Transcriptome Profiling of Escherichia coli B During Sequential Adaptation to T4 Phage and Iron(III) Stress

Background/Objective: Antimicrobial resistance poses a critical public health crisis, highlighting the urgent requirement to investigate bacterial evolutionary adaptations and pioneer alternative therapeutics. Consequently, bacteriophages and metal-based compounds are emerging as viable options to combat drug-resistant infections. Building on our finding that T4 phage resistance in E. coli B also confers adaptation to high iron(III), we used RNA-sequencing (RNA-seq) to explore bacterial gene expression in resistant and control populations. We analyzed samples from our five experimental groups-Ancestor (ANC), control (CON), phage-selected (Phage), iron(III)-selected (FE), and phage/iron(III)-selected (PF), to understand how these regimes drive transcriptional changes. Method: Total RNA was extracted using the TRIzol protocol, and sequencing libraries were prepared with the Illumina RNA Total Library Prep Kit. Sequencing was performed on the Illumina NextSeq 1000/2000 platform. Reads were aligned to the E. coli B ATCC 11303 reference genome, and pairwise comparisons between the five experimental groups were conducted to determine differential gene expression profiles. Results: Principal component analysis (PCA) showed that iron-adapted populations (FE and PF) separated distinctly from the Ancestor and control populations along PC1 (capturing 40% of the variance), while the phage-selected replicates were split, with one (Phage5) clustering with CON3 and two (Phage2, Phage4) falling closer to, but clearly separated from, the Ancestor. Differential expression analysis (P_adj < 0.05 and |log₂FC| ≥ 1) revealed extensive transcriptional rewiring, with 482 and 381 differentially expressed genes (DEGs) in the FE and PF populations, respectively, compared to the Ancestor, and 177 DEGs in the phage-selected population compared to the Ancestor. The direct pairwise comparison between the iron-selected and phage/iron-selected populations yielded zero DEGs, demonstrating that both iron-adapted populations converged on a near-identical gene expression profile regardless of their distinct genetic and evolutionary backgrounds. Conclusions: This study suggests that pmrB and arn pathway genes may serve as primary markers for resistance to iron stress. These results are significant because they demonstrate a coordinated, multi-gene defense mechanism in E. coli B against high iron(III) stress, in which the arn operon and eptA remodel lipid A and the outer membrane, while glycerol-3-phosphate metabolism and phage-shock/chaperone pathways are repressed.