Structural and dynamic investigations on the ccd system

Low-copy-number plasmids often possess poison-antidote systems, which guarantee preferential growth of plasmid-canying cells in a bacterial population by killing newbom bacteria that have not inherited a plasmid at cell division. In the proposed project we plan to carry out a structural and dynamic investigation of the components encoded in the ccd operon of the F sex factor plasmid post-segregational killing system. This comprises CcdB, a toxin targeting the essential DNA gyrase of Escherichia coli and CcdA, the unstable antidote that interacts with CcdB and neutralizes its toxicity. The scientific aims of the project are the NMR solution structure determination of CcdA (wildtype and R70K mutant) and CcdB as well as dynamical investigations using N-15 relaxation measurements. The reversibility of protein unfolding of CcdA at higher temperatures will be studied and folding intennediates structurally characterized. In addition we will attempt to characterize the CcdA-CcdB and the CcdA-(CcdB)-DNA complex by NMR either through complete structure determinations or chemical shift mapping. The structural and dynamical characterization of the Ccd killing module of the F plasmid will provide a deeper understanding of how plasmid stabilization and maintenance in bacteria is achieved and about differences between the multitude of known plasmid toxin-antitoxin modules. Apart from these fundamental biological questions, killing systems play an increasing role in biotechnology due to their use for stabilizing autonomously replicating vectors employed in recombinant bacteria. Ei addition, the solution structure and dynamical information about CcdB might help in the discovery and analysis of natural molecules with anti-gyrase activities such as CcdB or microcin B17, which hold promise for the design of new antibiotics.