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Conjugative transposons such as Tn916 are mobile genetic elements that move between different species and genera of bacteria by a mechanism that requires cell-cell contact (4, 22, 24). They are found in a wide variety of both gram-positive and gram-negative bacteria and are responsible for the spread of antibiotic resistance in many gram-positive pathogens. Most conjugative transposons encode resistance to tetracycline, carrying either a tetM or tetQ determinant that confers resistance by a ribosome protection mechanism (28). Many conjugative transposons also encode resistance to other antibiotics. One of the best-studied conjugative transposons is Tn916, which was first isolated from Enterococcus faecalis (8). Tn916 can move between many different gram-positive bacteria, including E. faecalis and Bacillus subtilis, and can transfer from gram-positive bacteria to gram-negative bacteria (18). Tn916 is an 18-kb element containing 24 open reading frames, one of which encodes resistance to tetracycline (tetM) (7).

Conjugative transposition of Tn916 has three stages: excision to produce a circular form of the transposon, conjugal transfer of the excised transposon from donor to recipient, and integration of the transferred transposon into the genome of the recipient (2, 25). Excision requires the activity of the products of two transposon genes, int and xis (19, 20, 29) (Fig. 1). Int is an integrase family recombinase (1). It is a bivalent DNA-binding protein whose C-terminal domain recognizes and binds to imperfect 26-bp repeats at each end of the transposon and to flanking host DNA (12). Its N-terminal domain binds specifically to short repeated sequences (called DR-2 repeats) 150-bp from the left end of the transposon and 90 bp from the right end (5, 12, 32). Int catalyzes DNA cleavage and strand exchange by making staggered cleavages 6-bp apart in the DNA at each end of the transposon to produce 5' OH termini (14, 31). Following strand exchange, the ends of Tn916 are joined to form the circular intermediate (25). The junction of the circular intermediate contains a 6-bp heteroduplex created by the joining of single stands of the 6-bp sequences, termed coupling sequences (2), that flank the transposon in the donor DNA.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Genetic map of Tn916. The gray background line represents transposon DNA, and the thick black arrows represent open reading frames. Four short reading frames, orf24 (to the right of orf23), orf12 (to the right of tetM), orf10 (to the right of orf6), and orf5 (to the right of xis), are not shown. The position of the origin of conjugal DNA transfer, oriT, is indicated between orf21 and orf20. The thin arrows indicate five promoters and their direction of transcription.

Xis is a small, basic sequence-specific DNA-binding protein that binds near the ends of Tn916, close to the binding sites for the N-terminal domain of Int (21). Xis is necessary for excision in vivo and in vitro at physiological salt concentrations (17, 20, 30). The mechanism of action of Xis is not currently understood.

Conjugal transfer of Tn916 requires a series of genes located in the right end of the transposon (orf23 to orf13 in Fig. 1) (26). During conjugal transfer of Tn916, genetic evidence suggests that a single strand is transferred from the donor cell to a recipient cell, where the complementary strand is synthesized (23). Tn916 oriT was identified as a segment of DNA that, when cloned onto a plasmid, results in mobilization of the plasmid by a Tn916 transposon (10). It is located between orf21 and orf20 (Fig. 1). The oriT of Tn916 is presumably where the DNA is nicked to initiate the transfer of a single strand of DNA, although this has not been demonstrated directly.

Expression of the tra genes of Tn916 is dependent on int and occurs from the Porf7 promoter (Fig. 1) (3). This suggests that the tra genes are only expressed upon excision of the transposon. This is consistent with the observation that Tn916, when resident on a nonconjugative plasmid, cannot mobilize the markers associated with the plasmid (6) and implies that conjugal transposition of Tn916 is regulated at the level of excision (15). However, if Int and Xis are expressed from a heterologous promoter, excision occurs at a high frequency but the frequency of conjugal transfer and integration remains unchanged, implying that excision is not sufficient for conjugal transfer to occur and that the transfer step in conjugative transposition might be regulated (16).

We found that within oriT is a DNA sequence that is similar to the DNA at the end of Tn916 to which the C terminus of Int binds. Binding of Int to oriT could prevent transfer of Tn916 and play a role in the regulation of conjugative transposition. We show that Tn916 Int binds specifically to oriT in vitro and that this binding is dependent on both the N terminus and the C terminus of Int.

Tn916 Int binds specifically to oriT. To assess the ability of Int to bind to oriT, we performed DNase I footprinting experiments. As a probe we used an agarose gel-purified BamHI-SalI restriction fragment containing oriT from plasmid pAM5160 (10). This fragment contains 466 bp of Tn916 DNA containing the entire functional oriT region. In all experiments, the oriT-containing DNA fragment was labeled at the SalI site with [-³²P]dTTP by using DNA polymerase I Klenow exonuclease-negative fragment (Promega). Int was purified as described (31). Footprinting experiments were performed as described by Lu and Churchward (13) except that the reactions were incubated for 1 h prior to DNase I treatment and were not treated with phenol-chloroform following treatment. The concentration of DNase used for all reactions was 1.3 ng/µl.

View larger version (151K): [in this window] [in a new window]   FIG. 2.   (A) Nuclease protection of radiolabeled oriT DNA by increasing concentrations of Int. Lanes 1 and 2, G and G+A Maxam-Gilbert sequence reactions. Lane 3, no DNase I. Lanes 4 and 9, DNase I, no Int. Lanes 5 to 8, increasing concentrations of Int. The vertical bar to the left of lane 4 indicates the protected region. (B) Competitor titration using unlabeled oriT fragment as competitor. Lane 1, Maxam-Gilbert G-specific sequence reactions. Lane 2, no DNase I. Lane 3, DNase I, no Int. Lane 4, DNase I, Int, no competitor. Lanes 5 to 8, increasing amounts of competitor DNA. The vertical bar to the left of lane 3 indicates the protected region. (C) Competitor titration using unlabeled nonspecific DNA fragment as competitor. Lane 1, Maxam-Gilbert G-specific sequence reactions. Lane 2, no DNase I. Lane 3, DNase I, no Int. Lane 4, DNase I, Int, no competitor. Lanes 5 to 8, increasing amounts of competitor DNA. The vertical bar to the left of lane 3 indicates the protected region.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Quantitation of DNA competitor titrations. (A) Panels 1 to 3 are enlargements of the protected regions from panels A to C of Fig. 2, respectively. Panel A1, lanes 4 to 9 of panel A from Fig. 2. Panel A2, lanes 3 to 8 of panel B from Fig. 2. Panel A3, lanes 3 to 8 of panel C from Fig. 2. The asterisks to left of panels A2 and A3 indicate the bands used for quantitation. (B) Left graph: competitor titration using unlabeled oriT fragment (solid-circles) and nonspecific fragment (open circles). Percent competition was determined by taking the ratio of the protected (upper) band and the unprotected (lower) band. For each competitor concentration, this ratio was then normalized, assuming that the value obtained from lane 1 of panels A2 and A3 with no Int was equal to 100% competition (no binding) and the value from lane 2 of panels A2 and A3 with no competitor was equal to 0% competition. Right graph: competitor titration using Int C-terminus-specific competitor (solid circles) and N-terminus-specific competitor (open circles). Quantitation and analysis were carried out as for the left graph.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Comparison of the Int-protected region of oriT with the ends of Tn916. Upper line: protected oriT sequence. The asterisks indicate unprotected bases (not distance along the sequence). Middle line: joined transposon ends in 5'-to-3' orientation. Lower line: complementary strand of the joined transposon ends in 5'-to-3' orientation. Vertical dashes indicate bases in the transposon end sequences that are identical to bases in oriT.

Both the C-terminal and the N-terminal DNA-binding domains of Int are necessary for binding oriT. In order to determine which domain of Int interacts with oriT, we used double-stranded oligonucleotides that are specific to the N-terminal and the C-terminal domains as competitors in the DNase I protection assays using the same molar excess of competitor as described above. The N-terminus-specific oligonucleotide was DR22, and the C-terminus-specific oligonucleotide was CL (11). Each of these oligonucleotides contains the region protected from nuclease cleavage by the N- and C-terminal domains of Int, respectively. The degree of competition was quantitated as described above. The right panel of Fig. 3B shows that both oligonucleotides abolished binding of Int to oriT when used as competitors. In addition, using DNase I protection, we were unable to see any binding of oriT by either the N-terminal or C-terminal domain of Int alone (data not shown), while these domains protect separate defined regions of the ends of Tn916. We conclude that both the N-terminal and C-terminal DNA-binding domains of Int are necessary for binding to oriT.

Specific binding of Int to oriT. The results described here show that Tn916 integrase binds specifically to a site within oriT that is similar to the ends of the transposon to which Int also binds (12). Comparing between experiments using different DNA substrates, the apparent affinity of Int for oriT is similar to that for the transposon ends and flanking bacterial DNA. Two protein molecules bearing the C-terminal domain of Int bind to the transposon end and flanking DNA (11). The binding of Int to the site within oriT differs in three ways from binding to the transposon ends. First, although a region of approximately the same length (~50 nucleotides) is protected in each case, the sequence comparison indicates that this region may only contain a specific binding site for a single Int molecule. Second, there are sites within the protected region of oriT that remain susceptible to DNase I cleavage. In contrast, the sequences at the transposon ends and flanking DNA are completely protected from DNase I cleavage (13). Third, Int binding to oriT is inhibited by oligonucleotides containing specific binding sites for both N- and C-terminal domains of Int, indicating that both domains contribute to the observed protection of oriT. This is despite the fact there are no sequences of DNA within the oriT fragment that are recognizably similar to the DR-2 repeats located near the ends of the transposon to which the N-terminal domain of Int binds (5, 12). In contrast, oligonucleotides containing specific N-terminal domain binding sites do not compete with binding of the C-terminal domain of Int to the transposon ends and flanking DNA (13).

Consequences of Int binding to oriT. Binding of Int to oriT is probably significant in vivo. Tn916 does not effectively mobilize a nonconjugative plasmid into which it is inserted (6). This can be explained by the observation that int, and thus presumably transposon excision, is required for expression of the transfer genes (3). However, there is apparently nothing to prevent the transfer genes from being expressed if Tn916 inserts downstream of an active transcriptional promoter. Binding of Int to oriT, if it inhibits oriT function, could provide a mechanism to prevent transfer from occuring prior to excision even if the transfer genes are transcribed, thus preventing transfer of only a portion of the transposon to a recipient cell. Int can potentially be expressed from Pint, Pxis, and Porf7 (Fig. 1) (3, 17), and all these promoters are constitutively active in all cells in the population (D. Muller and G. Churchward, unpublished results), indicating that Int is constitutively expressed in all cells, not just the minority of cells that act as conjugal donors of Tn916.

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