# Involvement of _E. coli_ 6S RNA in Oxidative Stress Response
## 2 Results
### Investigation of E. coli Strains with Complementation of the ssrS Gene  
Considering that _E. coli_ 68 RNA has been studied for years without any reported evidence for such an oxidative stress phenotype, we analyzed the phenotype of the MG1655 \(\Delta ssrS\) strain complemented with the wild-type \(ssrS\) gene expressed from a low-copy plasmid (p177_ssrS; see Section 4). The plasmid harbored the \(ssrS\) gene under the control of its native P1 promoter and the transcription terminator of the _E. coli_\(mpB\) gene (see Supplementary Data, Figures S3-S5, Table S1). The _E. coli_ MG1655 WT and \(\Delta ssrS\) strains were either transformed with plasmid p177_ssrS or the empty vector (p177_empty) resulting in complementation strains abbreviated as WT+\(S\), \(\Delta ssrS\)+\(S\), WT+\(0\) and \(\Delta ssrS\)+\(0\), respectively. Expression of 65 RNA in strains WT, WT+\(0\), WT+\(S\) and \(\Delta ssrS\)+\(S\), as well as the absence of 65 RNA in strains \(\Delta ssrS\) and \(\Delta ssrS\)+\(0\), was confirmed by Northern blot analysis (Figure S6). Stationary phase (24 h) levels of 6S RNA were comparable in strains WT,  
Figure 2: The lack of 68 RNA leads to reduced survival of _E. coli_ MG1655 cells in the presence of H\({}_{2}\)O\({}_{2}\). (**a**) Results of cell culture plating (serial dilutions indicated on the X-axis) after 30 min of incubation in the absence (left panel; LB control) or presence of 50 mM H\({}_{2}\)O\({}_{2}\) (central panel), 4 biological replicates in each case. A plating example after culture incubation with 50 mM H\({}_{2}\)O\({}_{2}\) is shown on the right. (**b**) Results of the zone of inhibition test for _E. coli_ MG1655 WT and \(\Delta ssrS\) bacteria grown in the presence of different H\({}_{2}\)O\({}_{2}\) concentrations (left panel, based on three biological replicates each); corresponding example agar plates are illustrated on the right: after streaking of cell culture dilution, a paper disk (5 mm diameter) soaked with 30% (_uv/uv_) H\({}_{2}\)O\({}_{2}\) was placed in the center (gray sphere) of the plate; the white area is the zone of no growth whose diameter was measured with a ruler after 24 h at 37 \({}^{\circ}\)C; 30% (_uv/uv_) H\({}_{2}\)O\({}_{2}\) corresponds to 9.79 M, 3.75% to 1.22 M.  
WT+0, WT+\(S\) and AssrS+\(S\), while 65 RNA levels in exponential (2 h) and transition (8 h) phase were somewhat lower in the WT and WT+0 than in the WT+\(S\) and AssrS+\(S\) strains. All strains showed essentially uniform growth behavior in standard rich (LB) medium (Figure S7). Comparative growth analysis in the presence of 5 mM H\({}_{2}\)O\({}_{2}\) confirmed the retarded outgrowth phenotype for the \(\Delta ssrS\) and \(\Delta ssrS\)+0 strains, whereas the \(\Delta ssrS\)+\(S\) complementation strain rescued this defect displaying growth behavior indistinguishable from that of the WT, WT+0 and WT+S strains (Figure 3). The same tendency of delay in growth of \(\Delta ssrS\) and \(\Delta ssrS\)+0 strains was observed for a number of individual experiments even in the presence of 2 mM H\({}_{2}\)O\({}_{2}\), although in the latter case the difference between strains was rather small (Figure S8). We also verified the stability of the \(\Delta ssrS\) phenotype in experiments where we started a new outgrowth in the presence of 5 mM H\({}_{2}\)O\({}_{2}\) from 24 h cultures grown under the same oxidative stress condition (Figure S9). The \(\Delta ssrS\) bacteria showed the outgrowth delay also in this second round of exponential growth, whereas the \(\Delta ssrS\)+\(S\) strain behaved as the WT. Thus, the observed phenotype is a functional feature of cells lacking 65 RNA and does not result from spontaneous selection of subpopulations resistant to H\({}_{2}\)O\({}_{2}\). Lack of 65 RNA Is Lethal for E. coli in the Presence of Elevated H\({}_{2}\)O\({}_{2}\) Concentrations  
In the experiments discussed so far, we adjusted fresh media to the respective H\({}_{2}\)O\({}_{2}\) concentration and then inoculated with an overnight culture grown in standard LB medium.