Regulation of the expression of nucS, a key component of mismatch repair system in mycobacteria

Abstract

Alterations in the expression of the mismatch repair (MMR) system can lead to transient hypermutation, accelerating the emergence of adaptive mutations under stress conditions, such as antibiotic pressure. While most bacteria and eukaryotes rely on the MutS and MutL protein families for MMR, many archaeal and actinobacterial species, including the major human pathogen Mycobacterium tuberculosis, lack these components. Instead, they utilize NucS, a structurally and evolutionarily distinct enzyme driving a non-canonical MMR pathway. Given the role of MMR in mutation control, understanding how nucS expression is regulated could be essential for uncovering the molecular basis of antibiotic resistance development in mycobacteria. In this study, we defined the promoter region and transcription start site of the nucS gene in Mycobacterium smegmatis. We found that nucS expression is growth phase-dependent in both M. smegmatis and M. tuberculosis, significantly decreasing during the stationary phase. This downregulation mirrors that of canonical MMR components, aligning with replication activity. Additionally, we present evidence that the alternative sigma factor {sigma}B may negatively regulate nucS expression during the stationary phase. We also identified candidate compounds capable of modulating nucS expression, underscoring its responsiveness to environmental cues. These findings deepen our understanding of mycobacterial stress adaptation and provide a groundwork for further investigation into the molecular mechanisms underlying antibiotic resistance. Strikingly, our work reveals a case of double convergent evolution: both canonical (MutS/MutL) and non-canonical (NucS) pathways have independently evolved not only the same DNA repair function, but also similar regulatory frameworks for genome integrity preservation under stress conditions. IMPORTANCEBacteria like Mycobacterium tuberculosis, which causes tuberculosis, develop antibiotic resistance through mutations. Most organisms use a MutS/MutL-based system called mismatch repair to correct DNA replication errors and prevent harmful mutations. However, some bacteria, including mycobacteria, rely on a unique repair protein called NucS. This study explores how the gene encoding NucS is regulated in mycobacteria. We found that less NucS is produced when bacteria stop growing, which mirrors what happens with other DNA repair systems. We also discovered that a regulatory protein called SigB may help reduce its production during this phase. In addition, we identified chemical compounds that influence how much NucS bacteria produce, showing that its levels respond to environmental conditions. Importantly, the study reveals that different DNA repair systems--despite being unrelated--have evolved similar ways to protect bacteria under stress. These findings help scientists understand how mycobacteria adapt and could aid in fighting antibiotic resistance.