Repression at a distance by the global regulator KorB of promiscuous IncP plasmids
High-Throughput Nucleotide Sequencing
Molecular Diagnostic Techniques
KorB protein (358 amino acids) binds to 12 highly conserved sequences on the RK2 genome and co-ordinates the expression of at least five operons encoding genes for stable inheritance and plasmid transfer. KorB represses the trfA, korA and klaA promoters where it binds 4 bp upstream of the -35 region (class I KorB operators, OB). We show here that KorB on its own can also repress the trbA, trbB, kfrA and kleA promoters where OB is between 80 and 189 bp away from the transcription start point (class II operator). A C-terminal deletion of 17 amino acids resulted in the loss of KorB's ability to repress through class II operator but not through class I operator. This deletion reduced multimerization of His6-tailed KorB protein in vitro and greatly reduced binding specificity for fragments containing OB sequences. At the trbBp region, where OB9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping the trbBp -35 region had no effect on the ability of KorB to repress trbBp. Nevertheless, gel retardation analysis showed that KorB binding is promoted by sequences upstream and downstream of OB9 and that KorB can form higher order complexes on DNA. However, DNase I footprinting suggested that RNA polymerase may interact directly with KorB bound at OB9 and implied that contacts between these proteins could be responsible for the action of KorB at a distance.