Browsing by Subject "Symbiosis"

Symbioses have shaped our modern world, providing for the air we breathe; for the plant and animal diversity we celebrate; and for the functioning of ecosystems from the tops of mountains to the ocean floor. Here I study symbiosis using fungal bacterial interactions as a model for understanding symbiotic dynamics. In this dissertation I present interpretations of experimental data about fungal bacterial interactions that lend insight into dynamics of symbiotic establishment and consequences of long-term endosymbiosis. More specifically, I examine the interactions of a plant-associated zygomycete, Mortierella elongata, and its interactions with several Betaproteobacteria in the Burkholderiales. I used genome sequencing, comparative genomics, physiological assays, and time-lapse microfluidic videography to ask the following questions; How are bacterial fungal symbioses initiated? How do bacteria and fungi communicate? What resources do these microbes share? Are long-term symbioses essential for one or both partners? What are the impacts of removing long-term endosymbionts for fungal host physiology? What are the effects long-term fungal endosymbiosis on bacterial genome content?

In chapter 1 I present lessons learned from genome sequencing of fungus Mortierella elongata and its primary resident endosymbiont, Mycoavidus cysteinexigens. I tested the hypothesis that genome reduction is a commonality of eukaryotic endosymbionts, and that characteristic genes and pathways are impacted by gene loss and inactivation in endosymbionts. I found that compared to its free-living relatives, M. cysteinexigens has a highly reduced genome and has lost genes coding for the biosynthesis of amino acids and intermediates of glycolysis, among other metabolic pathways. I describe a method for clearing fungi of endosymbionts using antibiotics. I report comparative physiological data for the cleared and uncleared strains and draw conclusions about the nature of their interactions based on the behavior of the fungal host lacking the endosymbiont. I tested the hypothesis that sharing of fungal fatty acids underpins this symbiosis, as suggested by the genome sequences of both microbes. I found that when cleared of endosymbionts, M. elongata grows more rapidly and accumulates fatty acids that are likely used by M. cysteinexigens when present.

In chapter 2 I investigate the transcriptional control of fungal-endosymbiont phenotypes. I continued working with the cleared and uncleared strains developed in chapter 1 and quantified transcript abundance in each isolate. I assigned functions to differentially expressed genes by identifying homologues in the fungal genetic model organism Saccharomyces cerevisiae. I layered on transcriptional data to the patterns that emerged from comparative analyses in chapter 1to better understand fungal response to endosymbiosis. I showed that differential expression of conserved genes underpin the increases in growth and altered metabolism in M. elongata when cleared of M. cysteinexigens. I found that endosymbiont presence is associated with toggling of metabolic programs that result in resources more or less bioavailable to M. cysteinexigens based on metabolic capability predicted by genome annotation. I found that genes with homologues in mating pheromone perception pathways are differentially regulated in cleared isolates of M. elongata, and that this aspect of clearing is shared by other isolates of M. elongata when cleared of their bacterial endosymbionts.

In chapter 3 I examine dynamics of pre-symbiotic signaling events between fungi and bacteria using Mortierella elongata and a free-living bacterium, Burkholderia BT03. Using microbial growth assays and a suite of conditioned medias I showed that growth stimulation is mutual for fungi and bacteria, and that signaling leading up to symbiotic phenotypes involves multiple bi-directional signal exchanges. I designed and used a microfluidic platform along with plate based and liquid culture systems to compare fungal growth rates in response to conditioned medias. By extrapolating rates from microbial growth assays including M. elongata, Burkholderia BT03 and related microbes, I inferred directionality, order, conditionality, specificity, and nature of signal exchange leading to microbial growth stimulation in this system.

As a whole this thesis explores how comparative microbial genomics and phenotypic assays can provide mechanistic insight into symbiotic establishment and the effects of long-term symbioses. The results presented here provide novel insights into biotic and abiotic factors dictating symbiotic establishment. Second, they suggest long-term endosymbionts of eukaryotic cells experience convergent gene loss. Lastly they emphasize that long-term endosymbionts strongly impact host metabolism, and that host-microbe metabolic intertwining is a commonality of many symbioses. The use of a systems biology approach to generate comparative genomics data on multiple levels that enable insight into the consequences of fungal bacterial symbioses is a novel contribution for the field.

Disease is an important driver of coral loss regionally and is projected to become more severe as temperatures increase around the world. Although there has been substantial research into the abiotic factors (e.g. temperature, nutrients) controlling coral diseases, we know significantly less about the biotic factors (i.e. species interactions) influencing disease dynamics. We examined how the species living on and within corals affect coral tissue loss from a white syndrome-like condition on Heron Island in the southern section of the Great Barrier Reef. We exposed Acropora aspera fragments in flow-through tanks to a fully crossed factorial experiment with three factors: the presence of a common symbiotic crab (Cyclodius ungulatus), contact with a common macroalgal complex, and simulated wounding mimicking fish predation. We found that crab presence increased coral survival from a white syndrome-type disease by over 25%, likely by removing macroalgae if present and by cleaning infected tissue. Conversely, contact with macroalgae dramatically increased coral mortality, with the chance of survival dropping to nearly 0 by the end of 25 days for corals that were in contact with algae. Wounding had no direct effect on coral health, but wounded corals with crabs did significantly better than corals with no wounding and crabs, which may be the result of coral-crab signaling. We suggest that A. aspera may produce nutrient-rich mucus when wounded, which attracts crab symbionts that help slow disease progression. These results suggest that incorporating biotic interactions into restoration designs may dramatically improve restoration outcomes and that adding beneficial symbionts may improve disease resilience at a local level.

Animal bacterial symbioses are pervasive and underlie the success of many groups. Here, I study ecological and evolutionary factors that shape interactions between a host and gut associates. In this dissertation, I interrogate interactions between the carpenter ant (Camponotus) and its associated gut microbiota to ask the following questions: What are the resident microbiota of the Camponotine gastrointestinal tract? How does persistent gut association affect rates of molecular evolution in gut symbionts? How are gut microbiota transmitted between social hosts? How does gut community composition and structure vary across host development? What evolutionary factors facilitate adaptation to the gut? How do the genomes of gut associates respond to selective pressures associated with persistent gut habitation? I use a combination of next generation sequencing, anaerobic isolate culturing, computational modeling, and comparative genomics to illustrate evolutionary consequences of persistent host association on the genomes of gut associates. In chapter one, I characterize the gut community of C. chromaiodes and describe two novel lineages in the Acetobacteraceae (AAB). I demonstrate rapid evolutionary rates, deleterious evolution at 16S rRNA, and deep divergence of a monophyletic clade of ant associated AAB. In chapter two, I design a novel molecular tool to prevent amplification of nontarget DNA in 16S based community screens. I then use this tool to characterize the gut microbiota of C. chromaiodes across several developmental stages and incipient colonies. I argue that highly similar bacterial profiles between a colony queen and offspring are indicative of reliable vertical transmission of gut bacteria. In chapter three, I isolate and culture two strains of AAB gut associates from C. chromaiodes, as well as an associate in the Lactobacillaceae, and perform whole genome sequencing. I use comparative genomic analyses to delineate patterns of genomic erosion and rampant horizontal gene transfer on AAB gut isolates that lead to genomes with mosaic metabolic pathways.

Taken together, this dissertation establishes a new model system for assessing evolutionary consequences of symbioses with gut bacteria. These results provide novel insights into the repercussions of bacterial adaptation to a host gut tract. They establish a foundation to interrogate questions unique to persistent extracellular gut symbionts. Finally, they delineate distinct forces shaping the functional capacity of symbiont genomes: gene loss through reductive evolution and gene acquisition via horizontal transfer from diverse community members.

Science has an inspiring capacity to change the world around us as it informs of the details governing life. From feats of engineering to medical breakthroughs, it has rapidly changed the way we live. Though it is woven into the fabric of all we do, there are still gaps between science and society—such as how science is shared from the academy and connects to communities. This has always fascinated me and the resulting body of this dissertation is three parts biology, exploring the symbiosis between the small aquatic fern, Azolla, and its cyanobacterial symbiont, Nostoc azollae; and one-part sociology, examining ways to reconnect science to the society it impacts so deeply and to which it owes so much. My hope is that within these pages you will find a new enthusiasm for plants—especially the tiny wonder, Azolla—and that you will see the value I do in strengthening the connection between science and the communities around it. With everything we do as scientists we have the power to affect the world around us, and it is our responsibility to think deeply about how we engage and join science and society together for the betterment of us all.

Chapters 2 and 3 focus on the biological aspects of the symbiosis between the aquatic fern, Azolla filiculoides, and its symbiont, N. azollae. It is not so coincidental that this symbiosis ended up being the subject of my research and has sweeping connections to societies around the globe—including its use as a green fertilizer in China for over 1500 years. Azolla is a small genus of aquatic fern with immense green potential to positively impact the globe. It owes this distinction to the nitrogen-fixing cyanobacterial symbiont, N. azollae, it harbors within specialized cavities within its photosynthetic leaves. These two partners have been living together for over 90 million years. There are many plant microbial symbioses, however, what makes the Azolla-Nostoc symbiosis unique among the others is that the cyanobiont is intimately associated with the fern perpetually throughout both organisms’ life cycles. The two are rarely—if ever—seen apart. This symbiosis has long captured the curiosity of scientists, who have explored various aspects in detail, such as what compounds are exchanged between the two partners, how the leaf cavities develop, and what structures are present within the cavities. Presently, we are at a stage to delve deeply into understanding the way this symbiosis thrives by exploring the genome, transcriptome, and connecting these to features of the leaf cavity.

Chapter 2 details a visual examination of the symbiosis using confocal microscopy. I used a clearing protocol coupled with confocal microscopy to image the leaves of Azolla filiculoides as the symbiosis develops to visualize the symbiotic cavity and labeled different cellular components with the fluorescent dyes calcofluor white and 4’,6-diamidino-phenylindole (DAPI). I imaged the cavity trichomes within the leaf pocket in whole leaves, as well as the trichomes at the apex that facilitate movement of the cyanobiont into the megaspores. These trichomes are the main plant structures that interact with the cyanobiont. The ultimate goal is to use this technique alongside the genomics and transcriptomics data (chapters 3 and 4) to identify the functions of the trichomes, eventually outlining the strategy A. filiculoides uses to engage in its symbiosis.

Chapter 3 takes a closer look at the putative symbiosis genes. I examined the expression of ammonium assimilation genes and their potential post-translational modifications; as well as how the larger pool of putative symbiosis genes may be transcriptionally regulated and what their functional categories are using gene ontology analysis. The RNA-sequencing analysis revealed 160 putative symbiosis genes. These genes included nutrient transporters for compounds like ammonium and molybdate, but did not include glutamine synthetase, glutamate synthase genes, or sucrose transporters. We also found that the nitrogen assimilation genes in A. filiculoides lack the post-translational modifications used in other plants to regulate their activity, leading to questions about how Azolla does this differently. This work provides the groundwork for establishing new ideas for how the Azolla-Nostoc symbiosis works, which factors are used to communicate between the two partners and what is used to regulate the exchange of nutrients, all of which allows their life cycles to be linked together.

This dissertation concludes with a departure from the biology of the Azolla-Nostoc symbiosis, and transitions into a survey of how science and society can be reconnected. In chapter 4, I detail three case studies addressing problems that keep science and society separated— (1) the inaccessibility of science to certain groups, (2) the inability of scientists to build trusting relationships with non-scientific audiences, and (3) the lack of innovative ways to engage the public about science. The first case study specifies programs I have been involved in to engage underrepresented minority students in the sciences. I also detail work to improve science to make it an inclusive environment for these students to succeed and thrive. The second case study focuses on training and preparing scientists to interact with the public. This is crucial to share science in ways that are accessible and resonate with people. I remark on my use of improvisational comedy as a way of making scientists more attentive to their audiences as they are presenting as well as improving their own body awareness. I also discuss work to introduce them to the basics of good science communication, and outreach opportunities to put this training into practice. In the third case study, I make a case for joining the arts and sciences as a means to powerfully connect science and people. This comes from a science and art exhibit that I launched with fellow graduate students, that has sparked a wave of other science-art minded endeavors. The combination of art, science, and community-engagement hit upon a way to captivate the public and pull them into the stories behind science. Together, these seek to be examples of how we can rejoin science and society in meaningful ways, allowing all people to share in science and see how science weaves into our lives.

Lichen-forming fungi employ a successful mode of nutrition as symbiotic partners with green algae and/or cyanobacteria (the photobiont). Nearly one fifth of all known fungi are obligate lichen formers, yet we know little of how they find compatible partners and establish long-lived symbiotic relationships. The combined growth of these symbionts forms a body (thallus) with emergent properties unlike either of the symbionts individually grown. Based on other well-studied eukaryotic systems, the development of a lichen thallus must rely upon the successful identification and collaboration of these two very different organisms. Identifying the molecular basis of microbe recognition and interactions remains one of the greatest challenges in studying symbiotic systems.

In this thesis, I determine the stage in which to begin looking for lichen symbiosis specific genes, and then examine mycobiont and photobiont genes that, when compared to the aposymbiotic state, are upregulated in the symbiotic state. Using the symbiosis between the mycobiont Cladonia grayi and the photobiont Asterochloris sp., as well as scanning electron microscopy observations of the earliest stages of contact between C. grayi and Asterochloris sp., I determined that the mycobiont undergoes a specific change in phenotypic growth in response to Asterochloris sp. This change is particular to the lichen symbiosis, and is not observed with algal shaped inanimate objects or algae other than Asterochloris. I then used this phenotypically defined stage that is exclusive to lichen symbiosis to begin studying the the genetic and molecular mechanisms underlying the development of a stratified lichen thallus. Using suppression subtractive hybridization to determine differential gene expression, fungal and algal libraries were made of upregulated genes in the first 2 stages of lichen symbiosis. The symbiotic expression levels of select genes were then verified using quantitative PCR. Lastly, a candidate gene for involvement in lichen symbiosis was transformed into Saccharomyces cerevisiae to test for protein function.

Further results of this study show that the fungal protein products of genes upregulated in lichen symbiosis show significant matches to proteins putatively involved in fungal self and non-self recognition, lipid metabolism, negative regulation of glucose repressible genes, an oxidoreductase, a dioxygenase, and a conserved hypothetical protein. Algal genes that are upregulated in lichen symbiosis include a chitinase-like protein, an amino acid metabolism protein, a dynein related protein, and a protein arginine methyltransferase. Furthermore, genes that are expressed in the early stages of lichen symbiosis are common varying metabolic pathways. Furthermore stages 1 and 2 of development are marked not by a drastic change in transcriptional products, but instead by an overall change in genes that are already expressed. Finally, the Cladonia~grayi Lip3was cloned in its entirety from genomic DNA and cDNA, was predicted to be secreted using signal peptide prediction software, and shown to be a functioning secreted extracellular lipase in yeast.

I conclude that many genes are involved in the interactions of symbionts and the development of a stratified lichen thallus, and that many more genes remain to be discovered. Furthermore, the possibility that genes exist in either symbiont that are specific to lichen symbiosis remains, and that their discovery awaits the creation of better genomic tools for \textit{Cladonia~grayi} and Asterochloris.