The structure of plasmid partitioning system and its functional components

Bacterial plasmids carry essential traits for their host, such as resistance genes, genes for bacterial infection and genes encoding for a conjugation apparatus. To guarantee the even distribution of plasmids apon cell division and a long term stability for many generations of a bacterial population stabilization systems have evolved. The aim of this project was the characterization of the stabilization system of the broad-host-range plasmid RP4. It consists of two divergent operons, encoding for five proteins. The first operon contains a resolvase (ParA) which has been shown to resolve plasmid multimeres. The second operon encodes a bacterial killing system, consisting of a toxin - antitoxin pair of proteins (ParE and ParD). This killing system promotes the survival of plasmid carrying cells by inhibiting the growth of cells, which have lost the plasmid. Within this project we have established the expression systems of the five proteins of this stabilization system in our laboratory. Three of these proteins could be produced in sufficient purity and quantity for biophysical studies and crystallization trials. The experiments were focussed on the resolvase (ParA) and the antitoxin of the killing- system (ParD). We could show, that the resolvase is a dimer in solution and binds to its specific DNA-site as a dimer. Microcalorimetric and biochemical experiments have shown, that the catalytic and the DNA-binding function reside in structurally independent domains. The antitoxin ParD is a dimer in solution, but it binds to its specific DNA-site as a tetramer. Using CD- spectroskopic und microcalorimetric methods we showed, that ParD is very thermostable and has a high refolding capacity (Oberer et.al., 1999). These features were used for improving the purification procedure. Because of the lack of diffraction quality crystals of ParD the structure determination with NMR-spectroscopic methods was initiated. The production of double labeled ParD protein (15N and 13C) enabled us to conduct NMR-experiments leading to the secondary structure assignment and 3D-structure solution of the antitoxin. The ParD-ParE killing system is of commercial interest because of its potential use as a selection marker in cloning and expression systems. Traditionally resistance genes were used as selection markers, contributing to the distribution of resistant bacterial pathogens. The development of alternative selection systems based on a killing system has been put into reality with two homologous systems so far.