Molecular interactions underpinning the segregation of TP228 multidrug resistance plasmid in Escherichia coli

Accurate segregation of newly replicated plasmids is essential for genetic stability throughout generations. The conjugative plasmid TP228, identified in Salmonella newport, confers resistance to several antibiotics and it is segregated during cell division thanks to its self-encoded parFGH partition system. This is composed of two trans-acting elements, ParF and ParG, and a centromeric region, parH. Interactions between the three components are essential for plasmid maintenance and are the main focus of this work. ParG is a dimeric protein composed of a folded domain and two unstructured tails. Alanine-scanning mutagenesis of the ParG tail has highlighted a number of residues, whose change impairs plasmid partitioning in vivo. These flexible regions were shown to play a fundamental role in binding to the partner protein ParF as well as conferring specificity to the interaction with the DNA. A combination of biochemical and biophysical techniques have been used to address which regions in the ParG tails are essential for the interaction with ParF and which play different roles. Surface plasmon resonance, together with microscale thermophoresis allowed quantification of the change in binding when residues are replaced in ParG. These results also allowed identification of positions in the tail that are supposedly required for binding to the DNA and structuring the tail at the initial stage of segrosome formation. Circular dichroism and electrophoretic mobility shift assays showed potential interaction of the amino acids in the middle of the tail with the site-specific DNA. On the other hand, cross-linking between ParF and ParG, in the absence and presence of parH, coupled to mass-spectrometry revealed a more complicated picture of the interaction among the segrosome components. Finally, a tandem affinity purification protocol gave new insights into the interaction network of the ParFGH system. Based on these findings, a more detailed mechanism of segrosome assembly and plasmid segregation is proposed