Advisor(s)

Thomas Atkinson
Kevin Dybvig

Committee Member(s)

Jan Novak
Janet Yother
Li Xiao
Moon Nahm
Todd Green

Document Type

Dissertation

Date of Award

6-1-2026

Degree Name

Doctor of Philosophy (PhD)

School

Joint Health Sciences (Interdisciplinary)

Department

Microbiology

Abstract

Mycoplasmas are cell wall-less bacterial symbionts with remarkably streamlined genomes. Mycoplasmas offer two intriguing model organisms for studying concepts of minimal life. Mycoplasma genitalium has streamlined its genome to 580-kbp while maintaining virulence to suit its niche of parasitizing the human urogenital tract. The J. Craig Venter Institute has synthesized and transplanted a 543-kbp genome into a recipient cell, resulting in a non-pathogenic organism termed JCVI-syn3A. The conservation of a biological process in M. genitalium and JCVI-syn3A strongly implies an essential function in mycoplasmas, and potentially all life. Despite common thought of mycoplasmas being the simplest cells, much of their basic biology remains poorly described. Protein glycosylation is a ubiquitous biological phenomenon, including that in mycoplasmas. This work outlines our current understanding of mycoplasma glycosylation and differential localization of cytoplasmic housekeeping proteins such as enolase to the cell surface in bacteria, commonly referred to as protein moonlighting. We then describe the conservation of a motif-free glycosylation process previously described in the murine pathogen Mycoplasma pulmonis in M. genitalium and JCVI-syn3A. Finally, we describe in these minimal genome bacteria the detection of known moonlighting proteins, such as elongation factor thermo unstable, in ultracentrifuged membrane material. We discuss how moonlighting proteins are modified with sugar-phospholipids in JCVI-syn3A and its Mycoplasma mycoides parent organism, with nearby leucine, isoleucine, lysine, and/or arginine residues to the modification site. This work lays the foundation for future analyses of protein moonlighting in bacteria and the basic glycobiology of minimal genome mycoplasmas.

Keywords

Glycobiology;Glycoproteomics;Glycosylation;Mass spectrometry;Protein moonlighting

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