Browsing by Subject "endosymbiont"

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.