The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons

Abstract

CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes.

Keywords: Conjugative transposon; Integrative and conjugative element; Recombination directionality factor.

Figure 1. Summary of CTnDOT regulatory cascade

Upon exposure to tetracycline translation of the tetQ-rteA-rteB operon is stimulated. The RteB protein increases the transcription of rteC. RteC then acts upstream of the excision operon and increases transcription of that operon. The proteins in the excision operon are involved in excision of the CTnDOT element from the chromosome, as well as transcriptionally activating the divergently transcribed mob and tra operons. (1 column figure)

Figure 2. Xis2d Binding

(a) The upstream tra region was divided into six different fragments (A-F) to test for Xis2d binding capabilities. The transcription initiation start site for the tra operon is designated as +1 in this paper, with all mentioned positions in reference to this point. Arrows indicate the location of the promoters and the direction of transcription. A * indicates that binding was detected with the indicated fragment. (b) Electrophoretic mobility shift assay (EMSA) containing each DNA fragment being tested with (+) and without (−) the presence of Xis2d. The concentration of Xis2d was 400nM. (C) EMSA containing each DNA fragment being tested with (+) and without (−) the presence of the Empty vector control. (2 column figure)

Figure 3. Xis2d Binding Dilutions

The three DNA fragments that interacted with Xis2d (Fragments A, C, and D) were tested with a range of Xis2d concentrations. The concentrations of Xis2d tested in this figure were 400nM, 150nM, 75nM, and 40nM. (1 column figure)

Figure 4. Xis2d Footprint

DnaseI footprint of Xis2d on the top strand of the 235bp DNA fragment from position −96 to −30 relative to the tra TIS. The x-axis represents the length of the DNA fragment. The y-axis represents the florescent intensity, with the top of the scale set at 1000. The NP chromatogram is the no Xis2d control and the subsequent chromatograms show experiments performed with 470nM, 300nM, 150nM, and 80nM Xis2d. The dashed boxes highlight where footprints were observed, and the * symbol refers to peaks of enhanced cleavage. (2 column figure)

Figure 5. Scanning Mutagenesis

(a) The 54bp region shown to be protected in the Xis2d footprint was sequentially mutated to the complement sequence to determine what regions of the fragment were important for binding. Underlined bases highlight the area that was mutated in each fragment. The percentage of DNA shifted for different concentrations of Xis2d (400nM and 100nM) is reported to the right of each fragment. (b) EMSAs of the DNA fragments being tested with 400nM and 100nM of Xis2d. The first lane of each EMSA is a DNA control and subsequent lanes correspond to each of the tested fragments. (2 column figure)

Figure 6. Xis2d Bending Assay

EMSA of Xis2d binding bending substrates. Fragment D was cloned between direct repeats containing multiple restriction enzyme sites: MluI (1), NheI (2), SpeI (3), EcoRV (4), SmaI (5), NruI (6), RsaI (7), BamHI (8). Cutting with each of the restriction enzymes results in the Xis2d binding location shifting within the total fragment while retaining the fragment length. Lane “D” is unbound DNA. Xis2d concentration was 400nM. (1 column figure)

Figure 7. Summary of Xis2d Results

The sequence from positions −204 to +31 relative to the tra transcription initiation site (+1) is shown. The transcription initiation start site (+1) is bold/italicized and all noted positions are in reference to this point. The double underline bases span the −7 and −33 boxes of the tra and mob promoters. The bases in bold type from positions −94 to −42 correspond to the bases that were protected in the footprinting assays when Xis2d was present. Of the protected bases those that are underlined correspond to areas where enhanced cutting was observed in the footprinting assays. The dashed box encircles areas where Xis2d binding was lost when the sequence was mutated and the solid line box indicates bases at the center of the bend induced by Xis2d binding. (2 column figure)

Figure 8. Xis2d and Exc Binding

Fragment D was incubated with Xis2d (0.09uM) and increasing concentrations of Exc. Lane “D” is DNA control. Lane “E” is the empty vector control. Lane 1 is Exc alone (6uM). Lane 2 is Xis2d alone (0.09uM). Lanes 3-8 have the same concentration of Xis2d (0.09uM), but have increasing concentrations of Exc added. Concentrations of Exc listed in the table above the EMSA. (1 column figure)

Figure 9. Xis2d Binds Dyad Symmetry

The dyad symmetry (XD) found in the intergenic region between the tra and mob operons compared to the Xis2d sites in the attR region (D1 and D2). The sequence shown for XD is from position −78 to −46. The underlined bases in the XD sequence comprise the dyad symmetry and the arrows designate the direction of the symmetry. (1 column figure)

Figure 10. Xis2d Consensus Sequence

Alignment of the left (XD-L) and right (XD-R) halves of the XD dyad symmetry and the D1 (D1-L and D1-R) and D2 (D2-L and D2-R) sites of attR. A summary of the number of times of each nucleotide appears at a given position is shown below the sites. A consensus sequence is shown at the bottom. (1 column figure)