Thermal Shifts Dominate Single-Gene Responses to Transcription-Targeting Antibiotics

Combined exposure to thermal and antibiotic stresses is ubiquitous. How cells adapt to these concurrent cues at the single-gene level remains unresolved. Here, we show that in Escherichia coli, both cold and heat shock responses dominate antibiotic stress responses across multiple levels of physiology. Using RNA-seq, we demonstrate that combined exposure produces transcriptomes that are largely indistinguishable from those induced by thermal shifts alone. Flow-cytometry measurements across intermediate temperatures and increasing ofloxacin concentrations further show that temperature-like responses are more prevalent outside the 30°C-39°C range. This dominance arises from temperature-driven changes in ATP levels and nucleoid organization, which in turn modulate DNA gyrase and RNA polymerase engagement with DNA. Combined exposure also induces SoxS- and MarA-mediated efflux systems more strongly than individual stresses. Machine learning analysis identifies CRP and σ³⁸ as key gene expression regulators under combined stress. Molecular dynamics simulations show that drug-target conformational plasticity is altered under combined conditions. Cross-species simulations across six bacterial species with distinct pathogenic profiles reveal that thermal modulation of antibiotic response is partially conserved across evolutionarily distant species. Together, the results identify temperature as a critical determinant of antibiotic response, suggesting that thermal shifts may facilitate bacterial adaptation to antibiotics.