# Involvement of _E. coli_ 6S RNA in Oxidative Stress Response
## 3 Discussion

Table 1: Stress response genes selected for qRT-PCR screening. it is a direct consequence of the lack of 6S RNA expression. This is the first reported lethal phenotype of 6S RNA-deficient bacteria in general. So far, phenotypes of 6S RNA knockouts were primarily observed under extreme alkaline or acidic conditions: _E. coli_[23], _B. subtilis_[19; 27], _Salmonella_[30]; salt stress: _R. sphearoides_[28], _B. subtilis_[27]; reduced viability upon exposure to nutrient starvation: _E. coli_[21], _B. subtilis_[19; 25; 29]; or reduced bacterial pathogenicity [31; 32]. Notably, the oxidative stress response is one of the most important defense mechanisms, especially in _E. coli_ and related _Enterobacterates_, including pathogenic species [50]. There are a lot of reasons for the endogenous formation of reactive oxygen species (ROS), but a number of oxidative stress factors also originate from the environment [51]. Moreover, the accumulation of ROS either for dietary reasons or due to antibiotic therapy widely impacts human microbiota in the gut [52]. The general defense of _E. coli_ against damage by oxidative stress is well-studied, including the key regulators OxyR and SoxRS, the alternative sigma factor RpoS and many other proteins that are conserved among Proteobacteria [36]. OxyR is a transcription factor that is activated through oxidation by H\({}_{2}\)O\({}_{2}\), which triggers the formation of an intramolecular disulfide bond [53]. Genes that are activated by oxidized OxyR include _katG_ (catalase-peroxidase), _dps_ (involved in DNA damage repair & iron storage), the _alpCF_ operon (alkyl hydroperoxide reductase, [AhpC\({}_{10}\)[AhpF]\({}_{2}\)) and _oxyS_ (see below) [54; 55; 56; 57]. SoxRS stands for a two-stage regulation system, where SoxR induces expression of SoxS that activates the expression of 20 or more genes. Overall, transcription factors OxyR, SoxR and SoxS were reported to regulate the transcription of 68 genes in 51 transcription units in _E. coli_ MG1655 cells [55]. Activation of these stress response regulons leads to increased expression of different H\({}_{2}\)O\({}_{2}\)-degrading enzymes, among them the major catalases KatG and KatE as well as alkyl hydroperoxide reductase AhpCF. Recent studies discovered some novel protein players as well, for example, the transcriptional regulator LsrR that directly inhibits _alpCF_ and _katG_ promoters [58]. NcRNAs also contribute to the oxidative stress response in bacteria [59]. The first described ncRNA regulator, MicF, for long considered to have only _ompF_ mRNA as a target, also affects the regulatory network involving the leucine responsive protein Lrp [60]. The most famous ncRNA in this context is OxyS--a H\({}_{2}\)O\({}_{2}\)-inducible (via OxyR) ncRNA that represses expression of transcription factors, such as NusG, FluLA and RpoS, which entails pleiotropic alterations in the expression of many other genes [59]. Similar to our 6S RNA knockout, deletion of OxyS in _E. coli_ was also shown to cause lethality after growth for 20 min at 5 mM H\({}_{2}\)O\({}_{2}\)[61]. Notably, OxyS is highly activated upon H\({}_{2}\)O\({}_{2}\) stress but is not transcribed under normal conditions [38]. By contrast, 6S RNA is constitutively expressed, with the highest levels in stationary phase, and is not activated in response to H\({}_{2}\)O\({}_{2}\) treatment, as shown in the present study (Figure S13). Nevertheless, despite being a global regulator of the transcription machinery, 6S RNA may well impact on transcription of specific genes involved in H\({}_{2}\)O\({}_{2}\) degradation. To explore this possibility, we analyzed the expression levels of 21 known players in the oxidative stress response by qRT-PCR in the \(\Delta\)_ssrS_ versus WT strain. While no substantial differences between the two strains were observed under standard growth conditions, we indeed saw a \(\sim\)2 to 3-fold downregulation of _soxS_, _alpC_, _sodA_ and _tpx_ mRNA levels upon treatment with H\({}_{2}\)O\({}_{2}\).