Researcher(s)
- Raghav Agarwal, Applied Molecular Biology & Biotechnology, University of Delaware
Faculty Mentor(s)
- Vijay Parashar, College of Health Sciences, University of Delaware
- Richard Knappenberger, College of Health Sciences, University of Delaware
Abstract
Swarming motility, the surface-based migration of hyper-flagellated bacterial cells, contributes to colonization and antimicrobial resistance in Bacillus subtilis. This process is governed by the transcriptional regulator DegU and its activator SwrA, which together control expression of the fla/che operon. While DegU forms tetramers upon DNA binding and SwrA stabilizes this oligomeric state, the precise molecular determinants of their interaction remain poorly understood. Our research aimed to characterize SwrA mutants predicted to affect hetero-oligomerization or interaction with DegU.
We expressed and purified several SwrA variants, including R74A, R84A, Y47A, and R42AN46A, using affinity and size-exclusion chromatography. Following purification, thermal stability assays revealed altered melting temperatures for select mutants, suggesting structural destabilization that may explain observed purification challenges. For the stable mutants, we initiated sedimentation velocity analytical ultracentrifugation (AUC) experiments to assess oligomeric state and stoichiometry. While early runs with wild-type SwrA and DegU confirmed DegU-SwrA hetero-oligomerization, mutant AUC data were inconclusive due to insufficient interaction between certain wild-type interactions, which we have since addressed through buffer optimization. More recent data have identified stable interactions between SwrA R74A and DegU but show an intermediary oligomeric state for the R84A mutant, suggesting less stable interaction between DegU and SwrA and the importance of R84 in the oligomeric interface.
Although full AUC analyses are ongoing, this research lays the groundwork for elucidating DegU-SwrA-DNA interaction interfaces and assembly. Future efforts will focus on optimizing mutant SwrA-DegU-DNA complexes and examining their oligomerization and stoichiometry. By identifying interaction-disrupting mutations, our findings may inform strategies to target swarming motility, an antibiotic resistance-linked behavior, without exerting selective pressure on bacterial viability.