## 3 Discussion
In this study, we selected SmR and SmD mutants derived in response to chronic oxidative stress using a relatively low concentration of hydrogen peroxide (20 mM) from the wild type, two Dps-disrupted strains, and a LysR family regulator-disrupted strain as parent cells of _D. geothermal_s (Table 1). The _rpsL_ point mutation P91H resulted in a MIC value of almost 50,000 \(\upmu\)g/mL, and when point and frameshift mutations were accumulated with mutations in _mthA_, the value increased to more than 50,000 \(\upmu\)g/mL in the \(\Delta\)_dgeo_0281_-originated SmR mutant. A Dps gene-disrupted mutant revealed slightly different MIC values of 20,000 \(\upmu\)g/mL because of different point mutations and their accumulation in the case of the \(\Delta\)_dgeo_0257_ SmR mutant. The point mutation K88E in the \(\Delta\)_dgeo_0257_ SmR mutant was related to the streptomycin interaction site in _rpsL_ of _E. coli_ (Figures 5 and S2B) [43]. The LysR family regulator-disrupted mutant included P91L in RpsL and had two SmD mutants. These P91L mutations in RpsL were already identified in _Salmonella typhimurium_[41]. In the absence of streptomycin, cell growth was stalled, whereas cell growth restarted following the addition of streptomycin at a concentration above 50 \(\upmu\)g/mL. This growth pattern of SmD was reported during early streptomycin commercial usage at the end of the 1940s [7, 8]. Both mutants exhibited a similar streptomycin MIC level of 50,000 \(\upmu\)g/mL. Evaluation of cell growth measurements with streptomycin showed that \(\Delta\)_dgeo_2840 S2 grew better than the \(\Delta\)_dgeo_2840_ S1 mutant. The complement strain C2840 exhibits different streptomycin-resistant MIC values after chronic hydrogen peroxide treatment with 20 mM and has mutations in K43, K88, and P91 on RpsL compared to parent strains (Figure S5). The complement strain shows a recovered growth pattern and hydrogen peroxide sensitivity (Figures S6 and S7). Based on our findings, we hypothesize that streptomycin interacts with the P91L residue and causes translation to restart and resume protein production, and consequently, cells are released from growth arrest. Other streptomycin-interacting residues, such as K43 and K88 of the streptomycin interaction sites in RpsL, thereby overcome translational inhibition and effectively restart protein synthesis. In the case of the thermophilic bacterium _T. thermophilus_, the K43 mutation leads to an SmR phenotype in the range of 500-2000 \(\upmu\)g/mL, and several P91 (which corresponds to the amino acid sequence in Figure S2A) mutations revealed an SmD phenotype with substitutions to arginine, glutamate, methionine, and tryptophan [21]. However, the P91A, P91C, and P91G mutants reveal an SmR phenotype. Thus, the charge and hydrophobic properties of the side chain of the amino acid residues in RpsL affect the growth phenotype of the mutant strains. Additionally, gene disruption by the transposition of an IS element affects streptomycin resistance in bacteria. In _E. coli_, high glucose concentrations affect IS_1_ transposition into _rpsL_[44], and in _T. thermophilus_ HB8, the spontaneous SmR strain has IS_th7_ integrated into _rsmG_[22], resulting in streptomycin resistance. IS_th7_ is a member of the IS427 group within the IS5 family. IS_th7_ produces a variable direct repeat sequence of 9 bp and has a transposase containing a DDE motif and a second ORF, termed OrfA [22]. In previous experiments on IS transposition, we discovered that the transposition of IS_2 ge6_, IS_pge7_, and IS_pge11_ into _dgeo_0524_ occurred in response to oxidative stress, which encodes a phytoene desatkarae for carotenoid biosynthesis, in the _dgeo_2840_ mutant, _Adgeo_0257_ mutant, and wild type, respectively [26; 27]. In this study, we also identified IS transposition into _rsmG_ in the _dgeo_2840_ mutant strain of _D. geothermals_. IS_pge6_ of the IS5 family integrated at the 58 th nucleotide of the _rsmG_ gene, resulting in an SmR phenotype with a MIC value of more than 10,000 \(\upmu\)g/mL streptomycin (Figure 1B and Table 1).