While it is clear that thermodynamics plays a nontrivial role in biological processes, exactly how this affects the macroscopic structuring of living systems is not fully understood. Thus, the objective of this dissertation was to investigate how thermodynamic variables such as exergy, entropy, and information are involved in

biological processes such as cellular metabolism, ecological succession, and evolution. To this end, I have used a combination of mathematical modelling, in silico simulation, and both laboratory- and field-based experimentation.

To begin the dissertation, I review the basic tenets of biological thermodynamics and synthesize them with modern fluctuation theory, information theory, and finite time thermodynamics. In this review, I develop hypotheses concerning how entropy production rate changes across various time scales and exergy inputs. To begin testing these hypotheses I utilized a stochastic, agent-based, mathematical model of ecological evolution, The Tangled Nature Model. This model allows one to observe the dynamics of entropy production over time scales that would not be possible in real biological systems (i.e., 106¬ generations). The results of the model’s simulations demonstrate that the ecological communities generated by the model’s dynamics have increasing entropies, and that this leads to emergent order, organization, and complexity over time. To continue to examine the role of thermodynamics in biological processes I investigated the bioenergetics of marine microbes associated with benthic substrates on coral reefs. By utilizing both mesocosm and in situ experiments I have shown that these microbes change their power output, oxygen uptake, and community structure depending upon their available exergy.

Overall, the data presented herein demonstrates that ecological structuring and evolutionary change are, at least in part, determined by underlying thermodynamic mechanisms. Recognizing how physical processes affect biological dynamics allows for a more holistic understanding of biology at all scales from biochemistry, to ecological succession, and even long-term evolutionary change.

Polymicrobial infection defines a combination of microorganisms, including viruses, bacteria, fungi, and sometimes parasites, which are simultaneously associated with an infected site. While the idea of polymicrobial infections is common nowadays, most clinical laboratories still focus on the culturing and identification of a single pathogen. The biological implications attributed to these singular microbial infections are deceptive, especially when considering the multiple complex interactions associated with polymicrobial infections. This dissertation used Cystic Fibrosis (CF) as a study system for polymicrobial infections in the lung. CF is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. CF affects multiple organs across the body, but pulmonary infection remains the main cause of morbidity and mortality. Studying CF lungs is challenging. Here a set of comprehensive methods was developed to simultaneously study the viral, microbial, and host genetics using metagenomics and metatranscriptomics approaches. Many of the bacteria found in the lungs of CF patients are of oral origin. Detailed genetic analyses showed that these bacteria adapt to the CF lungs by acquiring essential genes while losing non-essential ones. One example is Rothia mucilaginosa – an organism that is present in more than 80% of San Diego patients. De novo assembly of a near-complete R. mucilaginosa genome showed potential physiological adaptations through the acquisition of multiple genes. A survey of more than 20 CF patients in San Diego revealed that every patient harbors a unique microbial community, suggesting that CF patients require personalized medicine for the treatment of their lung infections. Despite these personalized differences in microbial taxonomical profiles across CF patients, the functional potential across these different microbial communities is highly similar. The conserved functional potential in CF microbes allows them to carry out aerobic and anaerobic respirations, as well as fermentation, highlighting the importance of anaerobes in the CF lungs. CF microbes regulate their metabolic activities in response to perturbations. In vitro community culturing of CF microbes showed that the anaerobes were sensitive to antibiotics commonly used in CF patients and their metabolic activities could be associated with the patient’s health. The role and importance of CF anaerobes and their survival mechanisms are illustrated in this study. In summary, this dissertation provides novel insights into polymicrobial infections in CF lungs and demonstrates the potential and advantages of coupling omics and clinical approaches for the study of other complex polymicrobial infections.

This dissertation work contains the following chapters: Chapter 1 revealed that bacterial pathogens of marine animals contain a higher abundance of prophages in their genomes than non-disease-causing host-associated bacteria. The work highlights the importance of horizontal gene transfer between bacteria and phages in the context of marine fauna, and reviews what is known about prophages in the context of marine diseases. Additionally, a comparison of the prophage-encoding genes between pathogens and non-pathogens, found that predicted prophage-encoded genes in disease-causing bacteria are enriched for carbohydrate and nitrogen metabolism, virulence factors, and membrane transport. Chapter 2 consisted of utilizing multi-omics and microscopy to characterize coral-turf algal interactions in situ that led to a working model of the ecology of these benthic holobiont interactions. I applied metagenomic sequencing in parallel with metabolomics to uncover the underlying bacterial, viral, and biochemical processes associated with coral-turf algal competition and reef decline. This work emphasizes the important role of host-associated bacteria and viruses in the ecological outcome of competing coral-algal interactions. In this context, the study fits into what was coined the “Algal Feeding Hypothesis”, which posits that changes in coral-algal interface communities are driven by bacteria that feed on algal-derived compounds. Chapter 3 focused on the molecular and microbial cartography of massive coral colonies using a multi-omics approach to understand the natural history of endangered massive coral colonies competing with benthic algae. In this work I combined 3D photography with metatranscriptomics, metabolomics, and metabarcoding to investigate all portions of the massive coral Orbicella faveolata visually and spatially in situ.

Viruses and microbes are foundational to the healthy functioning of coral reef ecosystems. However, dysregulation among viral and microbial communities owing to global and local pressures is driving widespread coral reef collapse. The objective of this dissertation was to investigate the drivers of this ecosystem degradation process, termed microbialization, on coral reefs and to identify mechanisms associated with ecosystem recovery from microbialized states.

To begin the dissertation, I review the biochemistry underlying coral reef function and describe how ecosystem energy is differentially stored in predominantly the macroorganisms on healthy coral reefs versus the microbes on degraded coral reefs as a result of positive feedback loops. In this review, I develop hypotheses concerning how microbes, organic matter, and oxygen interact to drive shifts to microbialized states and propose a tool to manipulate reef biochemistry to counter microbialization. To begin testing these hypotheses, I build midwater mesocosm tools called Coral Arks, develop protocols for designing, deploying, and monitoring them in situ, and demonstrate their structural integrity in the marine environment. Next, I use Coral Arks in a long-term field experiment, demonstrating that microbial ecology, water quality, and biogeochemistry are improved on Arks relative to seafloor sites at the same depth. Lower microbialization on the Arks enhanced survival and growth of transplanted corals and recruited diverse assemblages of reef organisms, including fish and benthic invertebrates. These findings highlight the role of microbes in shaping the coral reef abiotic environment and suggest microbialization can be countered by reducing organic matter inputs, enhancing dissolved oxygen, and reinstating viral predatory control over microbes. Lastly, I discuss the future of coral reef restoration, identify explicit goals for reinstating reef ecosystem services, and through a literature review, summarize potential interventions for manipulating coral reefs. Using this information, I propose a restoration framework based in control theory that uses Coral Arks as experimental platforms for identifying effective restoration interventions and scaling these interventions to a natural reef.

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations to the Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) gene, that leads to the buildup of thickened mucus in the lungs that are chronically infected by bacteria (often antibiotic resistant) and fungi. These chronic infections periodically flare in acute events called pulmonary exacerbations which leads to scarring of the lung and permanent decline in pulmonary function. This work presents a model of CF lung disease that incorporates the taxonomic diversity observed in the CF lung into ecological guilds based on their role in the CF sputum environment, resulting in the proposal of four functional groups called the Brewers (fermenting microbes), Drunkers (biofilm building consumers of fermentation products), Putrifiers (anaerobic biofilm dwelling bacteria) and Nihilists (lone wolf pathogens of the lung). Another model of the CF lung microbiome, found in this dissertation, predicts the succession of bacteria in CF sputum based on the concentration of succinate found in the sputum environment, which is known to be substantially higher than sputum from people without CF. The second model is based on the differential succinate flux values predicted by GEnome scale metabolic Models (GEMs) for the 29 most prevalent bacterial species in the CF lung, and observations of in vitro growth data of a mock community of four CF isolated strains of bacteria. The results of which support the existence of two distinct consortia of bacteria in the CF lung, consortium 1 made up of typical CF pathogens which can metabolize succinate and consortium 2 which contains bacteria associated with the human oral cavity that do not metabolize succinate and are likely inhibited by succinate at high concentrations. Lastly, with the aim to address the issue of treating antibiotic resistant bacterial infections in patients with CF or otherwise, this dissertation presents two engineered tailocins MobyWanKenobi and John Henry, that bind to and kill CF isolated strains of Stenotrophomonas maltophilia and both Staphylococcus aureus and Serratia odorifera respectively.

This work presents evidence that temperate and virulent viruses impact the ecology, evolution, and health state of their bacterial and eukaryotic hosts across ecosystems including the ocean, human gut, and urban environment.

Temperate viruses can switch between a lysogenic and a lytic lifestyle. In the lytic cycle, phage lyse the host cell shortly after infection to release the progeny into the environment. In the lysogenic cycle, phage integrate into the bacterial genome to replicate with it during cell division. Here, it is shown how bacteriophage, viruses that infect bacteria, are key drivers of evolution in the marine environment and presents ongoing work on a mechanism that regulates prophage lysogenic-to-lytic the switch in the human gut.

Part of this research was conducted amidst a global pandemic caused by a virulent virus; this inspired the assembly of a workflow that combines community building with molecular biology to identify environmental reservoirs of SARS-CoV-2 and other eukaryotic RNA viruses. Using these methods, SARS-CoV-2 RNA was detected on surfaces of the urban environment and the risk of infection by contact with contaminated fomites in San Diego County was predicted. The viral RNA community present on these surfaces was also characterized.

Lastly, with the aim to uncover novel virus-derived systems where bacteria interact with their eukaryotic host, this thesis presents the identification of a new phage tail-like contractile injection system in Bacteroides, that was found prevalent in the gut of healthy adults.