GENETIC ANALYSIS OF CELL ENVELOPE BIOGENESIS IN ESCHERICHIA COLI

Abstract

The <italic>Escherichia coli</italic> cell envelope is a multi-layered structure that serves as a selective permeability barrier, permitting influx of nutrients, preventing entry of potentially toxic molecules, and allowing efflux of waste products. The cell envelope consists of an inner and outer membrane that delimit the aqueous periplasm, which houses the peptidoglycan cell wall. The lipids, proteins, glycan chains, and peptides that make up the cell envelope structures are all transported and assembled by discrete pathways that are tightly coordinated with one another.

In this thesis, we focus on understanding the assembly and function of the LptDE subcomplex responsible for assembling the glycolipid lipopolysaccharide in the outer leaflet of the outer membrane. Through the use of bacterial genetics and biochemistry, we have isolated and characterized <italic>lptD</italic> mutants that affect assembly and/or function of LptD. Our work provides insight into the robust interaction between LptD and LptE, which is necessary for proper assembly and function of the subcomplex. We also shed light on how LptD interacts with LptA to complete the Lpt transenvelope bridge as well as how it interacts with LPS, suggesting a model for LPS transport and assembly.

In addition to learning about the Lpt pathway, <italic>lptD</italic> mutants reported here have contributed to our knowledge of other cell envelope processes as well. We present results that show BamD can bind to substrate, through the use of an LptD mutant substrate. Using a different <italic>lptD</italic> mutation, we show that accumulation of phosphatidic acid in the outer membrane alters the permeability barrier and increases resistance to vancomycin. Finally, through the analysis of a dominant mutation in the retrograde phospholipid transport pathway that was originally identified as a suppressor of an <italic>lptD</italic> mutation, we demonstrate that the outer membrane physically contributes to cell integrity to prevent lysis when the local peptidoglycan structure is weakened. Taken together, the results presented here illustrate that cell envelope biogenesis is a highly complex process that involves an elegant interplay between all of the pathways that contribute to its assembly.