in E. coli to identify genetic interactors with the gene yciB. The results identified a gene encoding an inner membrane lipoprotein, dcrB, as a synthetic lethal partner of yciB. Prior to this work, studies focused on these inner membrane proteins were few and did not present a clear picture of their genetic interaction or functionality in the cell. The goal here was to uncover the molecular basis of these genetic interactions, ultimately towards assigning a function to the individual gene products.

Characterization of the synthetic lethal interaction between yciB and dcrB is the primary focus of the second chapter of this thesis. Therein, an E. coli double mutant, yciB dcrB, is shown to be non-viable primarily due to a malfunction in the biogenesis of proteins destined to the outer membrane. Data reveal these proteins are trapped at the inner membrane, exerting toxic effects to the cell which result in the activation of a diverse set of cell envelope stress response signaling mechanisms as well as cell death. The results indicated an essential, synergistic role for YciB and DcrB in outer membrane protein biogenesis. Exploration of the possible roles for the individual proteins revealed a yciB mutant displayed defects in outer membrane permeability as well as a reduction in proton motive force at the inner membrane, suggesting a role for YciB in membrane energetics which are important towards cell envelope integrity.

In the third chapter of this thesis, results are presented which describe in greater detail the mechanistic basis of the synthetic defect in outer membrane protein biogenesis. Specifically, through genetic and biochemical analyses the removal of yciB and dcrB is shown to result in the impairment of the biogenesis of lipoproteins targeted to the outer membrane. A precise step in the maturation of lipoproteins, wherein lipoproteins are modified with the phospholipid derived molecule, diacylglycerol, is shown to be defective in a yciB dcrB double mutant, resulting in the toxic accumulation of outer membrane lipoproteins at the inner membrane. Overall, the results indicated a synergistic role for YciB and DcrB in lipoprotein maturation. It is hypothesized based on the results that yciB negatively impacts aspects of E. coli cell envelope physiology in a manner which renders dcrB essential towards biogenesis of outer membrane lipoproteins.

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