Date of Award

7-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Ocean Engineering and Marine Sciences

First Advisor

Andrew G. Palmer, Ph.D.

Second Advisor

Eric Guisbert, Ph.D.

Third Advisor

David Carroll, Ph.D.

Fourth Advisor

Brooke Wheeler, Ph.D.

Abstract

Group behaviors in microorganisms are often regulated by low-molecular weight molecules that enable them to sense population density, a signaling system known as quorum sensing (QS). QS in prokaryotes has provided insight into the role ‘social’ behaviors play in the bacterial world and refined our models of virulence, bacterial motility, and more. While less common, similar behaviors are observed in certain fungi, primarily yeasts, in which QS controls cell morphology, biofilm production, and other phenotypes. We have recently pursued the potential for QS to be more broadly distributed among unicellular eukaryotes, which could significantly alter our understanding of social behaviors in the microscopic world. Specifically, I have investigated the potential for QS in the globally distributed green algae, Chlamydomonas reinhardtii.

C. reinhardtii is a model for studying motility in unicellular organisms and is used in the biomanufacturing of biofuels and pharmaceuticals. Since motility is an integral response to changing environments, understanding the systems regulating motility is crucial. However, manually tracking a statistically significant number of cells is impractical. Therefore, we developed an automated method to observe C. reinhardtii motility, enabling us to identify individual cells and gather global data on their direction, speed, and size.

Our findings include a population-dependent increase in the swimming speed of C. reinhardtii. Through a series of experiments, we confirmed that this increase in swimming speed is due to cell density rather than age or nutrient availability. This phenomenon relies on the synthesis and detection of a low-molecular-weight compound, dubbed the Chlamydomonas swim speed factor (CSSF). Extracts containing CSSF increased swimming speeds in the closely related Chlamydomonas moewusii, and vice versa, indicating that this signaling system is conserved within the genus.

We next developed a refined procedure for preparing CSSF-containing extracts using solid-phase extraction (SPE), accelerating the discovery process for identification of this signal. By iteratively applying high-performance liquid chromatography (HPLC) to the extracts, coupled to image analysis with subsequent fractions, we identified extract fractions containing one or more CSSFs. Further analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) helped us identify terpenes/sesquiterpenes, fatty acids, sterols/phytohormones, and an as of yet unnamed molecule with a mass-to-charge ratio of ~171.12 as potential candidates for this signaling molecule.

The works collected herein establish new tools for evaluating and modeling motility in this model organism as well as a methodology for similar studies in other unicellular microorganisms. Understanding and controlling QS can have broad impacts on food production and safety, human health, biofuel production, waste management, and other important fields. Expanding our understanding of QS to include these unicellular eukaryotes offers valuable insights into the evolution and regulation of microbial "social" behaviors. Discovery of QS in C. reinhardtii and the closely related species, C. moewusii, impacts our understanding of microbial ecology and broadens the potential for discovery of QS among other unicellular eukaryotic species.

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