Daniel R. Sharda
Anna C. Karls
Scholarship of Discovery
RNA polymerase holoenzyme (Eσ) mediates transcription in eubacteria, and is composed of five constant subunits (α2ββ’ω) and a variable sigma (σ) subunit that is responsible for promoter recognition and initiation of transcription. An alternative sigma factor in Salmonella Typhimurium, σ54 (also called RpoN), is mechanistically different than classical σ70-type sigmas, requiring a different promoter consensus sequence, an activator, and ATP hydrolysis. The Rtc RNA repair operon lies within the regulon of RpoN in S. Typhimurium, but has no known physiological function. Previous work characterized similar systems in archaea and humans, which were determined to function in recovery from environmental stresses. Focusing on recovery from environmental stress as a function of the Rtc RNA repair operon, I hypothesized that nitrogen limitation, iron limitation and cell wall stress would induce expression of the Rtc RNA repair operon in Salmonella Typhimurium.
A plasmid encoding the quantifiable expression of LacZ under the control of the Rtc RNA repair operon was used to measure the impact of environmental stresses on expression of the operon. Cefotaxime as a cell wall stressor induced a four-fold increase in expression maximally at a dose of 40 μg/ml, nitrogen limitation exhibited a two-fold increase, and addition of 2,2’-bipyridyl as an iron chelator did not induce any significant increase in expression at 0.2, 0.3, or 0.5 mM. However, these three treatments all fell short of the positive control treatment with Mitomycin C (MMC), which had two and four-fold increases in expression compared to cefotaxime and nitrogen limitation treatments.
Induction of expression upon treatment with MMC, cefotaxime and nitrogen limitation displays the diversity of signals that induce the Rtc RNA repair operon. Induction with cefotaxime indicates the Rtc RNA repair operon may function to repair transcripts essential for metabolites involved in transitioning to anaerobic metabolism. Induction with nitrogen limitation suggests that the repair operon plays some role in adapting to low nitrogen conditions. However, not all sources of environmental stress were able to induce operon expression. Expression upon iron limitation was not observed and indicates a distinct difference in the response of rtcR between E. coli and Salmonella.
Gulledge, Caleb M., "Defining Environmental Stresses that Activate the RNA Repair Operon in Salmonella Typhimurium" (2016). Honors Program Projects. 71.