Binding analysis of the response regulator NarL protein to the promoter of the O6-methylguanine-DNA methyltransferase (ogt) gene in Salmonella Typhimurium.

BACKGROUND: Salmonella Typhimurium (STM) is a gram-negative bacterium that causes severe gastrointestinal disorders in both animals and humans. The regulation of DNA repair genes is critical for maintaining genomic stability of the bacteria. O6-methylguanine DNA methyltransferase (Ogt), plays a vital role in repairing alkylated DNA in STM; however, the transcriptional regulation of ogt gene remains poorly characterized. Furthermore, NarL is a transcriptional regulator, involved in the pathogenesis of STM under anaerobic condition. Therefore, this study investigated the interaction between NarL protein and the promoter region of the ogt gene. METHODS: In this study, narl gene was cloned in pET32a vector and NarL protein was expressed in Escherichia coli BL21 (DE3). Subsequently, the ogt gene promoter (pogt) was selected, amplified, cloned and its activity was evaluated. Electrophoretic mobility shift assay (EMSA), isothermal titration calorimetry (ITC), molecular docking were employed to elucidate the interaction between NarL protein and ogt promoter. Furthermore, the regulatory role of NarL in ogt gene expression was validated in vivo using RT-qPCR and β-galactosidase assay. RESULTS: This study resulted that NarL protein interacts specifically with the ogt promoter, as confirmed by EMSA and ITC, with ΔG of - 9.42 kcal mol⁻¹. Furthermore, RT-qPCR and β-galactosidase assays demonstrated that deletion of narl significantly (P ≤ 0.01) decreased ogt transcript levels and promoter activity than wild Salmonella Typhimurium, whereas exogenous supplementation of recombinant NarL protein restored the expression. These findings suggest that NarL plays a potential regulatory role in ogt gene expression in response to environmental signals. CONCLUSION: These findings highlight an interaction between NarL protein and the promoter region of ogt gene in Salmonella Typhimurium, linking nitrogen metabolism with the DNA repair pathway in STM, which may contribute to the bacterial survival under nitrosative stress.