Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd. / Master of Science / Many amphibian population declines around the world have been caused by chytridiomycosis, a skin disease. This disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). The skin of amphibians is also home to many bacteria that can provide important functions for the amphibian host, like preventing infection by Bd. To understand how these bacteria might provide protection, we examined the entire genomes of 40 bacterial isolates that reside on the skin of four amphibian species from Virginia, USA. These bacteria were previously tested for their ability to prevent Bd growth and 40 of them were chosen for sequencing based on selecting closely related isolates that varied in their ability to inhibit Bd growth. This allowed us to compare their genomes and generate hypotheses about possible genomic differences that could contribute to the variation in Bd growth inhibition. We identified sixty-five gene families that were present in all 40 bacteria. We also looked for sets of genes (biosynthetic gene clusters) that are known to produce secondary metabolites, which are compounds that can include antifungals. The two most abundant clusters we identified that had the potential to produce compounds that inhibit fungal growth were siderophores and Type III polyketide synthases. We then looked for genes that were not part of biosynthetic gene clusters that could produce specific compounds that can inhibit Bd growth, such as chitinase and violacein. We found variation in chitinase genes in several isolates that seemed to be associated with the ability to inhibit Bd growth. In addition, there were some differences in violacein genes that should be examined more in future studies. Overall, we suggest that using comparative genomic approaches can be valuable for identifying key bacterial functions in the microbiome.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/103961 |
| Date | 22 June 2021 |
| Creators | Wax, Noah David |
| Contributors | Biological Sciences, Belden, Lisa K., Jensen, Roderick V., Vinatzer, Boris A., Haak, David C. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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