Microorganisms exist ubiquitously on Earth, yet their functions and ecological roles remain elusive. Investigating these microbes is accomplished by using culture-dependent and culture-independent methodologies. This study employs both methodologies to characterize: 1) the genomic potential of the novel deep-subsurface bacterial isolate Thermanaerosceptrum fracticalcis strain DRI-13T by combining next-generation and nanopore sequencing technologies and 2) the microbiome of the artificial marine environment for the Hawaiian Bobtail Squid in aquaculture using next-generation sequencing of 16S rRNA gene. Microbial ecology of the deep-subsurface remains understudied in terms of microbial diversity and function. The genomic information of DRI-13T revealed a potential for syntrophic relationships, diverse metabolic potential including prophages/antiviral defenses, and novel methylation motifs. Artificial marine environments housing marine the Hawaiian Bobtail Squid (Euprymna scolopes) contain microorganisms that can directly influence animal and aquaculture health. No studies presently show if bacterial communities of the tank environment correlate with the health and productivity of E. scolopes. This study sought to address this by sampling from a year of unproductive aquaculture yield and comparing the bacterial communities from productive cohorts. Bacterial communities from unproductive samples show less bacterial diversity and abundance coupled with shifts in bacterial composition. Nitrate and pH levels between the tanks were found to be strong influences on determining the bacterial populations of productive and unproductive cohorts.

Ecosystems comprise large groups of highly interdependent organisms. Cnidarians, such as sea anemones and corals, are keystone species in many marine ecosystems, especially coral reefs. Each individual cnidarian also constitutes an ecosystem unto itself, a "holo- biont", consisting of the host animal and accompanying microbial symbionts. To interro- gate cnidarian holobionts, I used computational approaches to analyze the transcriptomes of three cnidarians and build mechanistic models of their microbial symbionts. In par- ticular, I analyzed and annotated the transcriptomes of the cauliflower coral Pocillopora damicornis, the lined sea anemone Edwardsiella lineata, and the starlet sea anemone Ne- matostella vectensis, providing information about the molecular functions expressed by these organisms, and allowing development of a corresponding set of public databases: PocilloporaBase, EdBase, and an updated version of StellaBase, that facilitate access to the corresponding datasets. Additionally, I developed a method to infer the phylogenetic antiquity of transcripts. This method also allowed me to identify transcripts from other organisms (e.g., microbes) belonging to the anemone or coral holobiont. In parallel – in order better to understand the microbial symbionts that share envi- ronments with cnidarian hosts, I also developed new computer-simulation approaches for modeling metabolic interactions between different microbial species. These approaches are based on genome-scale stoichiometric reconstructions of metabolic networks and on Flux Balance Analysis (FBA). In addition to contributing to the development and testing of a new FBA-based platform for modeling communities in structured environments (Compu- tation Of Microbial Ecosystems in Time and Space, or COMETS), I used this platform for specific in silico experiments on microbial symbiosis. In particular, I computed all pairwise interactions between 582 different prokaryotic models, and identified global patterns of pu- tative positive (cross-feeding) vs. negative (food competition) interactions in this matrix of species pairs. I found that about 7% of the pairs yielded a greater biomass when grown together than when grown separately as monocultures. Despite existing challenges, such as the limitations of gap-filling steps in model construction and the need for a better knowl- edge of nutrient composition in natural environments, this approach could in the future help forecast shifts in the coral holobiont under likely scenarios of marine environmen- tal changes. In general, this work demonstrates how the integration of high-throughput sequencing technology and mechanistic systems-biology simulations, can provide unique tools to analyze interactions between microbes, and to mitigate or reverse adverse changes in marine ecosystems.

Food-borne infections are a major source of mortality and morbidity. Salmonella causes the highest number of Food-borne bacterial infections in the US. This work contributes towards developing strategies to control Salmonella by (a) defining receptors used by Salmonella to adhere to and invade the host epithelium; (b) developing a host receptor based rapid detection method for the pathogen in food matrix; (C) and defining mechanisms of how probiotics can help alleviate Salmonella-induced cell death in the host epithelium. We developed a cell-cell crosslinking method to discover host-microbe receptors, and discovered three new receptor-ligand interactions. Interaction of Salmonella Ef-Tu with Hsp90 from epithelial cells mediated adhesion, while interaction of Salmonella Ef-Tu with two host proteins that negatively regulate membrane ruffling (myosin phosphatase and alpha catenin) mediated adhesion and invasion. We also showed the role of host ganglioside GM1 in mediating invasion of epithelial cells by Salmonella. Further we exploited pathogen affinity for immobilized gangliosides to concentrate them out of solution and from complex food matrices for detection by qPCR. A sensitivity of 4 CFU/ml (3 hours) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. Next we screened several probiotic strains for pathogen exclusion potential and found that Bifidobacterium longum subspp. infantis showed the highest potential for Salmonella enterica subspp. enterica ser. Typhimurium exclusion in a caco-2 cell culture model. B. infantis shared its binding specificity to ganglioside GM1 with S. ser. Typhimurium. Further, B. infantis completely inhibited Salmonella-induced caspase 8 and caspase 9 activity in intestinal epithelial cells. B. infantis also reduced the basal caspase 9 and caspase 3/7 activity in epithelial cells in absence of the pathogen. Western blots and gene expression profiling of epithelial cells revealed that the decreased caspase activation was concomitant with increased phosphorylation of pro-survival protein kinase Akt, increased expression of caspase inhibiting protein cIAP, and decreased expression of genes involved in mitochondrion organization, biogenesis and reactive oxygen species metabolic processes. Hence, B. infantis exerted its protective effects by repression of mitochondrial cell death pathway which was induced in the presence of S. ser. Typhimurium.

The composition and metabolic activity of the microbiome affect many aspects of health, and there is current interest in dietary constituents that may affect this system. The purpose of this study was to evaluate the effects of a mix of probiotics, a mix of prebiotics and a bioactive protein fraction on the microbiome, when fed to mice individually and in combination. Mice were fed the total western diet (TWD) supplemented with prebiotics, probiotics, and Tri-Factor (bioactive proteins) individually and in combination for four weeks. Subsequently, effects on the composition of gut microbiome, gut short chain fatty acids (SCFAs) concentration, gut inflammation and integrity of the mucosal barrier were analyzed. Ruminococcus gnavus was increased in mice gut microbiome after feeding prebiotics. Bifidobacterium longum was increased after feeding probiotics. Probiotic was associated with higher level of Clostridium neonatale. The treatments affected beta-diversity with exception of Tri-Factor, but not alpha diversity of microbiome. All treatments were associated with lower plasma zonulin, compared to the control group, indicating an effect on gut permeability. There were no treatment effects on cecal or fecal SCFAs, and the treatments did not affect gut inflammation as measured by fecal calprotectin.

Anglerfishes are easily one of the most popular deep-sea creatures due to their menacing appearance, extreme sexual dimorphism, parasitic mating approach, and eye catching bioluminescent lure. Unlike most bioluminescent fishes, which intrinsically generate light, female anglerfishes belonging to nine of the 11 families within the suborder Ceratioidei (deep-sea anglerfishes) have developed a symbiotic relationship with bioluminescent bacteria that are housed within the light organs. Previous molecular work had identified symbionts from two anglerfish species as novel and possibly unculturable taxa (Haygood et al., 1992), but nothing more has been revealed about the bioluminecent symbionts of ceratioids. As part of the Gulf of Mexico Research Initiative-funded DEEPEND project (Deependconsortium.org), the objective of this study is to characterize the escal microbiome of deep-sea anglerfishes and identify potential-symbiont taxa. A total of 36 anglerfish specimens were collected on DEEPEND cruises DP01 through DP04. These specimens consist of adult and larval individuals belonging to six of the families with the suborder Ceratioidei: Ceratiidae (n=22), Oneirodidae (n=7), Linophrynidae (n=3), Melanocetidae (n=2), Centrophrynidae (n=1), Melanocetidae (n=2), Gigantactinidae (n=1). DNA was extracted from esca, skin, fin, gill, gut, and caruncle tissues, as well as seawater. High-throughput sequencing of the 16S rRNA hypervariable V4 region was carried out using the Illumina MiSeq. Sequencing revealed five potential bioluminescent-symbiont taxa (OTU IDs: 9129, 9131, 160210, 523223, and 939811), which had the greatest relative abundance (25.2% - 98.7%) within 12 of 21 adult specimens. These taxa belong to the family Vibrionaceae and were found at greater than 10% relative abundance in the escal samples of adult anglerfishes belonging to the Ceratiidae and Melanocetidae families, but they were not found in high abundance in larval individuals of the same families. Sequencing of larval samples revealed five potential bioluminescent-symbiont taxa (OTU IDs: 136178, 176420, 523223, 837366, 939811) which were of greatest relative abundance (8.1%-67.1%) within nine of 13 specimens. Also members of the family Vibrionaceae, these taxa were found in high abundance in larval anglerfishes belonging to the Oneirodidae, Linophrynidae, Gigantactinidae, and Ceratiidae families. This study is the first to to examine the bioluminescent symbionts from seven different ceratioid families.

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Natural microbiota are essential to the health of living systems - from the human gut to coral reefs. Although advances in DNA sequencing have allowed us to catalogue many of the different organisms that make up these microbial communities, significant challenges remain in understanding the complex networks of interspecies metabolic interactions they exhibit. These interactions are crucial to community stability and function, and are highly context-dependent: the availability of different nutrients can determine whether a set of microbes will interact cooperatively or competitively, which can drastically change a community’s structure. Disentangling the environmental factors that determine these behaviors will not only fundamentally enhance our knowledge of their ecological properties, but will also bring us closer to the rational engineering of synthetic microbiomes with novel functions. Here, I integrate modeling and experimental approaches to quantify the dependence of microbial communities on environmental composition. I then show how this relationship can be leveraged to facilitate the design of synthetic consortia. The first chapter of this dissertation is a review article that introduces a framework for cataloguing interaction mechanisms, which enables quantitative comparisons and predictive models of these complex phenomena. The second chapter is a computational study that explores one such attribute – metabolic cost – in high detail. It demonstrates how a large variety of molecules can be secreted without imposing a fitness cost on microbial organisms, allowing for the emergence of beneficial interspecies interactions. The third chapter is an experimental study that determines how the number of unique environmental nutrients affects microbial community growth and taxonomic diversity. The integration of stoichiometric and consumer resource models enabled the discovery of basic ecological principles that govern this environment-phenotype relationship. The fourth chapter applies these principles to the design of engineered communities via a search algorithm that identifies environmental compositions that yield specific ecosystem properties. This dissertation then concludes with extensions of the modeling methods used throughout this work to additional model systems. Future work could further quantify how microbial community phenotypes depend on each of the individual factors explored in this thesis, while also leveraging emerging knowledge on interaction mechanisms to design synthetic consortia.