Bacteria-animal interactions play a widespread role in stimulating the life-cycle transitions of marine invertebrates, this interaction is critical for processes such as coral reef formation, sustaining fisheries stocks and biofouling. However, we know little about the mechanisms mediating this beneficial bacteria-animal communication. Pseudoalteromonas luteoviolacea (P. luteo) release an array of tailocins known as metamorphosis associated contractile structures (MACs) inducing tubeworm metamorphosis. It is currently unknown how these structures induce the cellular response associated with metamorphosis. We have discovered that MACs contain at least two cargo effector proteins, Pne1 and Mif1, which are present in the MAC structure. Pne1 is a nuclease toxin effector which is responsible for MACs ability to kill mouse macrophages and SF9 insect cell lines. Mif1 is an effector loaded within the MACs inner tube complex and is critical for the inductive phenotype of MACs observed in H. elegans larvae. Additionally, we have shown via purified recombinant protein, this cargo is sufficient to induce metamorphosis. Through biochemical analysis we have shown Mif1 shares functionality with a class of secreted effector lipases and exhibits lipase activity. Additionally, we have identified that Mif1 stimulates the upregulation of a lipid second messenger DAG and via pharmacological characterization of metamorphosis we have determined the conserved PKC pathway to be both necessary and sufficient to induce this metamorphosis. This has provided evidence for the bacteria stimulating the PKC signal transduction pathway via lipid second messengers to directly induce animal metamorphosis. These experiments are the first to show a bacterial protein is sufficient to induce the metamorphosis of any marine animal and yields insight into how bacterial stimulate animal development.

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.