Transcriptomic Profiling of Escherichia coli and Salmonella enterica Reveals Regulatory Roles of the 3' UTR-Derived sRNA UhpU Under Various Growth Conditions

To investigate the biological function and regulatory impact of the 3' UTR-derived small RNA (sRNA) UhpU, we performed transcriptomic analyses in both Escherichia coli and Salmonella enterica under multiple growth conditions. These include short-term pulse expression of UhpU in minimal medium supplemented with glucose-6-phosphate (G6P) or LB medium, as well as a comparative transcriptomic analysis of wild-type and ?uhpTU deletion strains during LB growth. This dataset provides insight into UhpU's regulatory roles in metabolic adaptation and its evolutionary diversification across species. Overall design: Three sets of RNA-seq experiments were conducted: 1.Pulse Expression in S. enterica During G6P Metabolism: The 72-nt Salmonella UhpU sequence was cloned downstream of the arabinose-inducible PBAD promoter and expressed in a ?uhpTU background strain. Cultures were grown in M9CA medium with 0.1% glucose-6-phosphate (G6P) to OD600 ~0.5, induced with 0.2% L-arabinose for 10 minutes, and RNA was extracted. An empty vector control was included for comparison. Non-stranded RNA-seq libraries were generated after DNase I treatment. 2.Pulse Expression in E. coli During LB and G6P Growth: The 265-nt E. coli UhpU sequence was similarly cloned and expressed in a ?uhpTU strain. Cultures were grown either in LB medium to OD600 ~1.0 or in M9CA with 0.1% G6P to OD600 ~0.5. UhpU expression was induced with 0.2% L-arabinose for 10 minutes. RNA was extracted and processed as above, with the empty vector serving as a negative control.3.Comparative Transcriptomics of Wild-Type and ?uhpTU E. coli Strains: To assess the endogenous role of UhpU, RNA was isolated from wild-type and ?uhpTU E. coli strains grown in LB (Miller) medium to OD600 ~1.0. Stranded RNA-seq libraries were prepared after DNase I treatment to compare transcriptomic differences attributable to the absence of UhpU under standard conditions.