# Involvement of _E. coli_ 6S RNA in Oxidative Stress Response
## 2 Results
### Investigation of E. coli Strains with Complementation of the ssrS Gene
To further characterize the discovered phenotype of 65 RNA-deficient cells, we tested several oxidative stress regimens. The phenotype was exacerbated when 20 mM H\({}_{2}\)O\({}_{2}\) (f.c.) was added to exponentially growing (OD\({}_{600}\)\(\sim\) 0.5) _E. coli_ cell cultures in flasks, resulting in the lethality of \(\Delta ssrS\) and \(\Delta ssrS\)+0 bacteria (Figure 4a). When adding only 10 mM H\({}_{2}\)O\({}_{2}\), the observed effect vanished (Figure 4b). At intermediate H\({}_{2}\)O\({}_{2}\) concentrations (15 mM and 17.5 mM), the growth delay of the knockout strains correspondingly increased (Figure S10). We were also able to reproduce this effect in the 96-well plate format. Considering the fact that OD\({}_{600}\) values are not comparable in flask and plate format we slightly modified the protocol. We first grew _E. coli_ pre-cultures in flasks in LB medium up to OD\({}_{600}\)\(\sim\) 0.5 (exponential phase) in the absence of H\({}_{2}\)O\({}_{2}\), and then diluted this culture 1:5 in media containing different amounts of H\({}_{2}\)O\({}_{2}\); the resulting suspensions were transferred to 96-well plates for growth monitoring. In this type of experiment 20 mM H\({}_{2}\)O\({}_{2}\) was very toxic to all of the strains, but 10 mM H\({}_{2}\)O\({}_{2}\) was only lethal for \(\Delta ssrS\) and \(\Delta ssrS\)+0 bacteria (Figures 4c and S11). The latter two strains were also considerably delayed in cell growth in the presence of 7.5 mM H\({}_{2}\)O\({}_{2}\) (Figure 4d). Moreover, survival of \(\Delta ssrS\)+0 bacteria was reduced in comparison to \(\Delta ssrS\) bacteria, which may be attributable to the consumption of resources for maintaining the plasmid. Higher H\({}_{2}\)O\({}_{2}\) sensitivity of cell  
Figure 3: Complementation of the 65 RNA knockout strain by a plasmid-borne \(ssrS\) gene rescues the growth defect. (**a**) Growth curves of _E. coli_ strains in the presence of 5 mM H\({}_{2}\)O\({}_{2}\) either during cultivation in flasks with manual monitoring of optical density (OD\({}_{600}\)) or (**b**) in 96-well plates using an automated scanning reader; based on 3 biological replicates in each type of experiment. cultures grown in 96-well plates is likely due to the specific experimental setup, probably resulting in slower H\({}_{2}\)O\({}_{2}\) reduction owing to less thorough aeration and stirring of media in comparison to culturing in flasks. In some independent experiments, we also observed a clear trend toward faster growth of the \(\Delta ssrS\)+\(S\) and WT+\(S\) strains relative to WT and WT+\(0\) under H\({}_{2}\)O\({}_{2}\) stress conditions (Figure S11). This suggests that elevated 6S RNA levels during exponential growth may confer enhanced protection against damage by H\({}_{2}\)O\({}_{2}\) stress. However, in most cases, this effect was rather small (Figure 4c) and not clearly reproducible in all performed experiments. Reduced viability of 6S RNA-deficient cells in the presence of 20 mM H\({}_{2}\)O\({}_{2}\) was also demonstrated by plating assays (Figure 5). A relative decrease in the number of \(\Delta ssrS\) colonies in comparison to the WT became evident 30 min after H\({}_{2}\)O\({}_{2}\) addition. After 1 h of H\({}_{2}\)O\({}_{2}\) treatment, where WT cells also showed reduced viability, \(\Delta ssrS\) bacteria gave rise to only very few colonies. After 48 h, WT cells had recovered from oxidative stress, whereas the \(\Delta ssrS\) culture was devoid of any viable cells (Figure 5a). In contrast, complementation strain \(\Delta ssrS\)+\(S\) fully restored the WT phenotype 30 min and 3 h post-stress induction (Figure 5b), whereas strain \(\Delta ssrS\)+\(0\) yielded even fewer colonies than \(\Delta ssrS\) bacteria 3 h post-induction (Figure 5b,c). To exclude any influence of unnoticed genome alterations in our previously constructed \(\Delta ssrS\) strain, we also tested three other well-described _E. coli_ WT and correspond  
Figure 4: Lack of 6S RNA leads to lethality of _E. coli_ in the presence of high concentrations of H\({}_{2}\)O\({}_{2}\).