Cooperativity between KorB and TrbA Repressors of Broad-Host-Range Plasmid RK2

Abstract

The KorB and TrbA proteins of broad-host-range plasmid RK2 are key regulators of the plasmid genes required for conjugative transfer. trbBp is the primary promoter responsible for expression of mating pair formation genes. We show that despite the targets for KorB and TrbA at trbBp being about 165 bp apart, 189 bp upstream of the transcription start point and overlapping the ؊10 region, respectively, these two proteins show up to 10-fold cooperativity for the repression of trbBp. Deletion analysis of TrbA showed that the Cterminal domain (CTD), which has a high degree of sequence conservation with the CTD of KorA, is required for this cooperativity with KorB. Western blotting demonstrated that the apparently mutual enhancement of repression is not due simply to elevation of repressor level by the presence of the second protein, suggesting that the basis for cooperativity is interaction between KorB and TrbA bound at their respective operators.The self-transmissible, broad-host-range plasmid RK2 (indistinguishable from RP4 and RP1) has been studied in great detail (17). Its replication, partitioning, and transfer functions show many features of interest, but its most striking aspect is the complex regulatory circuitry which coordinates expression of the genes for all these basic functions. The regulatory proteins responsible for this coordination are KorA and KorB, encoded in the central control operon; KorC, encoded in the klc operon; and TrbA, encoded before the trb operon. The study described in this paper focused on the interactions between KorB and TrbA at trbBp (Fig.

Region-Specific Insertion of Transposons in Combination with Selection for High Plasmid Transferability and Stability Accounts for the Structural Similarity of IncP-1 Plasmids

“…2), even though they did not disrupt any genes. The low stability of these two plasmids might be due to the disturbance of the tight regulation of the plasmids (28,49,61). In fact, in pBPTn/J-3, Tn21Km was inserted in one of the binding sites for the regulation protein KorB (insertion site and consensus sequence, TTTA2GCGG CTAAA).…”

Identification and Characterization of the Conjugal Transfer Region of the pCg1 plasmid from Naphthalene-Degrading Pseudomonas putida Cg1

“…We recognize that complex networks of transcriptional and translational regulatory systems often control plasmid conjugation (57)(58)(59)(60). For example, in the trb operon (the genes for mating pair formation start with trbB) of plasmid RK2, it is thought that translational control on trbB transcripts is exerted by the trbA promoter (responsible for steady-state transcription of the trb operon as well as trbA).…”

“…For example, in the trb operon (the genes for mating pair formation start with trbB) of plasmid RK2, it is thought that translational control on trbB transcripts is exerted by the trbA promoter (responsible for steady-state transcription of the trb operon as well as trbA). Furthermore, repression of the trbB promoter is exerted by both KorB and TrbA proteins (25,36,58,60). Thus, it is likely that conjugal transfer genes in enteric IncP␣ plasmids (e.g., RK2 and RP4) are controlled by a network of regulatory elements (regulators and DNA sequences) that reduce the metabolic burden on the bacterial host.…”

Order and Disorder in the Domain Organization of the Plasmid Partition Protein KorB

“…ParB binds to a specific centromerelike sequence (parS) on the chromosome forming a higher order nucleoprotein complex that is thought to pair the sister chromosomes/plasmids. ParA, an ATPase, binds to ParB and is thought to act as a motor, pulling or pushing two ParB-bound chromosomes apart to different poles of the bacterial cell (2)(3)(4).…”