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 C-terminal 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.

FIG. 1

Linear map of the RK2 plasmid showing the main features of its 60-kb genome: transposable elements Tn1 and IS21 (black blocks), antibiotic resistance markers as indicated in Materials and Methods, origin of vegetative replication (oriV), and two blocks of transfer genes, Tra1 and Tra2 (dark grey blocks), including origin of transfer oriT. Arrowheads for oriV and oriT indicate the direction of replication or transfer. Enlarged are the trfAp-trbAp-trbBp region with the trbA gene and the central control operon, Ctl. Arrows by trfAp, trbAp, and trbBp indicate transcription start points and direction of transcription. The arrow above korA indicates the single promoter which is responsible for transcription of all five cistrons in the central control operon.

FIG. 2

Sequence of the trfAp-trbAp-trbBp region. Coordinates refer to the complete sequence of RK2, available as GenBank accession no. LZ7758. −35 and −10 regions of relevant promoters are indicated. O_A, KorA operator; O_B, KorB operator; SD, Shine-Dalgarno sequence. Asterisks and alternative letters show mutations in either the putative degenerate KorB operator (first three, from left to right) or in the −10 region of the trbBp region (fourth and fifth). The primers used for amplification of promoter fragments and TrbA deletions by PCR (described in Materials and Methods) correspond to the following coordinates: 1, 18190 to 18212; 2, 18259 to 18278; 3, 18242 to 18263; 4, 18449 to 18431; 5, 18498 to 18477; 6, 18506 to 18489; 7, 18514 to 18493; 8, 18518 to 18501; 9, 18527 to 18508; 10, 18554 to 18534; 11, 18716 to 18735; 12, 18742 to 18759; 13, 18790 to 18776; 14, 18915 to 18896; and 15, 18397 to 18414. orf, open reading frame.

FIG. 3

Genetic analysis to determine whether the presence of both TrbA and KorB binding sites are necessary for cooperative repression of trbBp. Arrows indicate trbBp which is fused to xylE. M1, mutations in the putative degenerate KorB operator (Fig. 2); M2, mutation in the −10 region of trbBp which also reduces sensitivity to trbA repression (Fig. 2); O_B9, the known KorB operator; 103 and 104, EcoRI sites in a 35-bp segment left as a result of Tn1723 transposon insertion followed by deletion of the internal EcoRI fragment which is defined by EcoRI sites 15 bp from each end of the transpsoson (20). c.i., cooperativity in vivo index when KorB was expressed from pDM1.21 (pDM1.2 as control) and TrbA was expressed from pMZT24 (pGBT30 as control). Repressor expression was induced with IPTG as indicated in Table 2.

FIG. 4

Alignment of TrbA and KorA proteins of RK2. The underlining indicates the conserved C-terminal region. Identical amino acids are shown in bold. Arrowheads indicate the end points of deletions. For TrbA, the numbers indicate the number of amino acids removed from the C terminus. For the KorA deletions, the numbers indicate the distance from the N terminus, as described by Kostelidou et al. (11), as well as the number of amino acids removed from the C terminus (N-terminal distance/C-terminal residues removed). The other main feature is the TrbA helix-turn-helix (HTH) motif as predicted by Jagura-Burdzy et al. (7).

FIG. 5

Western blot analysis of C-terminally deleted TrbA derivatives, carried out as described in Materials and Methods. Lanes: 1, pMZT24 (TrbA wt); 2, pMZT46 (C-Δ15); 3, pMZT47 (C-Δ18); 4, pMZT48 (C-Δ33); 5, pMZT43 (C-Δ8); 6, pMZT44 (C-Δ11); 7, pMZT45 (C-Δ13); 8, pGBT30 (no insert).

FIG. 6

Western blot analysis of the relative amounts of TrbA and KorB alone and in combination with the second repressor, carried out as described in Materials and Methods. Cleared lysates used for cooperativity testing were run on SDS-polyacrylamide gel electrophoresis gels. (A) Rabbit anti-KorB as primary antibody. Lanes: 1, KorB and TrbA; 2, KorB only; 3, TrbA only. (B) Rabbit anti-TrbA as primary antibody. Lanes: 1, KorB and TrbA; 2, TrbA only; 3, KorB only. Antibodies were prepared as described in Materials and Methods.