The ecology and evolution of the extracellular vesicles of halophilic Archaea

Abstract

Extracellular vesicles (EV) are small, spherical structures that bud from the cell envelope. These particles are produced by cells across all three domains of life, marking them as a ubiquitous feature of cell biology. EVs transport proteins, lipids, nucleic acids, and other bioactive compounds in membrane-enclosed packages, which allows them to mediate a multitude of different functions. While much has been uncovered for bacterial and eukaryotic EVs, little is known about the functions and production of EVs from the archaeal domain. The work presented in this dissertation aims to explore the mechanisms of production, biochemical composition, and function of EVs produced by halophilic Archaea. In Chapter II, I present a standardized method for the isolation and purification of EVs from archaeal cultures. This method details the isolation of EVs from liquid cultures, the purification from other extracellular contaminants, and protocols related to the subsequent downstream analysis. By providing a standardized protocol, I aim to promote additional research into archaeal EV production that is comparable to each other. Using these methods in Chapter III, I characterize the biochemical composition of EVs from Haloferax volcanii and other halophilic Archaea. I uncover that halophilic Archaea produce EVs enriched with small-sized RNA with regulatory potential, which has not been demonstrated for archaeal EVs previously. Proteomic analysis reveals a conserved small GTPase that is crucial to EV production, which we have named archaeal vesiculating GTPase (ArcV). Structurally and functionally, ArcV bears similarities to the Arf-family of GTPases, which are responsible for regulating vesicle formation in the eukaryotic endomembrane system. We propose that this provides evidence for previously established hypotheses regarding the archaeal origins for the eukaryotic endomembrane system, and that small GTPase-dependent vesicle formation could have emerged earlier than previously considered. In Chapter IV, I further characterize EV-associated RNA from Halorubrum lacusprofundi, with and without virus infection. I observe that the RNA population exported in EVs changes upon viral stress, leading to the enrichment of specific RNAs. I also explore the effects on the cell transcriptome after incubation with EVs, suggesting that EVs are able to affect gene expression in the receiving cell. The characterization of archaeal EVs has implications on both microbial community dynamics and prokaryotic evolutionary history. The ability for EVs to induce changes in gene expression in the receiving cell suggests a regulatory mechanism that acts on a population-wide scale. Additionally, the presence of a small GTPase-dependent mechanism for EV production in Archaea requires us to reevaluate our current understanding of prokaryotic membrane remodeling mechanisms and the evolutionary history of eukaryotic-like features.