The microbiota is an important contributor to host health and fitness, impacting all aspects of life, from development and metabolism to immunity and behavior. Substantial work has been done to characterize the factors that shape the microbiota, and have demonstrated the importance of both environmental and host factors. However, much of the multifaceted relationship between the environment, the host, and the microbiota remains to be elucidated. My dissertation attempts to demonstrate the ways in which hosts can shape their microbiota through a taxonomic and functional evaluation of microbiotas within two experimental systems.

In chapter 1, I examine the relative contributions of the host and the environment to microbiota composition. Substantial work has been done to characterize the factors that shape the gut microbiota. Studies of the human gut microbiota have shown effects of geographical location, diet, and host genetics. However, the relative contribution of such factors in shaping the gut microbiota remains unclear, probably due to the large inter- individual variation. The nematode Caenorhabditis elegans offers a convenient model to characterize the contributions of the environment and the host to microbiota composition. While C. elegans is a well-characterized model organism, surprisingly little is known about its natural history, especially its interactions with microbes. To characterize the gut microbiota of C. elegans, I used 16S rDNA-targeted sequencing to measure the gut bacteria of worms grown in natural-like environments. By taking advantage of the availability of genetically-homogenous worm populations to reduce noise and average out inter-individual variation, and thus better discern shared features of the C. elegans microbiota, I demonstrate that the worm gut microbiota assembly is a deterministic process. My results suggest a dominant contribution of the host to microbiota composition, and further suggest a role for negative interactions between microbiota members.

In chapter 2, I further examined the contribution of host genetics to microbiota composition by identifying differences in microbiotas assembled in worms of different genotypes spanning 200-300 million years of nematode evolution. Using 16S rDNA- targeted sequencing, I demonstrate a significant contribution of host genetics to microbiota composition. However, experimental variables affected worm microbiota composition more than the worm genotype, and hindered my identification of host-specific taxa. In an attempt to overcome this, I isolated members of the Enterobacteriaceae core microbiota family from C. elegans and C. briggsae. This, however, did not identify phylogenetic distinctions between commensals of the two species. The functional evaluation of these gut isolates did reveal host-adaptation in the form of host-specific contributions to development, infection resistance, and lifespan. My results support the role of host genetics in shaping microbiota composition, and suggest that the extent of this contribution may surpass what could be deduced based on the commonly available phylogenetic resolution.

In chapter 3, I surveyed the roles of specific host factors in shaping the C. elegans gut microbiota by examining the gut microbiota of C. elegans mutants deficient in feeding and immune function. When selecting mutants, I considered selection during feeding and selection within the intestine as two general ways a host could shape its microbiota. Altered feeding could affect colonization by changes in food choice, food uptake, and food grinding. Immune responses can significantly affect the colonization of pathogens, and it is likely that these immunity and stress response pathways could affect the colonization of non-pathogenic bacteria. From my previous work, I amassed a collection of C. elegans gut isolates with representatives from almost all core gut microbiota families. Because my previous work did show that the environment does influence the microbiota composition to a degree, I created a synthetic community as a food source to control for environmental variation. My results suggest that changes within the intestinal environment can affect microbiota composition, and demonstrate a role of the TGF-β/DBL-1 pathway in shaping the gut colonization of Enterobacter. This specificity suggests that hosts can shape their microbiota through specific host-microbe interactions.

In chapter 4, I utilized the tomato plant and P. syringae system to examine the ability of the phyllosphere microbiome to confer resistance to P. syringae infection of tomato leaves. In plants, the phyllosphere (above ground) microbiome is likely to be important for resistance to pathogens that infect the aerial portions of plants; however, relative to the below-ground microbiome, these communities are understudied and we do not yet know how the protective effects of this microbiome might vary as a function of bacterial density or resource availability. My results suggest that the presence of a phyllosphere microbiome does decrease pathogen colonization success, as plants sprayed with a microbiome had significantly less pathogen growth than plants sprayed with sterile buffer. I additionally show that the dose of microbiome spray has an effect on protection, and that this effect is dependent on the composition of the microbial community, but not microbial diversity. Finally, my results suggest that microbe-microbe competition (resource community dynamics) may play an important role in protection, as the addition of fertilizer abolishes the observed microbiome-mediated protection. Together, my results may alter our understanding of microbiome-mediated protection within agricultural settings and the use of plant probiotics.

Animals have evolved sophisticated mechanisms to perceive stress or even anticipate it before it has caused damage. Ideally, stress responses are controlled to ensure that they are specific and not excessive lest they cause collateral damage to the host. Here I describe my work in Caenorhabditis elegans examining the initiation and consequences of stress responses. In chapters 2 and 3, I examine the mechanisms of initiation of C.elegans immune responses to Pseudomonas aeruginosa, focusing on pathogen recognition. In chapters 4 and 5 I describe a project that was initiated within the realm of host-pathogen interactions, but evolved to focus on age as a context determining the outcome of both biotic and abiotic stress responses.

Despite extensive knowledge of conserved signaling pathways in the C.elegans immune response, a question that has remained elusive is whether C.elegans can directly recognize pathogens, or if alternatively, they detect cellular damage caused by infection. I have shown that the immune response to P. aeruginosa can be dissociated from colonization and that potential damage from secreted molecules cannot induce a marker of the early immune response; in contrast, a non-pathogenic Pseudomonad can. This suggests that the response can be initiated at least in part by structural features of the bacteria. Furthermore, I have identified a family of genes in C.elegans encoding LysM domain proteins which are known to participate in recognition of microbial envelope components. Genetic and functional analyses suggest a potential role for family members in pathogen recognition in C. elegans.

In the second half of this dissertation, I show that while the C.elegans JNK homolog KGB-1 is stress-protective during development, its activation in adults compromises stress resistance as well as general lifespan. I go on to show that this phenomenon is mediated in part by KGB-1's age-dependent antagonistic modulation of the conserved FOXO transcription factor, DAF-16. Genome-wide analysis of the KGB-1 transcriptome revealed that KGB-1 regulates several genes in an age-specific manner, and pointed to another conserved transcription factor, FOS-1 as a mediator of KGB-1's effects. Interestingly, the phenomenon of the same protein having opposite effects dependent on age is reminiscent of the Antagonistic Pleiotropy theory for the evolution of aging. Our results shed light on molecular mechanisms underlying this long-standing theory.

Immunity and aging are complex processes under the control of different genetic pathways. These pathways must be coordinated in order to respond to various inputs, but how this is achieved is not fully understood. In this dissertation, I demonstrate two examples of proteins that integrate different genetic pathways into appropriate gene expression responses in Caenorhabditis elegans.

In Chapter 1, I examined the relationship between a conserved innate immune signaling pathway and a GATA transcription factor. GATA transcription factors play roles in development and tissue differentiation, but less is known about their roles in mature tissues. In C. elegans larvae, the GATA transcription factor ELT-2 regulates terminal differentiation of the intestine, but it is also expressed in the adult intestine, where it was suggested to maintain intestinal structure and function, and where it was also shown to contribute to infection resistance. To study the function of elt-2 in adults, we characterized elt-2-dependent gene expression following its knock-down specifically in adults. Microarray analysis identified two ELT-2-regulated gene subsets: one, enriched for hydrolytic enzymes, pointed at regulation of constitutive digestive functions as a dominant role of adult elt-2; the second was enriched for immune genes that are induced in response to Pseudomonas aeruginosa infection. Focusing on the latter, we used genetic analyses coupled to survival assays and quantitative RT-PCR to interrogate the mechanism(s) through which elt-2 contributes to immunity. We show that elt-2 controls p38-dependent gene induction, cooperating with two p38-activated transcription factors, ATF-7 and SKN-1. This demonstrates a mechanism through which the constitutively nuclear elt-2 can impact induced responses, and play a dominant role in C. elegans immunity.

In Chapters 2 and 3, I describe work characterizing the role of a transthyretin-like gene in influencing lifespan. The transthyretin-like (ttr) gene family of C. elegans consists of 59 genes that are related to vertebrate transthyretin, a protein that carries lipophilic molecules such as retinol and thyroid hormone through the blood and cerebrospinal fluid. Although levels of various ttrs increase during stress stimuli and aging, the functions of ttrs remain largely uncharacterized. We found that RNAi against one ttr, ttr-1, increased levels of the FOXO transcription factor DAF-16 and extended lifespan. In agreement, ttr-1 overexpression reduced lifespan. A ttr-1::gfp fusion protein was localized to the periphery of the spermatheca, the vulva, and the intestine. Analysis of genetic interactions between ttr-1 mutants and known regulators of lifespan revealed that ttr-1 disruption compromised lifespan extension following daf-2 knock-down (although DAF-16 nuclear localization was similar to that in wildtype animals), and abolished lifespan extension in eat-2 mutants, which exhibit slow pharyngeal pumping and represent a genetic model for dietary restriction. These results, in conjunction with its expression in the intestine and somatic gonad suggest that ttr-1 may play a role in orchestrating organismal responses to nutrient availability.