Dissertations, Theses, and Capstone Projects
Date of Degree
9-2025
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy
Program
Biology
Advisor
Anuradha Janakiraman
Committee Members
Stefan Pukatzki
David Jeruzalmi
Lars Dietrich
Natividad Ruiz
Subject Categories
Biology | Microbiology
Abstract
Escherichia coli and other Gram-negative bacteria are characterized by a complex double-membraned cell envelope that consists of an inner membrane, a thin peptidoglycan cell wall, and an outer membrane, between which exists the aqueous periplasm. This envelope architecture provides remarkable protection from the outside environment including resistance against antibiotics. Attempts to quell the rise of antibiotic resistance, therefore, require a concerted understanding of the biogenesis of the cell envelope. One class of proteins, lipoproteins, play important roles in the maintenance and function of the cell envelope. Lipoproteins are modified at the inner membrane and the mature proteins are tethered to either membrane via their acyl chains. The most abundant E. coli protein, Lpp, is an outer membrane lipoprotein that provides a crucial covalent linkage to the underlying peptidoglycan and contributes to envelope architecture, stability, and signaling across the envelope. When lipoprotein maturation is perturbed, Lpp can establish toxic covalent linkages of the inner membrane to the peptidoglycan. However, the precise molecular mechanism leading to cell death under these conditions is unclear. Lpp and its biogenesis have been extensively studied. Despite this, we know surprisingly little about whether critical accessory factors exist that facilitate or regulate Lpp modification, particularly in conditions of stress. Here, we report the identification and characterization of a regulatory mechanism that mitigates the formation of lethal linkages of inner membrane-stranded Lpp to the peptidoglycan.
Chapter 1 introduces the Gram-negative cell envelope and abundant lipoprotein Lpp, as well as Lpp's toxic effect on the cell at the inner membrane in conditions of lipoprotein maturation stress.
In Chapter 2, we discuss data that support a role for the inner membrane lipoprotein DcrB as a putative chaperone of Lpp transit in conditions of inefficient lipoprotein maturation. dcrB is essential in conditions of reduced lipoprotein maturation efficiency (chemical/environmental stress), and its lethality can be suppressed through abrogation of Lpp’s linkage to peptidoglycan from the inner membrane. Through a combination of genetic and biochemical approaches, we determine that DcrB directly binds Lpp and that its presence leads to both a decrease in Lpp toxicity at the IM and to alterations in Lpp’s oligomeric states. We therefore posit a chaperone-like role for DcrB in preventing premature oligomerization of Lpp at the inner membrane. Our results suggest that retention of Lpp in its monomeric form also avoids aberrant links to the peptidoglycan.
In Chapter 3, we report data from our examination of the current model for DcrB and Lpp interaction at the inner membrane by investigating the molecular mechanism that underlies Lpp-mediated toxicity from this cellular location. Alteration of Lpp oligomerization through mutational analysis in the presence and absence of DcrB suggests that a stable trimeric structure indeed contributes to increased toxicity at the inner membrane. Furthermore, shortening or increasing the length of Lpp polypeptide correspondingly decreases its toxicity at this location, which suggests that the distance between the inner membrane and the peptidoglycan cell wall is a substantial factor in cell death.
In Chapter 4, findings from Chapters 2 and 3 are briefly summarized and discussed, and we suggest that future experimentation based on this work should focus on detailed examination of the physical interaction between DcrB and Lpp, and a more complete investigation of how both Lpp trimeric stability and length contribute to its toxicity at the IM.
Chapter 5 outlines the Methods utilized throughout this thesis (Chapters 2, 3, and Appendix A), along with strains, plasmids, and primers from each section.
Appendix A includes preliminary findings on a putative role for DcrB in cell division, and Appendix B examines the architecture of the Pseudomonas aeruginosa biofilm.
Taken together, our results are consistent with a model in which DcrB prevents toxic Lpp-mediated linkages to the peptidoglycan from the inner membrane by preventing premature oligomerization of Lpp, under conditions when Lpp accumulates at the inner membrane. This function of DcrB represents an important point of regulation of Lpp maturation at the inner membrane.
Recommended Citation
LaMarre, Emily J., "Biogenesis of the Abundant Lipoprotein Lpp During Lipoprotein Maturation Stress in Escherichia coli" (2025). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/6457
