## 3 Role of Small RNA in Stress Response
### RyhB  
RyhB is a 95 nt sRNA that plays a key role in iron homeostasis and virulence in several pathogenic and non-pathogenic strains of bacteria including _E. coli_[138], _Salmonella_[139], and _Vibrio_[108] (Figure 2). It regulates targets through translational repression and mRNA degradation, often mediated by Hfq and RNase E. These include genes involved in the TCA cycle, respiration, and siderophore synthesis. RyhB is directly repressed by Fur [140]. Under iron-limiting conditions, RyhB was shown to play an iron-sparing response by inhibiting the translation of non-essential protein that uses Fe or Fe-S as a co-factor, thereby ensuring that the essential proteins can ligate to Fe-containing co-factors to drive key metabolic processes needed for bacterial survival [140] (Figure 2a,b). Previous works have suggested that the deletion of _fur_ did not impact virulence, but that the loss of _ryhB_ or both _fur_ and _ryhB_ impaired bladder colonization in the murine model of infection [105]. Although the role of Fur and RyhB have been well characterized in the non-pathogenic strains of _E. coli_, their contribution to the pathogenic strains remained elusive. To address this, using genomic and molecular biology tools like ChIP-seq, RNA-seq, and Proteomics, we have characterized the Fur and RyhB regulon in UPEC strain CFT073, revealing a complex interplay among iron acquisition, stress responses, and metabolic adaptation [140]. Fur directly and indirectly regulates a wide array of genes, including those involved in siderophore biosynthesis, iron uptake, and stress mitigation pathways, with RyhB modulating some components posttranscriptionally. Unique to CFT073, Fur-regulated pathways such as the siderophore aerobactin biosynthesis impose a metabolic burden, leading to increasing amino acid demand, which may be mitigated by external amino acid uptake. We found that the absence of Fur, a condition that mimics an Fe-limiting environment, induced the RpoS-mediated stringent stress response pathways in CFT073. This activation of the stringent stress pathways may provide the pathogen with an anticipatory response, where iron limitation could also be an indicative of other nutrient limitation, as the pathogen is often encountered in the nutrient-limited urinary tract (Figure 2b). This regulatory network, distinct from that in commensal strain MG1655, underscores the role of Fur and RyhB in optimizing iron and other nutrient acquisition and virulence in nutrient-limited host niches, offering insights into UPEC pathogenesis and potential targets for controlling infections [140]. Additionally, RyhB was also shown to play a key role in virulence of an avian pathogenic _E. coli_ strain by regulating the expression of genes involved in biofilm formation, adhering to brain microvascular endothelial cell line, and contributing to meningitis development in the murine model of infection [106]. Figure 2: **The role of small RNAs in stress responses. ** (**a**) The regulation of RyhB under iron-replete conditions: Under Fe-replete conditions, the ferric uptake regulator (Fur) binds to the iron (red circles), becomes active, and represses the transcription of _ryhB_ as well as iron uptake systems, preventing unnecessary iron acquisition. A few components of this figure were created using Biorender. (**b**) The regulation of RyhB under iron-limiting conditions. ** Under iron-limiting conditions, Fur becomes inactive, leading to the de-repression of _ryhB_ expression. RyhB facilitates iron-sparing responses by selectively repressing the translation of non-essential iron-dependent proteins while allowing the synthesis of essential Fe- and Fe-S cluster-containing proteins. In UPEC strain CFT073, RyhB also contributes to an anticipatory stress response, equipping the pathogen for survival in the nutrient-limited environment of the urinary tract. (**c**) The role of OxyS in oxidative stress response: OxyS is induced under oxidative stress conditions by the transcriptional regulator OxyR.