Insights into the Thermotolerance of an Escherichia coli RNA Polymerase Mutant

When exposed to abiotic stresses, Escherichia coli responds by activating various stress-mitigating pathways. Initiation of stress responses partially relies on the RNA polymerase (RNAP) to transcribe genes necessary to tolerate various stresses, including nutritional deprivation and heat exposure. Consequently, RNAP mutations impacting transcription can have pleiotropic effects on the cell physiology and the ability to tolerate stress. Previously, while investigating antibiotic-resistant mutations arising in the absence of major antibiotic efflux pumps, four mutants containing alterations in the RNA polymerase beta subunit gene (rpoB) were isolated (Cho & Misra, 2021). Of the four mutants, one (RpoB58) was found to be thermotolerant, permitting homogenous, stable growth at temperatures up to 47°C, whereas the parental rpoB wildtype (RpoB-WT) was only able to do so up to 45°C. Additionally, RNA-Seq analysis indicated that the RpoB58 mutant had a ‘stringent’ profile that is normally seen under nutritionally deprived conditions. To better understand the regulatory pathways used to confer stress tolerance, this thesis sought to further characterize and investigate the intracellular mechanisms contributing to the thermotolerance conferred by the rpoB58 mutation. The RpoB58 mutant was found to be significantly more tolerant to both continuous heat stress (up to 47°C) and short-term heat (55°C) and ethanol (25%) exposure. Additionally, the RpoB58 mutant tolerated the absence or depletion of major heat shock chaperones DnaJ and DnaK that normally play key roles during temperature stresses by reducing protein misfolding. RNA-Seq data and reporter gene assays showed reduced expression of genes involved in protein synthesis. A similar reduction in the expression of protein synthesis genes was observed when cells were grown in growth-limiting minimal media. Interestingly, growth in minimal medium rescued the ΔdnaJ defect like the rpoB58 mutation. Based on these data, it was proposed that a decrease in protein synthesis, whether caused by rpoB58 or the growth medium, would result in less growth-inhibiting protein misfolding and aggregation, especially at higher growth temperatures where proteins are susceptible to denaturation and aggregation. As a result of these investigations, a possible mechanistic insight was provided as to how the rpoB58 mutation confers thermotolerance.