# Ribosomal Hiberation Factor Links Quorum-Sensing to Acid Resistance in EHEC
## 3 Results
### Acid-Triggered SdiA-RMF-RpoS Signaling Axis Governs Ribosomal Stress Adaptation  
At neutral pH (7.0), _sdiA_ maintained basal _rmf_ expression (\(\Delta\)_sdiA_: 22.5% downregulated), but AHL showed no effect (WT + AHL: 0.98 vs. WT, NS) (Figure 5A). Under acid stress (pH 2.5, 2 h), AHL and SdiA displayed synergy effects. _rmf_ induction by AHL required SdiA, achieving 2.1-fold upregulation (WT + AHL: 2.10 vs. WT 1.00). Irreversible SdiA dependence: \(\Delta\)_sdiA_ reduced _rmf_ by 75% (0.25, \(p\) \(<\) 0.01), and AHL failed to rescue (\(\Delta\)_sdiA_ + AHL: 0.63, still 37% downregulated) (Figure 5B).  
Regarding RpoS functional compensation, the deletion of _rpoS_ suppressed _rmf_ expression by 44%, but exogenous AHL treatment fully restored _rmf_ levels via SdiA-dependent signaling, achieving expression comparable to the wild-type in the \(\Delta\)_rpoS_ background  
Figure 5: pH-dependent transcriptional circuitry of the SdiA–RMF–RpoS regulatory axis. **(A)**_sdiA_ modestly regulates _rmf_ at neutral pH. No significant changes in _rmf_ expression among WT, WT + AHL, \(\Delta\)_sdiA_, or \(\Delta\)_sdiA_ + AHL (_p_ \(>\) 0.05). **(B)**_sdiA_ indispensability for acid-induced _rmf_ expression. At pH 2.5, _rmf_ decreased by 75% in \(\Delta\)_sdiA_. **(C)** Compensatory AHL signaling overcomes _rpoS_ deficiency. \(\Delta\)_rpoS_ reduced _rmf_ by 44% at pH 2.5, but AHL administration restored WT-level expression. **(D)** RMF-independent _rpoS_ regulation at pH 7.0. Minimal _rpoS_ change in \(\Delta\)_rmf_ versus WT (_p_ \(>\) 0.05). **(E)** Acid-specific _rmf_ induction. pH 2.5 elevated _rmf_ expression 2.9-fold versus pH 7.0. **(F)** RMF sustains basal _sdiA_ transcription. \(\Delta\)_rmf_ decreased _sdiA_ by 22% at pH 7.0. **(G)** RMF enables acid-enhanced SdiA activation. \(\Delta\)_rmf_ reduced AHL-induced _sdiA_ by 65% at pH 2.5, restricting signaling capacity to 13.8% of maximum. **(H)** RMF maintains RpoS homeostasis. Neutral pH caused 22% rpoS reduction in \(\Delta\)_rmf_ (_p_ \(<\) 0.05). **(I)** Acid-stress synergy reveals RMF–RpoS codependence. Lethal acidity (pH 2.5) induced catastrophic 85% _rpoS_ downregulation in \(\Delta\)_rmf_. Data represent mean \(\pm\) SD; n = 3 biological replicates. * \(p\) \(<\) 0.05, **_p_ \(<\) 0.01, *** \(p\) \(<\) 0.001, NS: not significant.  
(Figure 5C,D). Moreover, _rmf_ induction exhibited strict acid dependence: pH 2.5 triggered a 2.8-fold upregulation (2.80 vs. pH 7.0 baseline) (Figure 5E).  
We further investigated cross-regulatory circuitry where RMF modulates SdiA activation. Under acid stress, AHL-induced _sdiA_ expression increased 4.5-fold (5.80 at pH 2.5 vs. 1.30 at pH 7.0), demonstrating pH-gated signal amplification. However, _rmf_ reduced basal _sdiA_ expression by 65% (0.35 vs. WT 1.00), severely limiting AHL responsiveness to 13.8% of maximal activation (Figure 5F,G).  
Crucially, RMF regulated RpoS abundance in a pH-dependent manner: at a neutral pH (7.0), _rmf_ reduced _rpoS_ expression by 22% (Figure 5H), while acid stress (pH 2.5) exacerbated this defect, causing 85% downregulation in _rmf_ (0.15 vs. WT 1.00) (Figure 5I).  
Collectively, these data support a pH-gated regulatory triad: AHL \(\rightarrow\) SdiA \(\rightarrow\)_rmf_\(\rightarrow\) RpoS, wherein ribosomal modulation by RMF is required to maintain stress sigma factor activity during acid adaptation.