Molecular analysis of the pRA2 partitioning region: ParB autoregulates parAB transcription and forms a nucleoprotein complex with the plasmid partition site, parS

Abstract

The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes. The pRA2 par region was effective in stabilizing both pRA2 and F mini-replicons. Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC. Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required. Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60-fold and that ParA had little effect on transcriptional activity. Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon. Based on this information, putative -10 and -35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity. The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system. Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized. Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography. Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region. DNase I footprinting revealed that ParB not only binds to the conserved sequence 5′-TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS. It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS.