Genetic tools to explore function in marine symbiotic bacteria
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Genetic tools to explore function in marine symbiotic bacteria

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My PhD research explores which bacterial products can trigger marine invertebrate metamorphosis, an irreversible transformation from free-swimming larvae to settled juvenile on the seafloor. We reviewed the existing literature for bacteria-stimulated metamorphosis in marine invertebrates (Chapter 1) to provide context for the research sections (Chapters 2-4). Taken together, this dissertation showcases the utility of using bacterial genetics to explore the molecular mechanisms underlying bacteria-induced metamorphosis and other symbiotic interactions.In Chapter 2, we work with a tractable marine bacteria, Pseudoalteromonas luteoviolacea and show that it encodes the gene cluster for two different products capable of stimulating metamorphosis in different animals. We generated deletion mutations for one or both of the products and then tested their effects on two marine invertebrate models, tubeworms and Hydractinia. We find that the different metamorphosis-inducing products are highly specific, and do not influence metamorphosis universally across different animals. In Chapter 3, we further explore one of the metamorphosis-inducing products from bacteria, a chemical compound called Tetrabromopyrrole (TBP). While Pseudoalteromonas luteoviolacea produces some TBP, other strains such as Pseudoalteromonas sp. PS5 produce more TBP and influence robust coral metamorphosis. To explore the genetic link between TBP production and coral metamorphosis, we established PS5 as a genetically tractable strain. We generated an in-frame deletion of a single gene, bmp2, and showed that the mutant strain could no longer 1) produce TBP and 2) induce metamorphosis. These results suggest that biofilms of PS5 produce enough TBP to influence coral metamorphosis in vivo and can be used to inform decisions for coral probiotics. In Chapter 4, we build on modular plasmid toolkit platform to enable higher throughput genetic manipulation in diverse marine bacteria. We develop methods for genetically engineering marine bacteria using preexisting tools and parts, while adding new parts (i.e. promoters) to explore function in marine bacteria. We successfully transformed 10 strains across 2 proteobacterial classes, 4 orders and 7 genera. We developed a new method to visualize invertebrate microbiomes after the induction of metamorphosis, revealing that tubeworms ingest inductive bacteria after they undergo metamorphosis. These outcomes enable the exploration of fundamental questions surrounding marine host-microbe interactions.

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This item is under embargo until June 17, 2024.