Mutational analysis of the global regulator KorA of broad-host-range plasmid RK2

KorA protein encoded in the central control region of IncP plasmid RK2 binds to seven operators on the plasmid genome and acts as a global repressor of genes for replication and stable inheritance functions. At trfAp, the promoter for plasmid replication genes, KorA also causes derepression of trbAp, the promoter for trbA, encoding another global regulator (TrbA), which controls genes required for conjugative transfer. Both KorB, a second global repressor encoded in the central control region, and TrbA also act in the trfAp-trbAp region to down-regulate trfAp, but neither of these extra repressors allows derepression of trbAp. To initiate a functional dissection of KorA, we used random mutagenesis and a positive selection system to identify korA mutants which no longer repressed trfAp. Nine single amino acid changes were obtained, which did not affect polypeptide length or apparent stability. These clustered either in the N-terminal region of the protein (region I) or in the putative HTH motif (region II). No changes were obtained in the C-terminal region (region III). Four truncated KorA proteins, with deletions either from the N-terminal or the C-terminal end, were also screened together with the single mutants. Both the band-shift assay with trfAp DNA and the in vivo promoter-probe assays with either trfAp or trbAp showed that none of the region II mutants could bind to DNA and repress the promoter. The region I mutants with a conservative amino acid substitution retained some DNA binding and repressor activity, as well as the ability to dimerise. However, an in vivo system to detect trans-dominance of the mutants indicated that one region I point mutant together with the two N-terminally truncated mutants had lost their dimerisation ability. Deletions into the basic C terminus of KorA did not abolish dimerisation. The results implicate region I in dimerisation, region II in DNA binding and region III in a yet unspecified role, possibly interaction with other proteins such as KorB.