# Ribosomal Hiberation Factor Links Quorum-Sensing to Acid Resistance in EHEC
## 3 Results
### Ribosome Hibernation Underpins Quorum Sensing-Mediated Acid Survival in EHEC  
RNA sequencing of EHEC O157:H7 EDL933 exposed to lethal acidity (pH 2.5) with 100 uM 3-oxo-C6-AHL identified 83 significantly dysregulated transcripts (Figure 2A), comprising 36 upregulated and 47 downregulated genes (\(|\)log\({}_{2}\)FC\(|\)\(>\) 0), while 3896 genes remained unaffected. Some ribosome-related differentially expressed genes are listed in Table 1.  
Figure 1: Quorum sensing-dependent enhancement of acid resistance in EHEC. **(A)** Exogenous AHL augments acid survival. Stationary-phase wild-type EDL933 was subjected to pH 2.5 with 0, 10, or 100 uM 3-oxo-C6-AHL for 1–3 h. Survival rates (relative CFU = CFUh/CFU0) were quantified through colony enumeration (n = 3 biological replicates; mean \(\pm\) SEM; \({}^{*}p\)\(<\) 0.05, \({}^{**}p\)\(<\) 0.01, \({}^{***}p\)\(<\) 0.001 versus 0 uM at corresponding time points). **(B)** Endogenous AHL production confirmed by cross-streaking. WT/_pyenl_ (left plate) activated violacein biosynthesis in CV026, verifying functional signal synthesis. Controls: C6-HSL-positive control, EDL933 WT-negative control. **(C)** Endogenous AHL signaling promotes acid tolerance. Survival kinetics of WT versus _yenl_-expressing strain (WT/_pyenl_) under pH 2.5 exposure. Relative CFU data demonstrate significant protection by _yenl_-derived AHL (\(p<\) 0.01 versus WT at matched durations). Figure 2: Transcriptomic profiling and functional validation of acid stress response in EHEC. (**A**) Differential expression analysis. Volcano plot identifies significantly altered transcripts between AHL-treated and untreated groups. _X_-axis: log\({}_{2}\) fold-change; _Y_-axis: \(-\)log\({}_{10}\) (adjusted _p_-value). Red points: up-regulated genes (log\({}_{2}\)FC \(>\) 1, FDR \(<\) 0.01); green points: down-regulated genes (log\({}_{2}\)FC \(<\) \(-\)1, FDR \(<\) 0.01); blue points: non-significant genes. (**B**) Gene ontology enrichment. Terms are categorized by biological process (red bars), cellular component (green bars), and molecular function (blue bars). Ribosomal pathways show peak enrichment (\(-\)log\({}_{10}\)[padj] \(=\) 12.1 for cellular components). (**C**) KEGG pathway analysis. Significantly enriched pathways include ribosome assembly (boxed in red; \(-\)log\({}_{10}\)[padj] \(=\) 10.5) and pathogenic _E. coli_ infection. (**D**) Technical validation of RNA-seq data. Log\({}_{2}\) fold-change comparison between sequencing and RT-qPCR results for selected ribosomal genes (_rmf, rniA, rpsL, rplM..._) Error bars: \(\pm\)SD. (**E**) Acid resistance impairment in _rmf_ mutant. Wild-type (WT), _Armf_ deletion mutant, and genetically complemented strain (\(\Delta mrf/prmf\)) were challenged at pH 2.5 for 1–3 h. Survival rates (relative CFU = CFU\({}_{\rm h}\)/CFU\({}_{0}\); mean \(\pm\) SEM; n \(=\) 3) reveal significant attenuation in \(\Delta mrf\) (\(**\)\(p<\) 0.01, ***\(**\)\(p<\) 0.001 versus WT at corresponding time points). Functional enrichment analyses converged on ribosome pathways. GO terms: Ribosomal structure dominated cellular components (peak \(-\)log\({}_{10}\) (_padj_) = 12.1), translation topped biological processes (8.2), and ribosomal constitution led molecular functions (10.3) (Figure 2B). KEGG pathways: Ribosome assembly (\(-\)log\({}_{10}\) (_padj_) = 10.5) showed \(>\)6-fold stronger enrichment than secondary pathways (e.g., RNA degradation: 4.0) (Figure 2C). Technical validation via RT-qPCR of 13 key ribosomal genes demonstrated (Figure 2D). Coordinated induction of hibernation factors _rmf_ (log\({}_{2}\)FC = 2.9 \(\pm\) 0.2) and _raiA_ (2.1 \(\pm\) 0.3). Suppression of structural ribosomal proteins (_rpsL_: \(-\)3.5 \(\pm\) 0.1; _rplM_: \(-\)4.2 \(\pm\) 0.2). Exceptional consistency between sequencing and qPCR datasets. Genetic ablation of _rmf_ yielded catastrophic acid vulnerability (Figure 2E). After _rmf_ deletion, survival plunged 4.4-fold at 1 h, 7.3-fold at 2 h, and 8.0-fold at 3 h vs. the wild-type.