# Ribosomal Hiberation Factor Links Quorum-Sensing to Acid Resistance in EHEC
## 4 Discussion  
This study establishes ribosomal hibernation as the indispensable mechanism connecting QS to acid resistance in EHEC. We demonstrate that optimized exogenous QS signaling (100 uM 3-oxo-C6-AHL for 2 h) and endogenous _yenl_-derived AHL robustly induce the ribosomal modulation factor RMF, which serves as the non-redundant executor of bacterial survival under extreme acid stress. Genetic evidence confirms that the RMF-dependent formation of 100S ribosome dimers is essential for resisting lethal acidity (pH 2.5), with _rmf_ deletion causing catastrophic acid sensitivity (17-fold reduction) and abolishing QS-mediated protection against diverse stressors (including 13.7-fold increased starvation mortality). Crucially, we elucidate a pH-gated regulatory circuit where SdiA senses AHL signals to activate _rmf_ specifically under acidic conditions, while RMF reciprocally sustains RpoS activity (85% _rpoS_ suppression in _rmf_ at pH 2.5) and enhances SdiA-mediated signaling. This self-reinforcing loop represents a sophisticated adaptation mechanism that transcends conventional stress-response models. These findings provide mechanistic insights into EHEC's exceptional resilience, partially addressing long-standing questions regarding its ability to withstand gastric acidity despite lacking native AHL synthase. Unlike _Vibrio cholerae_--which requires high infectious doses (>10\({}^{9}\) CFU) due to limited AR capacity--EHEC's low infective dose (\(\approx\)10\({}^{2}\) CFU) appears to be substantially facilitated by RMF-mediated ribosomal optimization [31]. While prior research attributed acid resistance primarily to gad complexes or chaperone systems [4], our integrated data suggest that ribosomal reprogramming constitutes a major effector mechanism downstream of QS. This supports an emerging paradigm where ribosomal hibernation actively fortifies pathogens against host assaults beyond its classical role in nutrient stress adaptation. Mechanistically, our data suggest that RMF's role extends beyond ribosome stabilization to potentially influence global transcriptional regulation [14]. The observed severe RpoS deficiency in _rmf_ mutants offers an alternative perspective to conventional paradigms positioning RpoS as the apex stress regulator [12; 32]. We found that _rmf_ ablation substantially compromises RpoS functionality under acid stress, consistent with RMF's putative upstream hierarchical position. This functional reorganization appears to be pathogen-specifically calibrated: whereas non-pathogenic K-12 strains primarily utilize ppGpp-dependent RMF induction [33], EHEC seems to employ acid as the dominant trigger, potentially reflecting its ecological niche adaptation. Such specialization could permit EHEC to exploit microbiome-derived AHLs via SdiA as a pre-emptive strategy for gastric challenges, showing distinctions from _Salmonella_'s direct acid-sensing strategies. Our work further proposes a sophisticated energy allocation dynamic. While RpoS coordinates active defense by transcribing effector proteins (_gadA/hdeA_)--an ATP-intensive process--RMF-driven hibernation likely conserves energy by minimizing translational expenditure [5; 9; 15; 34; 35]. These potentially complementary strategies may exhibit dynamic synergy under combined QS and acid stress. During acute acid exposure (0-2 h), RMF rapidly initiates ribosome dimerization (2.9-fold _rmf_ upregulation within 2 h, as measured in our assays), suggesting a prioritization of immediate energy conservation. As stress persists (>2 h), RpoS-driven effector genes become progressively predominant, potentially enabling long-term survival through active repair mechanisms. This temporal division of labor could explain why \(\Delta m\)_r_/ concurrently loses acid resistance and RpoS functionality: we speculate that the absence of dormancy may lead to continuous energy expenditure on futile translation, possibly exhausting resources required for RpoS activation.