Madeline O'Connor

Summer 2022 UROP: Bacterial antibiotic persisters have been the subject of much study in the medical research field. Genetically antibiotic-resistant bacteria can result in chronic bacterial infections; however, even bacteria that are susceptible to antibiotics may persist. Termed as persisters, they are transiently tolerant to antibiotics by changing their phenotype. Rhizobia are a suitable model for antibiotic persisters because they yield high proportions of dormant cells in the face of starvation by differentiating into high-poly-3-hydroxybutyrate (PHB) dormant bacteria. The time it takes bacterial cells to come out of dormancy and resume proliferation, termed dormancy depth, is unknown for many bacterial species, including rhizobia. This proposal centers on investigating the dormancy depth for rhizobial cells after periods of starvation. We hypothesized that dormancy depth is not constant in a species and increases with longer periods of stress. This was tested through starvation of rhizobial cultures and examination through optical density for cell populations, Nile-red staining for PHB to determine the rhizobial phenotype, and microscopy over time. The cells died unexpectedly in the third week of starvation, so dormancy depths over time could not be determined. Spring 2023 Directed Research: Many species of bacteria, including antibiotic resistant varieties, can survive stress by changing their phenotype to allow dormancy. This is particularly interesting in terms of species that are not resistant to bactericidal chemicals in the traditional sense of possessing modifications such as different receptors in the cell wall, but are transiently tolerant instead. It has been found that dormant E. coli cells typically have one or two protein tangles, whose function has been hypothesized to suppress cell metabolism and reproduction characteristic of dormancy. Whether the protein aggregates are composed of newly synthesized proteins or existing proteins in the cell is unclear. Additionally, it is not known if other protein functions are necessary for dormancy to occur. In this project, we are seeking to determine if normal protein synthesis is a necessary component for rhizobial cells to use the dormant mechanism to survive periods of stress. This was accomplished through time-lapse photography of sinorhizobium cultures plated on TY media after each week of starvation over 3 week periods. Half of these cultures were exposed to non bactericidal concentrations of chloramphenicol, a drug that inhibits 70S ribosomes to halt protein synthesis. We hypothesize that normal protein synthesis is needed for dormancy to occur in rhizobia because it allows the cell to limit metabolism and reproduction through aggregation while keeping the necessary proteins for survival.When protein synthesis is inhibited, we predict that there will be a smaller proportion of dormant cells and a decreased rate of survival, determined through cell counts, in cultures compared to when protein synthesis is normal. At a concentration of 25 mg/L chloramphenicol, there was no statistically significant difference in cell counts between cultures. Further investigation with differing concentrations of chloramphenicol would be beneficial to validate the results of this experiment that protein synthesis is not crucial for bacterial dormancy.