## 2 Results
### Detection of IS Transposition  
We discovered an SmR mutant as a novel selectable biomarker for IS element transposition that was isolated from the _Adego_2840_ strain (Figure 3). The selected mutant _SrsmG_ showed a longer PCR fragment with _dgeo_2335_ only (Figure 3, in lane 9 of _dgeo_2335_). The IS_Dge6_ element was integrated into the _rsm_G gene at the 58 th nucleotide (Figure 4A). Further, we determined that the element is a member of the IS5 family, containing a DDE-motif transposase that is 335 amino acids in length, as well as a conserved terminal inverted repeat (TIR) sequence of 'AGACCGCTGCAAAcaAGGGGC', and a direct repeat (DR) sequence of 'TCA'. In our previous study, we showed that IS_Dge6_ actively transposed into and disrupted phytoGene desaturase, an enzyme required for carotenoid biosynthesis, in the _Algoe_2840 mutant strain [27]. The other SmR mutations revealed reddish colors in culture, indicating functional carotenoid synthesis and thus the absence of IS integration. Based on these findings, the _Algoe_2840 strain-specific IS5 family element IS_Dge6_ transposed into _rsmG_ and was detected through SmR selection by oxidative stress.  
Figure 1: The selection of streptomycin-resistant (SmR) and -dependent (SmD) clones by chronic oxidative stress of wild-type, _Algoe_0257_, _Algoe_0281_, and _Algoe_2840 parent strains. (**A**) Growth comparison on TGY medium containing 50 ug/mL streptomycin among parent (P) and mutant (S1-S4 and _rsmG_) strains. The red box indicates an Sm-dependent phenotype. (**B**) Minimum inhibitory concentration (MIC) values using the disc diffusion method for all selected mutants within the range of 10 to 250 ug/mL streptomycin for parent strains and 10,000 to 100,000 ug/mL streptomycin for Sm-resistant mutant strains on TGY medium.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & **Strains** & **Phenotype** & **MIC** & _dgoo_1873_ & _dgoo_2335_ & _dgoo_0776_ & _dgoo_0847_ & _dgoo_0915_ \\  & & **(log/mL)** & **(typal)** & **(rsmG)** & **(rm/k)** & **(rm/k)** & **(int/k)** & **(unso)** \\ \hline  & S1/S3/S4 & SmR & 50,000 & 272 nd C \(>\) A & - & - & - & S3: 1371 st C \(>\) A \\ WT & S2 & SmR & 50,000 & 272 nd C \(<\) A & - & - & - & (Arg \(>\) Arg) \\  & S1 & SmR & 50,000 & 272 nd C \(<\) A & - & - & - & - \\  & S2 & SmR & 50,000 & 2791D & - & 84 th deletion & - & - \\ \(\Delta\)dgoo_0257 & S1 & SmR & 20,000 & 282 nd A \(>\) G & 679 th & - & - & - \\  & & (K888) & deletionframeshift & & & 1371 st C \(>\) A \\  & & & & & & (Arg \(>\) Arg) \\ \(\Delta\)dgoo_0281 & S1 & SmR & 50,000 & 272 nd C \(>\) A & - & 102 nd G \(>\) C (Arg \(>\) Arg) & 627 th deletion & 1462 nd G \(>\) C \\  & & & & & & & (Gly \(>\) Arg) \\  & & & & & & 1491 st G \(>\) A \\  & S1 & SmD & 25,000 & 272 nd C \(>\) T & - & - & - & - \\  & & & (W91L) & - & - & - & - \\ \(\Delta\)dgoo_2840 & S2 & SmD & 25,000 & 272 nd C \(>\) T & - & - & - \\  & & & & (W91L) & - & - & - & - \\  & & & & & & & \\  & & & & & & S8 th ISync6 & - & - & - \\  & & & & & & transposition & - & - \\ \hline \hline \end{tabular}
\end{table}
Table 1: Detection of mutations in four streptomycin-resistant related genes and an antibiotic uptake gene, their streptomycin MIC values, and their phenotypes.  
Figure 3: PCR detection of insertion sequence (IS) transposition in four Sm resistance-related genes in wild-type (WT), \(\Delta\)_dgeo_0257, \(\Delta\)_dgeo_0281, and \(\Delta\)_dgeo_2840 mutants. Lanes: M, size marker; 1, WT; 2–5, WT S1–S4; 6, \(\Delta\)_dgeo_0257_S1; 7–9, \(\Delta\)_dgeo_2840_S1, S2, and \(\mathrm{\it{SrsmG}}\); and 10, \(\Delta\)_dgeo_0281_S1. The arrow indicates a larger PCR product caused by IS integration in _dgeo_2335_ (_rsmG_).  
Figure 2: Comparison of growth profiles among \(\Delta\)_dgeo_2840 parent and three Sm-resistant mutant strains (**A**) in antibiotic-free TGY medium and the SmD phenotype of \(\Delta\)_dgeo_2840_S1 and S2. The arrow indicates the point at which 50 \(\mathrm{\upmu g/mL}\) streptomycin was added (**B**).