The hierarchy of KorB binding at its 12 binding sites on the broad-host-range plasmid RK2 and modulation of this binding by IncC1 protein.

IncC and KorB proteins of broad-host-range plasmid RK2 are members of the ParA-ParB families of proteins needed for stable partitioning of bacterial chromosomes and plasmids. KorB also functions as a global regulator of expression of RK2 genes. It recognises and binds to a palindromic operator, O(B), found 12 times on RK2 DNA (O(B)1-O(B)12). We performed detailed studies on the binding of KorB to the 12 operators and showed that they fall into three groups (A, B, C) based on the binding strength of KorB. The highest affinity site is O(B)10, which occurs in the promoter transcribing genes for replication, trfAp. Purified IncC1 potentiated KorB binding to all O(B) sites except O(B)3, a site involved in partitioning. Using O(B)10 as a test system, we showed that IncC1 increases the stability of the KorB-DNA complex. The 5 bp sequences flanking the 13mer O(B) site were found to affect KorB binding and IncC1 potentiation activity. Study of hybrid operators indicated that flanking sequences on one side only were sufficient to specify the difference between O(B)10 and O(B)3. Replacement of adenine by guanine at positions -8 and -10 from the O(B)10 centre of symmetry was needed to convert it from the highest-affinity group (A) to the medium-affinity group (B) on the basis of KorB binding. These changes also eliminated potentiation by IncC1. The -8 and -10 positions from the centre of O(B)3 symmetry are occupied by guanines and this may provide part of the specificity of IncC1 behaviour on KorB binding. Studies on a series of synthetic operators suggested that KorB contacts O(B) flanking sequences, and that IncC1 may alter the conformation of multimeric KorB so that it is better able to make these contacts, thus stabilising the complexes once formed.