Poster abstracts
Poster number 58 submitted by Vy Nguyen
Characterizing and Comparing D-Amino Acid Toxicity Phenotypes in E. coli Strains
Ngoc Tuong Vy Nguyen (Department of Biology, Department of Chemistry, Earlham College), Dawt Sang (Department of Biology, Department of Chemistry, Earlham College), Landry Cox (Department of Biology, Department of Chemistry, Earlham College), Samia Kassim (Department of Biology, Department of Chemistry, Earlham College), Lexie Kuzmishin Nagy, Ph.D. (Department of Biology, Department of Chemistry, Earlham College)
Abstract:
Aminoacyl-tRNA synthetases charge tRNAs with the corresponding amino acid, generating aminoacyl-tRNAs for translation.1,2 Although there are two conformations of amino acids, L- and D-, only L-amino acids are used in translation. Mischarged D-aminoacyl-tRNAs cause D-amino acid toxicity by decreasing the pool of free tRNA available for translation.3 In E. coli and many other organisms, D-aminoacyl-tRNA deacylase enzymes (DTD) protects against D-amino acid toxicity by editing tRNAs mischarged with D-amino acids.4 Similarly, rhizobia encode the enzyme ProXp-x, which preliminary data has shown to edit tRNAs mischarged with non-proteinogenic amino acids, including alpha-aminobutyrate and some D-amino acids in vitro. While efforts are underway to characterize the mechanism of substrate selection, ProXp-x’s editing has not been assessed for its ability to protect against D-amino acid toxicity in vivo. The ultimate goal of this research is to determine if ProXp-x protects against D-amino acid toxicity, like DTD, in the well-studied E. coli model. We hypothesize that ProXp-x, by virtue of its editing activity, when transgenically expressed in E. coli delta-dtd strains will prevent D-amino acid toxicity upon exposure to exogenous D-amino acids. Currently, we are establishing phenotypic differences between WT (BW25113) and delta-dtd E.coli strains (Keio Collection Knockout E. coli).5,6 We compared the growth and viability of these strains in the presence and absence of different concentrations of L- and D-amino acids using growth curve assays, spotting assays, and colony forming unit (CFU) assays. These results will aid us in determining how different conditions of amino acid concentrations can affect growth, and establish conditions for the ProXp-x rescue assays.
References:
1. Bacusmo, J. M., Kuzmishin, A. B., et. al (2018). RNA Biology, 15(4–5), 576–585. https://doi.org/10.1080/15476286.2017.1353846
2. Ibba, M., & Söll, D. (2000). In Annual Review of Biochemistry (Vol. 69, Issue Volume 69, 2000, pp. (617–650). Annual Reviews. https://doi.org/10.1146/annurev.biochem.69.1.617
3. Shahjee, H. M., et. al (2002). Journal of Biosciences, 27(5), 515–520. https://doi.org/10.1007/BF02705049
4. Routh, S. B., et. al (2016). PLOS Biology, 14(5), e1002465. https://doi.org/10.1371/journal.pbio.1002465
5. Baba, T., et al. (2006) Mol Syst Biol; 2, 2006.0008. DOI: 10.1038/msb4100050.
6. Sahu, S. R., et. al (2023). Bio-Protocol, 13(21), e4872. https://doi.org/10.21769/BioProtoc.4872
Keywords: tRNA, editing, D-amino acids