Date of Award

9-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Ocean Engineering and Marine Sciences

First Advisor

Andrew Palmer

Second Advisor

David Carroll

Third Advisor

Julia Grimwade

Fourth Advisor

Alan Leonard

Abstract

Microbial communities significantly impact the processes of the eukaryotic hosts they colonize. In many cases, symbiotic organisms both pathogenic and mutualistic coordinate their efforts through a process called quorum sensing (QS). QS is driven by low-molecular weight signals that regulate gene expression at threshold inducer concentrations, allowing phenotypic switching to be driven by cell density. These quorum sensing signals are subject to eukaryotic perception, and quorum associated phenotypes can directly influence the growth and development of eukaryotes. Eukaryotic perception of quorum sensing signals has been chiefly studied in plants, and to a lesser extent mammalian systems, yet no delineated model or system exists for the unicellular eukaryotes, with whom cohabitate often playing niche roles. Understanding if and how these eukaryotes detect and influence quorums is a crucial piece of the microbiome dialogue. The plant-like unicellular eukaryote, the model algae Chlamydomonas reinhardtii, which produces microbial quorum regulating compounds and co-exists with many QS bacteria in both soil and aquatic communities, was investigated as a model for quorum sensing. To examine if C. reinhardtii could sense and respond to quorum sensing, we established methods for microplate culture and phenotypic analysis. This system was used to measure the effects of quorum sensing compounds on growth and viability. Unlike plants, C. reinhardtii, is not stationary, such as plants, and exhibits cellular motility. Methods were therefore developed to also accurately measure C. reinhardtii motility, and to quantify the effects of quorum sensing signals on this process. Although the motility methods established were a significant improvement from preexisting manual methods, C. reinhardtii generally did not exhibit any phenotypic changes upon exposure to autoinducers. The most active autoinducer was that from Sinorhizobium meliloti, which reduced C. reinhardtii viability as scored through esterase activity and membrane integrity, yet did not notably alter growth. Experimental measures of cell viability in biological research typically examine individual variables, for example, esterase activity is used as a metabolic indicator. These variables are naturally modulated in living cells for many reasons, however, and do not necessarily reflect viability modulation, but potentially an adaptation to new conditions. It is therefore important to interpret viability measurements in the context of cell growth. Without a decrease in cell concentration, it was surmised that the quorum sensing autoinducers may be subject to degradation, which was confirmed through liquid chromatography mass spectrometry (LCMS), highlighting an exuded factor. However, minimal media growth studies determined that autoinducer supplements rescued reduced growth phenotypes. Furthermore, coculture with Sinorhizobium meliloti improved growth of both species under different initial culture densities. These studies indicate that C. reinhardtii is a strong negative regulator of quorum sensing autoinducers, that growth is stable after direct exposure to autoinducers, and under competitive conditions (nutrient limitations or coculture), growth can be improved through exposure to autoinducers or co-culture. These findings highlight similarities between plants in degradation and mimicry to remodel the quorum sensing landscape. As many eukaryotic quorum sensing interactions are not reproducible from autoinducer signals alone, the need to explore co-culture studies is evident. Unicellular eukaryotes, such as C. reinhardtii in this study, may play equal if not more significant roles in regulating microbial quorums than traditionally examined eukaryotes, such as plants.

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