Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2.

Abstract

Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon. Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids. The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer. Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively. Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC. For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255. The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218. Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC. This IncC-KorB contact differs in location from the ParA-ParB/SopA-SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant.