A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids

The kor regulon of broad host-range, incompatibility group P (IncP) plasmid s uses the KorA, KorB, and KorC repressors to regulate expression of genes for replication, conjugation, segregation, and host range. One operon, kilC , encodes the KorC repressor and two genes of unknown function (klcA and kl cB). The predicted sequences of the 51.1 kDa KlcB protein, the 11.3 kDa Kor A repressor, and another small (13.5 kDa) regulatory protein, TrbA, show a highly related 35 amino acid residue segment (V-L-P domain). We found that induction of the klcB gene is toxic to Escherichia coli host cells harborin g an IncP plasmid. We confirmed a model in which the V-L-P domain of KlcB i nteracts directly with the V-L-P domain of KorA to derepress KorA-regulated operons, thereby allowing expression of toxic genes. First, a lacZ reporte r fused to the kleA promoter, which is regulated by KorA and KorC, revealed that klcB induction specifically releases KorA-repression but has no effec t on KorC repression. Second, induced expression of the V-L-P domains from KorA or KlcB is sufficient to release KorA-repression at the kleA promoter. Third, purified GST-KlcB fusion protein interacts specifically with His-ta gged KorA. Fourth, fusion of the V-L-P domains of KorA and TrbA and full-le ngth KlcB polypeptide to the DNA-binding domain of bacteriophage h represso r leads to the formation of functional, dimeric repressors, and mutations t hat alter conserved residues of the V-L-P domain adversely affect dimerizat ion. Fifth, crosslinking experiments demonstrated that the V-L-P domain of KorA is able to dimerize in solution and form heterodimers in mixtures with full-length KorA polypeptide. These findings show that the V-L-P domain is a protein-protein interaction module that is likely to be responsible for dimerization of KorA and TrbA, and important for KlcB dimerization. We spec ulate on the possible significance bf KlcB-KorA heterodimers in IncP plasmi d maintenance. (C) 2001 Academic Press.