## 4 Materials and Methods
### Quantitative Real-Time (qRT)-PCR  
To determine the target gene expression levels of two translational elongation factor (EF-Tu) genes (_dgeo_0646 and _dgeo_1869_) and a chemical modifier gene of streptomycin (_dgeo_1202_), specifically in the _dgeo_2840_ strain, we performed qRT-PCR analysis. We prepared samples at two growth phases with an OD\({}_{600}\) of 2.0 for the early exponential growth phase and 4.0 for the late exponential growth phase among the wild type, \(\Delta\)_dgeo_2840, and both SmR mutants of \(\Delta\)_dgeo_2840. The grown cells were harvested, washed with 0.9% NaCl solution, and resuspended. The cell wall was disrupted by phenol extraction solution, followed by DNA digestion by DNase treatment. Total RNA was extracted using a RiboEx extraction kit from GeneAll (Seoul, Korea) from the wild type and mutants grown to an OD\({}_{600}\) of 4. The extracted RNA was purified using a Qiagen RNeasy kit (Hilden, Germany). Using 1 \(\upmu\)g of RNA in 8 \(\upmu\)L of distilled water, cDNA was synthesized using the PrimeScript(tm) 1 st strand cDNA synthesis kit from TaKaRa (Kusatsu, Japan) with 6-mer random primers and the following conditions: 60 \({}^{\circ}\)C for 5 min, 4 \({}^{\circ}\)C for 3 min, 30 \({}^{\circ}\)C for 10 min, 42 \({}^{\circ}\)C for 60 min, and finally, 95 \({}^{\circ}\)C for 5 min. In the second step, 4 \(\upmu\)L of 5\(\times\) buffer, 4.5 \(\upmu\)L of RNase free water, 1 \(\upmu\)L of RTase, and 0.5 \(\upmu\)L of RNase inhibitor were added. The amount of cDNA was measured using a Nano-Drop spectrophotometer (DeNovix, Wilmington, DE, USA) and normalized. Next, qRT-PCR was performed using each target gene primer set and cDNA with TB Green(r) Premix Ex TaqMan(tm) from TaKaRa (Kusatsu, Japan) and a CFX96(tm) Optics Module from Bio-Rad (Hercules, CA, USA). Data were normalized using wild-type GAPDH expression as described previously [45]. All statistical analysis was performed using Prism(tm) ver. 6. The \(t\)-test was used to identify differences between samples. A \(p\)-value less than 0.05 was considered significant.  
The supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ijms23179764/s1](https://www.mdpi.com/article/10.3390/ijms23179764/s1)]([https://www.mdpi.com/article/10.3390/ijms23179764/s1](https://www.mdpi.com/article/10.3390/ijms23179764/s1)).  
Conceptualization, C.L. and S.-J.L.; methodology, C.L., Q.Y., E.S. and T.T.; validation, Q.Y., E.S. and T.T.; data curation, C.L., Q.Y. and E.S.; writing--original draft preparation, C.L. and S.-J.L.; writing--review and editing, Q.Y., E.S. and S.-J.L.; project administration, S.-J.L.; funding acquisition, S.-J.L. All authors have read and agreed to the published version of the manuscript.  
This study was supported by a grant from the National Research Foundation of Korea funded by the Korean government (MSICT) (2020R1F1A10700779 and 2022R1A2C1010233).  
Not applicable.  
The authors thank the NRF of Korea through the Ministry of Science and ICT of the Korean government for their research funding support. Q.Y. thanks Kyung Hee University for a foreign student scholarship.  
The authors declare no conflict of interest.