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 (Padj < 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.