Identification of operators and promoters that control SXT conjugative transfer

Abstract

Transfer of SXT, a Vibrio cholerae-derived integrating conjugative element that encodes multiple antibiotic resistance genes, is repressed by SetR, a lambda434 cI-related repressor. Here we identify divergent promoters between s086 and setR that drive expression of the regulators of SXT transfer. One transcript encodes the activators of transfer, setC and setD. The second transcript codes for SetR and, like the cI transcript of lambda, is leaderless. SetR binds to four operators located between setR and s086; the locations and relative affinities of these sites suggest a model for regulation of SXT transfer.

FIG. 1.

RT-PCR analysis of the s086 transcript. (A) Schematic representation of the open reading frames at the 3′ end of SXT. Thick arrows represent open reading frames. Promoters are designated by bent arrows. The thin arrows indicate the positions of the primers used for RT-PCR. (B) Amplification of a transcript via RT-PCR with primers specific for s086 and setC. cDNA was produced with a primer specific to the 3′ end of setC. Lane 1, sample amplified after RT; lane 2, control PCR on RNA that was not reverse transcribed; lane L, molecular weight markers.

FIG. 2.

Annotated sequence of the intragenic region between s086 and setR. The likely translation start codons are in boldface and underlined. The putative s086 Shine-Dalgarno sequence is shown in gray. Putative −10 and −35 regions are shown in boldface. Bent arrows indicate the putative s086 and setR transcription start sites that were defined by 5′ RACE. For these experiments, an overnight culture of Jo193 cells was diluted 1:100 into fresh Luria-Bertani medium. Cells were then grown to an OD₆₀₀ of 0.3, and mitomycin C was added at a concentration of 200 ng/ml for a final hour of growth. mRNA was prepared with the RNeasy Mini kit (Qiagen, Md.). 5′ RACE was performed as described by the manufacturer (Invitrogen). The arrows show the substituted bases in the P_R −10 region. Boxed regions show the 14-bp regions with dyad symmetry that likely represent SetR binding sites. The lines above and below the sequence show the regions that were protected from DNase I cleavage and correspond in style to lines depicted in Fig. 3. Lines above the sequence denote regions protected on the top strand, and lines below the sequence denote regions protected on the bottom strand. Although protection was observed, the boundaries of OL could not be resolved on the bottom strand.

FIG. 3.

DNase I protection by SetR of the region between s086 and setR. Regions of protection are denoted by vertical lines beside the gel. The vertical line styles correspond to the lines shown in the schematic in Fig. 2. DNase I protection assays were performed as previously described (14). Briefly, end-labeled DNA probes were generated by PCR with one 5′-radiolabeled primer and a second nonradiolabeled primer. Binding reactions were carried out in 40 μl containing 120,000 cpm of the labeled DNA fragment and purified SetR-H₆ under the same conditions as previously described gel shift experiments (4). Binding was carried out for 10 min at 4°C. Samples were then brought to room temperature, 0.4 U of DNase I (Ambion, Austin, Tex.) was added, and the mixture was incubated for 30 s. The G+A sequencing ladders were generated as previously described (15). Dried DNA pellets were resuspended in a formamide loading buffer and loaded on 6% sequencing gels. The gels were dried and exposed to autoradiographic film. Lane 1 in each panel is probe DNA with no added SetR-H₆. SetR-H₆ concentrations were as follows: lane 2, 120 nM; lane 3, 180 nM; lane 4, 210 nM; lane 5, 300 nM; and lane 6, 600 nM.