Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Elucidating the Effect of the Antibiotic Tetracycline on the Regulatory Function of the Guanidine-Sensing ykkCD Riboswitch

The primary objective for one of several project avenues spearheaded by the Fernandez lab is to develop an assay - ideally one more feasible for small-to-medium sized and/or undergraduate-only scientific research institutions - for evaluating the concomitant binding of tetracycline (tet antibiotic) and guanidine (guanidinium cation at physiologic pH) to the guanidine-sensing bacterial riboswitch ykkCD. Thus more generally a guanidine-I riboswitch, this noncoding regulatory RNA segment has been shown to also recognize the translation-inhibiting antibiotic via a high-affinity aptamer domain. Resultant conformational change in the regulator prompts translation-wise upregulation of genes downstream from it, among which - of those known - most encode membrane-bound transporters (e.g., SMR efflux pumps) responsible for expelling such toxins. This phenomenon establishes a major underpinning of the seemingly ever-evolving issue of antibiotic resistance in bacteria, further evincing that, aside from having to "rely on" incomparably slower multigenerational evolution of purely novel tactics, modern bacteria quickly adapt and employ their diverse detoxification machinery to render various traditional therapeutic agents largely ineffective. A binding assay is planned to decipher how proximal or distal one ligand-riboswitch binding region is to/from the other and to investigate relevant thermodynamics & kinetics, through which it should be distinguishable whether or not the influence of tet-ykkCD association on guanidine recognition is statistically significant. Soil bacterium Bacillus subtilis is the RNA source herein, whereas intestinal bacterium Escherichia coli is the replication vehicle/host (vector). Past prescribed assays call for cumbersome procedures, including radioisotope-labeled RNA utilization and tedious gel sequencing. The assay under development therefore seeks to employ more universally accessible methodology, ideally both isothermal titration calorimetry (ITC) and surface plasmon resonance spectroscopy (SPR), for tet-influenced guanidine-regulator binding affinity and efficiency determination.