Abstract

Outer membrane vesicles (OMVs) are small proteoliposomes generated by active protrusion of the outer membrane (OM) in Gram-negative bacteria during growth. Over 60 years of experimentation has ascribed numerous functions to OMVs produced by different bacteria. However, the mechanism(s) by which OMVs are produced is poorly understood. Due to this, the field remains controversial because many regard OMVs as byproducts of cell lysis and question their physiological relevance. Previous studies in Pseudomonas aeruginosa and related species confirmed that these microbes undergo programmed cell death that results in the stochastic release of vesicles and cellular contents. On the contrary, Bacteroides spp. have been shown to produce large quantities of OMVs that contain select protein cargo in the absence of cell lysis. This discrepancy indicates that OMV production may not be a universal process amongst Gram-negative bacteria. In this work, we aim to better our understanding of OMV biogenesis and regulation Bacteroides thetaiotaomicron VPI-5482 (Bt). We developed a novel molecular toolkit to enable the visualization and quantification of OMVs in vitro. To identify genes that impact OMV biogenesis and regulation, we adapted these tools to enable the high-throughput screening of a transposon mutant library. Genes uncovered during our screen were found to regulate OMV production, alter cargo selection, and even preclude OMV biogenesis. Many of these genes exhibit unique characteristics and are only found in Bacteroidota. Our investigations center the role of a new family of dual membrane-spanning anti-sigma factors (Dma) that modulate OMV biogenesis and are important for in vivo fitness of Bt. These studies represent the first systematic analyses to identify and characterize genetic factors involved in OMV biogenesis in Bt. Our results greatly improve the field’s understanding of OMV biology and will guide future studies regarding their role and importance in these organisms.

Committee Chair

Mario Feldman

Committee Members

Andrew Kau; David Rosen; Janice Robertson; Juliane Bubeck-Wardenburg

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

12-12-2025

Language

English (en)

Author's ORCID

0000-0002-3576-7901

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