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Title:
Analyzing the Genetics of Uric Acid Degradation by the Bacterium Acetobacter fabarum
Creator:
Hiruni Dodangoda
Peter D. Newell

Notes

Abstract:
The fruit fly Drosophila melanogaster is widely used as a model animal to study the influence of resident microorganisms (or microbiota) on health and disease. Acetobacter fabarum is a bacterium found in the gut microbiota of wild Drosophila and is currently being used to identify bacterial genes that impact host health. Prior research identified genes responsible for uric acid degradation by A. fabarum, prompting the hypothesis that gut bacteria break down this compound. This is significant because uric acid is the main nitrogen waste product of Drosophila and its accumulation can can cause renal disease in flies as well as humans. In this study, we tested the impact of two mutations in A. fabarum on the degradation of uric acid. One mutation was in uricase – the enzyme predicted to break down uric acid, and the other in an oxidoreductase gene from the same genomic locus. We found limited uricase activity in all strains of A. fabarum tested, including both mutants and the wildtype strain. This prompted investigation of alternative approaches to testing our overarching hypothesis. Preliminary results suggest genetic manipulation of a different species of Acetobacter may prove more successful as a way to test the effect of bacterial uric acid degradation on Drosophila health.
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Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Peter Newell.

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SUNY Oswego Institutional Repository
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SUNY Oswego Institution
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All applicable rights reserved by the source institution and holding location.

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Analyzing the Genetics of Uric Acid Degradation by the Bacterium Acetobacter fabarum Hiruni Dodangoda & Peter Newell SUNY Oswego Dept. of Biological Sciences ! The gut microbiota within organisms can not only have a great impact on the metabolism of the organism but can also influence a wide range of traits. ! Acetobacter fabarum is a gut bacterium that resides in Drosophila (1) . Previous research suggests it can degrade uric acid (UA) (2), a major excretory waste product of its host. ! This study utilized genetic manipulation to test the function of predicted uricase and oxidoreductase genes in UA degradation. ! We also tested the ability of several other Acetobacter species to degrade UA in an agar plate assay. ! Mutants with an inactive uricase gene ( UriKO ) will not be able to degrade UA, while mutants with an inactive oxidoreductase gene ( OxiKO ) may or may not, as this gene is uncharacterized. ! Acetobacter species isolated from Drosophila will possess uricase activity, leading to dissolution of UA crystals in UA agar plates. Genetic Manipulations: ! OxiKO and UriKO strains needed to be cured of antibiotic resistance plasmids prior to testing. First, a MIC experiment was conducted to identify the appr o priate antibiotic concentration. Second, a cycolserine enrichment was performed to isolate bacteria that lack tetracycline resistance. ! The mutant strains were then grown in plates containing Uric Acid along with the wildtype as a control (data not shown). Testing additional strains for uricase activity : ! Due to low uricase activity in the Acetobacter strain we were studying, we tested additional isolates using a UA plate assay in which dissolution of UA crystals indicates activity (Fig. 1). Figure 1 . UA plate assay . Figure 2 . Transposon mutants Alternative approach : Isolate mutants of Acetobacter # 50 ! The plate assay showed low uricase activity of wildtype A. fabarum (Fig. 1; OSW_54) but high activity for Acetobacter strain 50 (OSW_50). Therefore, we began the process of isolating uricase mutants of strain 50 using transposon mutagenesis (Fig. 2). Introduction Hypotheses Experiments Results ! Genetic and phenotypic data suggest successful isolation of OxiKO and UriKO mutants. However, low uricase activity for wildtype of this strain made further study difficult. ! UA plate assay showed significant uricase activity for Acetobacter strains 42, 46, 50 and 53. ! Trial genetic manipulations of Acetobacter 50 were successful, suggesting that isolating uricase mutants in this strain will be feasible. Conclusions ! Acetobacter gut bacteria isolated from Drosophila fruit flies show uricase activity in an agar plate assay. ! Transposon mutagenesis successfully produced mutant bacteria for strain #50. ! Future research will target the uricase gene of Acetobacter #50 to test if it is required for UA degradation. ! UA is a major waste product in Drosophila as well as humans (3). Future experiments will test the impact of microbial uricase activity on Drosophila health. References & Acknowledgements: 1. Sommer AJ, Newell PD. Metabolic Basis for Mutualism between Gut Bacteria and Its Impact on the Drosophila melanogaster Host. App l Environ Microbiol. 2019;85 2. Winans NJ, Walter A, Chouaia B, Chaston JM, Douglas AE, Newell PD. A genomic investigation of ecological differentiation between free living and Drosophila associated bacteria. Mol Ecol. 2017;26 3. Lang S, Hilsabeck TA, Wilson KA, Sharma A, Bose N, Brackman DJ, et al. A conserved role of the insulin like signaling pathway in diet dependent u ric acid pathologies in Drosophila melanogaster. PLoS Genet. 2019;15: e1008318 Thank you to SCAC Challenge Grant for funding!