## 3 Results
### Stability of Rsm sRNA  
If most sRNAs indeed regulate translational efficiency, their turnover in varying environments deserves a closer examination. For this, we examined the stability and steady state level of Rsm sRNAs by quantitative real-time reverse transcription polymerase chain reactions (qRT-PCR). We used two different media: TSB/10 and undiluted TSB medium. To determine the in vivo stability of Rsm sRNAs, exponentially phase and late stationary phase growing bacteria were treated with rifampicin to block any further initiation of transcription. Samples were taken at 1, 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min and DNase-treated total RNA was used for qRT-PCR (Table 2, Figure S2). The half-life of each Rsm sRNA was calculated, revealing that these sRNAs are not equally stable. Overall, the half-lives of RsmY and RsmZ are much longer than those of RsmX-1 and RsmX-2 (Table 2, Figure S2).  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Medium**} & \multirow{2}{*}{**Phase**} & \multicolumn{6}{c}{**Half-Lives in WT (in Min)**} \\ \cline{3-6}  & & \(\mathbf{OD_{60 nm}}\) & **RsmX-1** & **RsmX-2** & **RsmY** & **RsmZ** \\ \hline \multirow{2}{*}{TSB} & Expo & 1.5 & \(12\pm 1.1\) & \(5\pm 0.6\) & \(8\pm 0.2\) & \(37\pm 2.6\) \\  & Stat & 4.5 & \(23\pm 5.4\) & \(16\pm 1.5\) & \(45\pm 5.2\) & \(53\pm 0.9\) \\ \hline \multirow{2}{*}{TSB/10} & Expo & 0.5 & \(7\pm 1.5\) & \(5\pm 1.6\) & \(8\pm 1.7\) & \(26\pm 3.6\) \\  & Stat & 0.9 & \(37\pm 4.4\) & \(40\pm 8.6\) & \(\geq\)60 & \(\geq\)90 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Stability of Rsm sRNAs. Half-lives of _P. brassicacarum_ NFM421 RsmX-1, RsmX-2, RsmY and RsmZ. Experiments were conducted in triplicates.  
Figure 5: The expression level of _rsmX-1_, _rsmX-2_, _rsmY_ and _rsmZ_ depends on nutrient availability. Activity of a _lacZ_ reporter fused to the promoter regions of _rsmX-1_, _rsmX-2_, _rsmY_ and _rsmZ_ were measured in the _P. brassicacarum_ wild-type strain and are reported as Miller units of \(\beta\)-galactosidase activity (\(\pm\) standard deviation). The cultures were grown for 24 h at 30 \({}^{\circ}\)C before being tested and the tables below each graph indicate the OD\({}_{600}\) of the cultures. Experiments were conducted in triplicates.  
The half-life of the four Rsm sRNAs increased during growth to reach their maxima during the stationary phase. For example, RsmX-1 and RsmX-2 stability increased \(\sim\)8-fold in TSB/10 in the stationary phase compared to the exponential phase. RsmY and RsmZ were observed to be highly stable with a half-life of at least 60 min (Table 2, Figure S2).  
Moreover, the choice of nutrients condition alters their stability; the four Rsm sRNAs were observed to be more stable in TSB/10 medium at stationary phase (e.g., half-life of RsmY and RsmZ is \(>\)60 min and \(\sim\)50 min in TSB/10 and TSB, respectively). A similar effect was observed for RsmX-1 and RsmX-2, whereas, they both exhibited half-lives of 30-34 min in TSB/10 medium, which were reduced in TSB medium to 23 and 16 min, respectively.  
In _P. brassicacarum_, we hypothesize a correlation between the number of GGA motifs and the stability of the four Rsm sRNAs transcripts conferred by RsmA/E. RsmZ, which showed the highest stability, possesses the highest number of GGA motifs (10 with 4-8 exposed); in contrast, RsmX-1 and RsmX-2 have the lowest number of GGA motifs (6 with 3-4 exposed) and were less stable. It should be noted that RsmY is moderately stable, although it only contains 7 GGA motifs (4-5 exposed) (Table 3).