Development of Dysgonomonas as a Model Genus to Study Microbial Interactions in the Hindgut of the Lower Termite Reticulitermes flavipes and Beyond

The genus Dysgonomonas is nestled within the order Bacteroidales, and unlike the other anaerobic genera within this order, Dysgonomonas are facultative anaerobes. Dysgonomonas are traditionally recognized from occurrence in human disease and dysbiosis. In the last decade, Dysgonomonas spp. have been isolated or detected by 16S rRNA gene surveys in several types of insects, most notably termites and xylophagous cockroaches. Dysgonomonas are also found in many industrial processes such as anaerobic digesters and sludge- or wastewater-fed microbial fuel cells. Many of these environments contain lignocellulose and other complex polysaccharides, and the content of their genomes corroborates their adaptation to these locales. The genomes of Dysgonomonas are well-adapted to degrade diverse components of lignocellulose, as the number and diversity of polysaccharide utilization loci (PULs) suggests. Novel Dysgonomonas spp. continue to be described, particularly in association with xylophagous cockroaches and termites, but there is a lack of understanding of their function in these systems. Dysgonomonas are associated with the termite hindgut wall and also with single-celled eukaryotic protists which are obligate symbionts of these environments. The lack of growth media, sequenced genomes and genetic tools preclude in-depth studies of their roles in these systems. This dissertation describes the process for which a foundation was built to understand the function of Dysgonomonas spp. in diverse environments including the hindgut of the model lower termite Reticulitermes flavipes. Complex, defined and minimal growth media were developed to enable robust and reproducible growth under aerobic or anaerobic conditions. Most Dysgonomonas strains required L-cysteine, ferric hemin, thiamine, biotin and cyanocobalamin as growth supplements, regardless of oxygen condition. These media were used to isolate novel strains from worker and alate termites and to isolate auxotrophic transposon mutants. Genomes from four strains of Dysgonomonas corroborated the observed metabolic auxotrophies and mirrored the diversity of PULs seen in related strains. Finally, a genetic toolkit was created for targeted and untargeted mutagenesis and in vivo reporter expression. These tools allowed the creation of a xylose-utilization deficient mutant, a mariner transposon library and a GFP reporter strain that allowed previously undescribed visualization of protist predation of bacteria in the lower termite hindgut.