ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen associated with raw produce such as sprouts, where it encounters fluctuating nutrient, temperature, and oxidative stress. This study elucidated the global transcriptional adaptations of E. coli O157:H7 EDL933 during colonization and persistence on alfalfa sprouts under simulated commercial sprouting and refrigerated storage. RNA sequencing was used to compare STEC grown in tryptic soy broth (D0), colonizing fresh sprouts for 5 days (D5), and held during sprout refrigeration for additional 3 days (D8). Among 5,002 expressed genes, 782 and 826 were differentially expressed (DEGs; |log2FC| ≥ 2, adjusted p < 0.05) at D5 vs. D0, and D8 vs. D0, respectively, whereas 11 genes only differed in the D8 vs. D5 comparison, indicating that the STEC transcriptome was largely stabilized by the time sprouts reach maturity, with refrigeration inducing minimal further shifts. Transition of STEC from nutrient rich broth to sprout tissues induced a profound metabolic reprograming, characterized by the up-regulation of de novo amino acid biosynthesis (e.g., hisA–H, leuABCD, and ilv operons), to compensate for nitrogen limitation within sprout tissues. STEC further adapted to sprout environment by inducing high-affinity uptake systems for potassium (kdpA/B/C/F), phosphorus (pstSCAB), and sulfur (tauABCD), alongside enterobactin-mediated iron acquisition. To survive sprout microenvironment, STEC elevated the transcription of acid resistance (gadA/C, cadA/B), oxidative stress defense (sodA, katE/G), and envelope protection (degP, pspA–D, baeS) genes. Although initial colonization at D5 involved the induction of flagellar components, the transition to a sessile biofilm-associated lifestyle at D8 was marked by the repression of motility genes and the strong induction of curli fimbriae (csg) and colanic acid (wca) loci. Functional enrichment at D5 and D8, compared to D0, indicated coordinated stress regulation and translation. The major transcriptional trends observed in RNA seq were confirmed using RT-qPCR. Collectively, these findings highlight a multi tiered molecular adaptation strategy, enabling STEC adaptation and persistence in sprout environment, thus, providing insights that could be translated into STEC risk mitigation during sprout production
