Examining Roles of Residues of Plasmid TP228 Partitioning Proteins ParF and ParG in Interaction through Crosslinking of Mutant Cysteines

Stable inheritance is vital for the survivability of plasmids, especially those that are low copy number. Low copy plasmids that utilise a partitioning system show greater genetic stability. Type I partitioning systems, also called ParABS systems, consist of three core components: ParA, the ATPase, ParB, the plasmid binding protein and parS, ParB’s cis-binding site. The ParFGH system of the TP228 plasmid, found natively in Salmonella enterica serovar Newport, is one such system. It utilises a ParF meshwork that transports ParG bound plasmids to the midpoints of daughter cells during cell division. While the mechanism of the system is well understood, the interaction interface between the ParF and ParG proteins is not well characterised and presents a potential target for therapeutic treatment by preventing successful interaction of these two proteins in bacteria. This study focussed on mutations of the ParF and ParG proteins to replace residues of interest with cysteines. Constructed plasmids encoding these mutants were transformed into BL21(DE3) Escherichia coli. These proteins were overproduced and purified. A series of reactions were constructed containing combinations of either wild-type or mutated protein and cofactors before being treated with a BMOE crosslinking agent to elucidate on the physical interactions occurring between residues of interest in the reactions. These methods were successful in generating proteins but issues in stability and purity, particularly in ParF proteins, prevented visualisation of data on SDS-gels following crosslinking. Little to no appreciable conclusion regarding the interaction site of ParF and ParG was possible. This study did provide solutions to failings in the methods used to purify these proteins and presented some potential solutions to improve purification and storage of ParF. It was also possible to generate experimental improvements that would clarify data gained in similar studies on the interaction of this partitioning system.