# Involvement of _E. coli_ 6S RNA in Oxidative Stress Response
## 2 Results
### Screening for Oxidative Stress Response Genes Affected by 65 RNA  
To identify possible 65 RNA targets that may contribute to reduced viability of the \(\Delta ssrS\) strain, we analyzed the expression levels of a set of genes known to be involved in oxidative stress response by qRT-PCR (Table 1). As a first step, we compared the WT and \(\Delta ssrS\) strain under standard growth conditions. All tested genes showed no significant changes in expression levels upon lack of 65 RNA (Figure 6a). The only difference was a trend toward weak upregulation of genes _oxyS_, _tpx_, _osmC_, _btuE_ and _guAD_ in the \(\Delta ssrS\) strain. Next, we analyzed expression levels of these genes in the exponential phase of cell growth after exposure to 20 mM H\({}_{2}\)O\({}_{2}\) (setup as in Figure 4a). To minimize RNA degradation at 20 mM H\({}_{2}\)O\({}_{2}\) and to preempt progressing lethality of the \(\Delta ssrS\) strain, we withdrew cell aliquots after 10 min of H\({}_{2}\)O\({}_{2}\) treatment. At this time point, WT and \(\Delta ssrS\) bacteria showed similar numbers of viable colonies (Figure 5a). Total RNAs isolated from both cell cultures before the addition of H\({}_{2}\)O\({}_{2}\) were used as negative controls. Figure 5: _E. coli_ cells with deletion of the \(ssrS\) gene show decreased viability in the exponential phase in the presence of 20 mM H\({}_{2}\)O\({}_{2}\) compared to cells expressing 65 RNA. Viability of _E. coli_ cells was monitored by plating of serially diluted culture aliquots withdrawn at different time points along the growth curves shown in Figure 4a. Representative individual experiments of different sets of strains grown in parallel: (**a**) WT and \(\Delta ssrS\) only; (**b**) WT and \(\Delta ssrS\) in comparison to complementation strain \(\Delta ssrS\)+\(S\); (**c**) growth of the four complementation strains. The strongest activation upon H\({}_{2}\)O\({}_{2}\) treatment was demonstrated for the _oxyS_ and _katG_ genes (\(\sim\)10\({}^{2}\)-fold), however, no difference between the WT and _AsnrS_ strain was observed (Figure S14). Moderate upregulation under oxidative stress was evident for genes _crp_, _osmC_, _yuaA_, _uspF_ and _uspG_. Other genes demonstrated either essentially no sensitivity to H\({}_{2}\)O\({}_{2}\) treatment (e.g., _katE_, _btUe_, _uspE_, _gapA_) or even downregulation of mRNA levels, e.g., _oxyR_, that is in line with previous observations [35]. However, mRNA levels of four genes among the total setup were revealed to be lower in the _AsnrS_ versus WT strain under H\({}_{2}\)O\({}_{2}\) stress (Figure 6b). Namely, expression levels of _soxS_ (activator of superoxide response), _alpC_ (alkyl hydroperoxide reductase C), _sodA_ (superoxide dismutase) and _tpx_ (thiol peroxidase) mRNAs were \(\sim\)2-3-fold decreased in the absence of 6S RNA. Figure 6: Relative expression levels (qRT-PCR) of selected mRNAs and non-coding RNA OxyS in \(E\). _coli_ WT and _AsnrS_ bacteria. (**a**) Standard conditions (in the absence of H\({}_{2}\)O\({}_{2}\)). No significant changes in expression levels were detected for any of the analyzed genes (standard t-test). (**b**) Genes with dysregulated mRNA levels in _AsnrS_ bacteria under H\({}_{2}\)O\({}_{2}\) stress. Total RNA samples were isolated either before (control, light grey bars) or 10 min after treatment with 20 mM H\({}_{2}\)O\({}_{2}\) (dark grey bars). Statistical analysis was performed by the two-way ANOVA test, _p_-values: ***-\(\sim\)0.001; ***-\(\sim\)0.01; ***-\(\sim\)0.05; n.s.—not significant. Relative amounts of mRNAs were normalized to 16S rRNA and mRNA levels of the WT strain in the absence of H\({}_{2}\)O\({}_{2}\) were set to “1”. Comparative Proteomic Analysis of E.coli 6S RNA Knockout and WT Cells under Oxidative Stress Conditions  
As previously performed for _B.