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!