Function of the MksBEFG plasmid defense system in Corynebacterium glutamicum

• Applicant: Professor Dr. Marc Bramkamp
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545418655

Bacterial cells face constant threats from foreign DNA invasion, triggering an ongoing arms race against mobile genetic elements such as phages, transposons, and plasmids. While in recent years a plethora of anti-phage systems have been discovered, plasmid defense mechanisms remain less explored. Recently, homologs of the structural maintenance of chromosome (SMC) complexes have been implicated in plasmid copy number control and termed Wadjet systems (JetABCD/MksBEFG/EptABCD) that are present in various organisms. Comprising a MukBEF-like SMC complex acting as a DNA sensor and likely utilizing loop-extrusion on DNA, Wadjet complexes also feature a novel nuclease (JetD/MksG) responsible for plasmid DNA cleavage. We have identified and characterized the MksBEFG system in Corynebacterium glutamicum. Structural data revealed MksG as a topoisomerase VI homolog, cleaving DNA in a divalent cation-dependent manner. While similar systems from Escherichia coli and Pseudomonas aeruginosa have been analyzed recently studied structurally and biochemically, studying an endogenous system in C. glutamicum provides unique insights into cellular regulation. Preliminary work shows spatial localization of the corynebacterial system through polar interaction with the scaffold protein DivIVA. Additionally, MksBEFG discriminates between different endogenous plasmids in C. glutamicum, raising questions about target identification and activity regulation. Therefore, the proposed research focuses on two main goals: i) Identifying molecular interactions of the MksBEFG complex with plasmid DNA and ii) Investigating the regulation of the Mks complex through spatial organization and the identification of putative accessory factors. This work program aims to provide crucial insights into the functionality of the Wadjet/Mks plasmid defense system within the biotechnologically important C. glutamicum. Given the dependence on plasmid systems for genetic manipulation and expression in C. glutamicum, this research not only contributes to a comprehensive understanding of DNA identification and restriction by the Wadjet system but also holds practical implications for strain and plasmid generation in biotechnological applications.

• DFG Programme: Research Grants