Molecular mechanisms and dynamics of the segregation of plasmid pB171 from an enteropathogenic strain of Escherichia coli

The stable inheritance of bacterial low copy number plasmids is mediated by dedicated active segregation systems. The Escherichia coli B171 enteropathogenic strain specifies virulence factors that cause diarrhea and are encoded by the low copy number plasmid pB171. The plasmid contains two partition cassettes. The system under investigation is the par2 module, which consists of three elements: two genes, parA encoding an ATPase and parB encoding a centromere-binding protein, and two partition or centromere sites, parC1 and parC2, which harbour direct repeats that are recognised by the ParB protein. An initial and crucial event in plasmid segregation is the binding of the centromere-binding protein site-specifically to the centromere site to assemble a higher-order nucleoprotein complex structure.

In this work, in vivo experiments have shown that the parC2 centromere is fundamental for plasmid stability. ParB is a dimer in solution. The crystal structure of the C-terminal DNA-binding domain of ParB was solved at 1.93 Å resolution and revealed that the protein folds into a dimeric ribbon-helix-helix motif similar to that of the Arc/MetJ transcriptional repressors superfamily. The solution nuclear magnetic resonance showed that the ParB N-terminal domain is unstructured and highly dynamic. The secondary structure prediction showed that the central region of ParB N-terminal domain forms a -helix structure. ParB binds parC1 and parC2 with high affinity and cooperativity. Nuclear magnetic resonance experiments identified two ParB regions that recognise DNA: the C-terminus and the tip of the N-terminal tail. Replacement of residues in the tip of ParB N-terminal tail provided insights into the role played by these residues in plasmid stability. Interestingly, chemical cross-linking coupled with mass spectrometry revealed that the centromere site and ATP enhance ParA-ParB complex formation and showed that the tip of ParB N-terminal tail interacts with the partner ParA. Based on these findings, a model of nucleoprotein complex assembly is proposed.