Aminoacyl-tRNA synthetases (aaRSs) are ancient and universally essential enzymes that pair tRNAs with the corresponding amino acids. To enhance accuracy, many aaRSs employ an editing activity that hydrolyzes incorrectly activated amino acids. Editing significantly decreases the frequency of mistakes in vitro, although the physiological role of editing in cells remains unknown, as aaRS editing functions are dispensable under certain conditions and absent in some cell types.
Through a combination of genetic and physiological tests, we will determine under what conditions aaRS editing is required for viability. Specifically, we will test the hypothesis that editing is required under conditions of extreme slow cell growth.
Determining mutation frequency and mechanism of aaRS mutation increase
We have been studying the effects of misacylation caused by the editing deficient phenylalanine aaRS, PheRS. Specifically, we disable the editing domain without disrupting acylation activity. By measuring changes in resistance to antibiotics, we determined an increase in mutation frequency. Currently, we are exploring this mechanism behind this increase not only in PheRS but also IleRS, the isoleucyl aaRS. IleRS has similar editing functions that will allow us to determine whether these effects are consistent in other aaRSs.
Increase in mutation frequency of pheT mutant observed in exponential phase but not log phase.
Understanding cellular pathways that are affected by aaRS editing
Aminoacyl-tRNA synthetases charge tRNAs with their cognate amino acid. To mitigate mischarging of chemically similar amino acids, some contain an editing function. Isoleucyl-tRNA synthetase (IleRS) distinguishes between isoleucine and valine. Editing in tRNA synthetases is highly conserved, but the requirement for editing depends greatly on cellular physiology and environmental conditions. In Bacillus subtilis, disrupting editing but not charging in IleRS inhibits spore formation by delaying Spo0A, the first transcription factor in the sporulation cascade. However, the mechanism for this delay is not known.
To determine this pathway, the mRNA of B. subtilis with a wild type ileS gene will be compared to that with the ileS(T233P) mutant allele lacking the editing function. As the spoIIGB gene is downstream of Spo0A, its activation is known to be delayed in the ileS(T233P) editing-deficient strain. Therefore, the strains used in this study are deleted for spoIIGB. This allows gene expression to be specifically analyzed in the sporulation cascade upstream of Spo0A. The next steps are sequencing the mRNA from ileS wild type and mutant strains in order to identify differences in gene expression and the consequences of these changes. Through this research, the cellular mechanisms that cause physiological defects in cells with a tRNA synthetase editing deficiency can be discovered.
Expression of a Spo0A-activated gene is delayed in the ileS (T233P) mutant.
Biofilm associated infection therapy and orthopedic implants
Through a novel integrated approach our collaboration with the Billi Lab explores the reduction of the incidence of infection in orthopedic implant applications. It also clearly demonstrates that the combination of gallium treatment with Pulse Electromagnetic Field (PEMF) could aid biofilm-associated infection therapy due to improved Ga efficiency.
The aim of this study was to develop a new class of gallium (Ga)-doped chitosan (CS) coatings fabricated by electrophoretic deposition (EPD) in staphylococcal infection therapy.