INTRODUCTION

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Many strains of Bacteroides species carry large self-transmissible elements called conjugative transposons (CTns). Many of these CTns are related to an element called CTnDOT. CTnDOT carries a tetracycline resistance gene, tetQ, and an erythromycin resistance gene, ermF (2, 23, 25). A schematic view of CTnDOT is shown in Fig. 1. Intercellular transposition of CTns is thought to occur by the following steps. The CTn initiates conjugal transfer by excising from the chromosome to form a circular intermediate. This intermediate is nicked at the transfer origin (oriT), and a single-stranded copy is transferred to the recipient cell, recircularized, and integrated into the recipient's genome (15, 17, 21). The region of CTnDOT that mediates the nicking and transfer reactions has been located and sequenced (11; G. Bonheyo, D. Graham, N. B. Shoemaker, and A. A. Salyers, submitted for publication), but virtually nothing is known about the excision and integration steps. In particular, there is only indirect evidence for the existence of the hypothetical circular intermediate that presumably forms during the excision reaction and later integrates in the recipient. In this paper, we present direct evidence that the circular intermediate is formed during the excision process and is the form that integrates into the chromosome.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Schematic map of the integrated forms of CTnERL and CTnDOT. The chromosomal DNA flanking the integrated element(s) is shown as dotted lines. The 13-kbp region containing ermF that is contained on CTnDOT but is not on CTnERL is indicated by the patterned rectangle and arrow below the line. The regulatory region of the elements contains the tetQ-rteA-rteB-rteC gene cluster that encodes proteins that are induced by tetracycline and that regulate activities of CTnDOT and CTnERL. Adjacent to this region is the oriT-mob and the transfer region that contains the transfer genes (traA to traQ). A sequence of traQ cloned on an insertional vector was used to clone the CTnDOT-chromosome left end junction from BT4107N1 on an SstI fragment. A PvuII-SspI fragment was subcloned and sequenced. The CTnDOT right end-chromosome junction was cloned from the same strain on a 6.2-kbp EcoRI fragment.

Previously, integration and excision of CTns have been studied in detail only in the case of Tn916, a CTn first found in enterococci (5, 7). Analysis of integration and excision events mediated by Tn916 led to the proposal of a novel model for integration and excision (3). During excision, staggered cuts are made 6 bp from either end. Then, each single-stranded 6-bp overhang is joined to the other end of the CTn to create a circular form in which six bases of double-stranded but unpaired DNA separate the ends of the CTn (5, 21). The 6-bp segments are called coupling sequences. At some point after excision, the 6-bp region is resolved in favor of one coupling sequence or the other (12). In natural hosts of Tn916, this resolution seems to occur very quickly in the donor cell, but it could also occur during the conjugal transfer step. After conjugal transfer of a single-stranded copy of the CTn, integration occurs in the recipient. The integration process does not duplicate the target site. Recently, the integrase of Tn916 and an Xis protein have been purified and shown to bind DNA adjacent to the ends of the CTn (10, 15).

The CTnDOT-type elements differ in a number of ways from Tn916. First, Tn916 integrates relatively randomly, whereas CTnDOT appears to integrate site selectively into about seven sites on the Bacteroides chromosome (2). Second, there is no sequence similarity between CTnDOT and Tn916 in any of the regions so far sequenced. Thus, it was not clear whether CTnDOT would excise and integrate similarly to Tn916 or by a different mechanism. A third difference between CTnDOT and Tn916 is that transfer of CTnDOT is stimulated 1,000- to 10,000-fold by tetracycline (19), a fold increase much higher than the tetracycline stimulation reported for Tn916 transfer (5). The tetracycline-dependent stimulation of CTnDOT transfer is mediated by proteins encoded by three regulatory genes: rteA, rteB, and rteC (18, 29). No regulatory genes analogous to the rte genes of CTnDOT have been found on Tn916. Previous studies of the transfer region of the Bacteroides CTns showed that transfer of a plasmid that contained the CTn transfer region was constitutive (11; Bonheyo et al., submitted). In this paper, we show that excision is a tetracycline-regulated step in transfer.

Also found in Bacteroides species are integrated elements that are much smaller than the CTns. These elements, exemplified by NBU1, Tn5520, and Tn4555, have been called mobilizable transposons because they rely on the CTns for transfer functions. The integrases of NBU1, Tn5520, and Tn4555 have been sequenced, and all are members of the lambda family of integrases (27, 32, 33). NBU1 excision more closely resembles excision of phage lambda in that there is an att site formed by the joined ends that integrates into an identical site in the chromosome. Excision of Tn4555 is more similar to that of Tn916 (31). Although the mobilizable transposons rely absolutely on the CTns for transfer, there is so far no evidence for any significant sequence similarity between them and the CTns. In this paper, we show that the integrases of two Bacteroides CTns are not closely related to those of the mobilizable transposons, although they are also members of the lambda integrase family.

	MATERIALS AND METHODS

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Bacterial strains, conjugations, and DNA manipulations. Bacterial strains used in this study are listed in Table 1. Methods of DNA extraction, cloning and Southern blotting analysis, and conjugation protocols have been described previously (9, 16, 20, 24).

Cloning of the CTnDOT right end. Previously, we fortuitously trapped a CTn from Bacteroides uniformis, CTnXBU4422, on a plasmid (25). The ends of CTnXBU4422 hybridize with the ends of CTnDOT, and so we used the CTnXBU4422 right end as a hybridization probe to assist in cloning of the CTnDOT right end. A 2.2-kbp DNA fragment containing the CTnXBU4422 right end from plasmid pXBU1 (25) was used as a probe in Southern blots to identify the DOT right-end fragment. Chromosomal DNA from BT4107N1, which contains CTnDOT, was digested with EcoRI, and a 6.2-kbp fragment hybridized to the probe. Restriction fragments around 6.2 kbp were recovered from a low-melting-point agarose gel, cloned into the EcoRI site of pUC19, and transformed into Escherichia coli. Colony hybridization was used to detect positive colonies using the CTnXBU4422 right-end fragment probe. The 6.2-kbp EcoRI fragment on pUC19:DRJ that hybridized to the probe was sequenced partially to locate the right end. The end was located initially by comparing the sequence of the clone with the sequence of the XBU4422 right end (2) and later to the sequences of the CTnDOT joined ends (DJE) and the BT4107N1 integration site. A 2.5-kbp region of the CTnDOT right end was double-strand sequenced. Sequencing was performed by the University of Illinois Biotechnology Genetic Engineering Facility with an Applied Biosystems model 373A version 2.0.1S dye terminator automated sequencer.

Cloning of the CTnDOT left end. Initially, we tried to clone the CTnDOT left end by using the XBU4422 left-end fragment as the probe but were not successful. Therefore, we decided to clone by using a Bacteroides suicide plasmid to integrate into a sequenced region that was next to the left end of CTnDOT. We would then clone out the plasmid with as much of the adjacent left-end region CTnDOT as possible into E. coli. The vector used was pNLY3, a pUC19-based vector containing the oriT of RK2 for mobilization out of E. coli and IS4351-cat, which is expressed in both E. coli and Bacteroides hosts (Table 1). The region of homology used for the integration was the internal region of traQ (Bonheyo et al.) which by Southern blots appeared to be >15 kbp from the CTnDOT left end (Fig. 1). A 368-bp fragment of traQ was amplified by PCR and cloned into pGEM-T (Promega, Madison, Wis.). The fragment was sequenced to determine orientation and subcloned into pNLY3.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Cloning of the CTnDOT left junction. The integrated insertional shuttle vector pNLY3 containing 368 bp of traQ, pNLY3:traQ', is shown integrated into the traQ region of CTnDOT in BT4107N1. The Bacteroides transconjugant was selected as Cmr. Southern blots verified the insertion into traQ. The DNA from the transconjugant was digested with SstI, which was determined to cut one time in the distal end of the vector and in the chromosome beyond the end of CTnDOT, using the left-end probe from CTnXBU4422. The digested DNA was first ligated and then used to transform E. coli. A large unstable plasmid, pNLY3:DOT-LE, that contained about 15 kbp of addition CTnDOT left end (LE) and chromosomal DNA at the left junction (JL) was isolated from a Cmr Apr transformant. A 5.5-kbp PvuII-SspI fragment was a CTnDOT-chromosome junction fragment that contained the CTnDOT left end (DOT JLE) and the chromosomal left end junction (Chr-JL) and was subcloned onto pUC19 (pDOT5B/SstI) for sequencing.

Detection of the joined ends of the excised CTnDOT. The sequences obtained from the ends of the integrated CTnDOT in BT4107N1 were used to design primers DRJ/R1729F and DLJ/INW-1/R241R (Table 2). These primers are directed out of the ends of the integrated element and cannot form a PCR product unless the element excises and forms a circular intermediate. Ten independent cultures of BT4107N1 were grown overnight with and without tetracycline and tested for the production of PCR-amplified excision product; 10-µl aliquots of different overnight cultures were used directly in PCR. The cells were spun down and resuspended in the volume of water needed for the PCR. The cycling conditions were as follows: (i) 5 min at 95°C; (ii) 25 to 35 cycles of 1 min at 94°C, 1 min at 51°C, and 2 min at 72°C; (iii) final extension of 5 min at 72°C. The PCR products were cloned onto pGEM-T and sequenced.

Comparison of the left and right ends of CTnDOT and CTnERL. Two primers (ERL-F1 and DLJ/INW-1/R241R [Table 2]) were used to PCR amplify the CTnERL joined ends. DNA from tetracycline-induced cells of strain BT4104, which contain a CTnERL insertion in the chromosome, was used as a source of template in a PCR. The PCR product was cloned onto pGEM-T and sequenced.

Disruptions in intERL and orf2. The conjugative transposon CTnERL was nearly identical to CTnDOT at their right ends, and the joined ends of CTnERL could also be detected by PCR. This similarity allowed us to make disruptions in the putative int gene and orf2 (open reading frame 2) in the right end of CTnERL to test for their effect on CTn excision. We could then use erythromycin resistance (Em^r) as the selection marker for the insertions. Insertions were then made in the integrase gene (intERL) and orf2ERL in BT4004. Disruptions in the integrase gene were made by cloning the 423-bp PCR-amplified internal fragment of intERL into pCQW1 (6), a plasmid that replicates in E. coli but not in Bacteroides hosts. Similarly, a 193-bp fragment of orf2 was cloned into pCQW1. The pCQW1 clones, pCQW1-int and pCQW1-orf2, were mobilized into BT4004 and were shown to integrate into the correct region of CTnERL by Southern blots. The resulting BT4004 Em^r transconjugants with the insertions in CTnERL were then tested for the ability of CTnERL to excise and form a circular intermediate as detected by PCR amplification of the ERL joined ends (EJE).

Use of inverse PCR to clone the left junctions of CTnDOT inserted in different sites (DOT-JS). Initially, we cloned and sequenced the DOT left and right junctions from one CTnDOT insertion strain, BT4107N1. The integration site in this strain was designated DOT-JS 2 and was PCR amplified by primers DRJ/R2700R and DLJ/U487F (Table 2). The only obvious sequence similarity between the CTnDOT ends and the integration site was a 10-bp sequence (5 bp away from the left end of the integrated CTnDOT) with high sequence identity to a 10-bp sequence present in the CTnDOT right end. To determine whether a similar sequence was found adjacent to the left ends of CTnDOT inserted in different sites, we examined the left-end junction sequences of four other strains, identified by pulsed-field gel analysis to have CTnDOT in different NotI bands (2). Inverse PCR, with out-directed primers anchored inside the left end of CTnDOT, was used to obtain left junctions from each of the four strains (BT4107-1, BT4107-3, BT4107-4, and BT4107-5). Specifically, chromosomal DNA from the four Bacteroides strains was digested with NlaIII and then ligated at a DNA concentration of about 2 µg/ml to favor monomeric circularization. After ligation, the circularized DNA molecules were used as templates in typical PCRs. The inverse PCR primers were DLJ/INW-1/R127R and DLJ/U430F (Table 2). The resultant PCR products were cloned into the pGEM-T vector and sequenced. The cloned junction sites were designated DOT-JS 1, DOT-JS 3, DOT-JS 4, and DOT-JS 5, respectively.

Construction of miniature versions of CTnDOT. To identify the gene or genes necessary and sufficient to mediate integration of the circular form of CTnDOT into the chromosome, we PCR amplified three different CTnDOT joined ends (DJE) products, using DNA from tetracycline-induced BT4107N1 cells as a template. The sequences of the primers used to produce the various PCR products are shown in Table 2. The smallest DJE PCR product (primers DRJ/R1729F and DLJ/INW-1/R241R) was 1.1 kbp and contained only the N-terminal region of the putative integrase (intDOT). The 2.3-kbp DJE product (primers DRJ/R593F and DLJ/INW-1/R241R) included the entire intDOT and the 2.6-kbp product (primers DRJ/R343F and DLJ/INW-1/R241R) included the intDOT plus a second ORF (orf2) downstream of intDOT (see Results). All three DJE PCR products were first cloned into pGEM-T and then subcloned into the Bacteroides suicide vector pGERM (Fig. 3). These different pGERM:DJE plasmids (Table 1) were transformed into E. coli strain S17-1 and mobilized into BT4001, a strain that does not contain any known conjugative transposon, to test for the ability to integrate into BT4001 chromosome. The vectors that were capable of integrating were tested with and without pAMS9 (29) providing the tetracycline-regulated regulatory genes (rteA, rteB, and rteC) for the ability to excise using PCR amplification to detect the joined ends of the excised circular form.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Construction of the CTnDOT minielements pDJE1.1 and pDJE2.3. The Bacteroides shuttle suicide vector pGERM that contains the Bacteroides selection marker ermG and the oriTRK2 cloned on pUC19 was used to construct the CTnDOT minielements to test for integration in Bacteroides hosts. The smallest minielement constructed contained a 1.1-kbp PCR product that included the CTnDOT joined ends (DJE fragment or attDOT) produced from the excised circular form of CTnDOT, subcloned into pGERM from pGEM-T. The smallest PCR product subcloned into pGERM that integrated normally into B. thetaiotaomicron target or attB sites was the 2.3-kbp DJE product that included attDOT and intDOT from the right end of CTnDOT cloned into pGERM. This CTnDOT minielement construct was called pDJE2.3. The map of an integrated pDJE2.3 is shown at the bottom. The element-chromosome junction at the left is labeled attL, and the junction at the right is labeled attR. The locations and orientations of the genes on the integrated plasmid are indicated.

Comparison of DOT minielement junction sequences (DME-JS) with DOT-JS. To determine if the CTnDOT minielement has the same site selectivity as the wild-type CTnDOT, it was necessary to clone and compare their integration sites (attB). Sixteen Bacteroides Em^r transconjugants with insertions of the CTnDOT minielement were isolated from 16 individual filter matings. Chromosomal DNA from these transconjugants was digested with KpnI, which does not cut inside the CTnDOT minielement. The digested DNA fragments were ligated and transformed into E. coli with selection for Ap^r. Plasmids in the transformants contained the minielement plus DNA adjacent to both ends of the integrated minielement. These DME-JS sites were sequenced using the primers DRJ/R2242F (right junction) and DLJ/INW-1/R127R (left junction).

	RESULTS

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Sequence features of the ends of CTnDOT. The sequence of the left end of CTnDOT did not contain any ORFs larger than 600 bp within the first 2 kbp from the end. Of the few smaller ORFs in this region, none had any amino acid sequence similarity to known integrases. The sequence of the right end, however, had an ORF whose first possible start site was 329 bp from the right end. Advanced Blastp (1) analysis of the deduced amino acid sequence of this ORF showed low sequence similarity to known integrases, such as the integrase of phage P2 (26% identity) and the integrases of the Bacteroides mobilizable transposons NBU1 (24% identity [27]), NBU2 (34), Tn5520 (49% identity [33]) and Tn4555 (26% identity [32]). The putative integrase gene was 1,233 bp in length. Previously, we had obtained a small amount of sequence near the ends of a cryptic CTn, CTnXBU4422, but had not identified any potential integrase gene. The integrase gene of CTnDOT is the first such gene from a Bacteroides CTn to be sequenced and characterized.

View larger version (59K): [in this window] [in a new window]   FIG. 4.   Sequence comparison of the C-terminal regions IntDOT and IntERL to related integrases. The box I and box II regions in the C-terminal ends of the lambda family of site-specific integrases as defined by Nunes-Düby et al. (14) are shown. The conserved arginine (R) in box I and the HRY triad in box II are in boldface and boxed. The GH doublet in box II that is also highly conserved is shown in boldface. The integrases are grouped: IntDOT, IntERL, IntTn5520 (33), and IntTn4555 (32) are at the top, followed by integrases from two other Bacteroides mobilizable transposons, NBU1 (27) and NBU2 (34). The third group contains members of the lambda family of integrases that come from other organisms: YqkM (Bacillus subtilis), P2 integrase, and XerC (Lactobacillus leichmannii). The shading indicates the regions of identity in the Int proteins relative to the IntDOT sequence shown in the top line. The locations in the integrase amino acid sequences of the amino acids involved in the alignments are shown at the ends of the sequences.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Model for the integration of CTnDOT into Bacteroides target sites. (A) Circular intermediate of CTnDOT after conjugal transfer and prior to integration with the known regions of the element indicated. The attDOT in the DJE is enlarged to show the 23-bp imperfect inverted repeats (IRRight and IRLeft), the 10 bp of high similarity to a Bacteroides target site (BTattB), and a 5-bp coupling sequence derived from its last site of integration. Details of the 10-bp alignment of the attDOT region and some attB sites are shown in panels B and C. Following staggered cuts flanking the coupling sequences in the attDOT and attB, the CTnDOT (or minielement) integrates into the target site. The 10-bp consensus sequence in the CTnDOT right end in the circular form is shown above the attB sequences in panels B and C, with the coupling sequence indicated by N's. The sequences in panel B are from the chromosomal target sites prior to integration of the circular form of the CTnDOT (DOT-JS). The sequences in panel C are from the chromosomal site prior to integration of pDJE2.3 (DME-JS). A conserved sequence GTANNTTTGC (10-bp region) was derived from a comparison of the CTnDOT right end and its target sites. The minielement target sites DME-JS 5-2 and DME-JS 8-2 are the same as the wild-type CTnDOT sites DOT-JS 1 and DOT-JS 2, respectively. After conjugal transfer and integration of pDJE2.3 (not shown), the coupling sequence from pDJE2.3 was found at the left side of the integrated element, adjacent to the 10-bp attB sequence, in DME-JS 3, 5-2, and 9-5 and the right side of the integrated pDJE2.3 in DME-JS 8-2 and 11 (sequence not shown). Parentheses indicate that the site was the same as the site above it.

Sequence features of the integration site. Since CTnDOT appeared to integrate site selectively, we expected to see some sequence similarity between one or both ends of CTnDOT and the integration sites. Comparisons of the sequences of the ends of CTnDOT with the sequences of the integration sites revealed some sequence similarity. One candidate for a recognition sequence was a 10-bp sequence that was immediately adjacent to the site where CTnDOT entered the chromosome and at the right end of the circular form of the element (Fig. 5A). In the integrated form, this target 10-bp sequence was 5 bp from the left end. Using inverse PCR, we obtained sequences adjacent to the left end of CTnDOT from insertions in four other chromosomal sites. Similar 10-bp sequences were seen in all four cases (Fig. 5B). Comparison of the four different insertion site sequences (DOT-JS 1 to DOT-JS 5) allowed us to define a consensus sequence that may be responsible for the site selectivity of the insertion process. The fact that this region is so small fits with the likelihood that there are multiple sites in the chromosome that could serve as integration sites for CTnDOT (2).

The integrase gene and the joined end sequences are sufficient for integration into the Bacteroides chromosome. To determine whether the putative integrase gene near the right end of CTnDOT was sufficient for integration in Bacteroides, we constructed a CTnDOT minielement which contained the joined ends of CTnDOT and the putative integrase gene (Fig. 3). The pGERM-based minielement, pDJE2.3, was mobilized into BT4001 with selection for Em^r. Approximately 10³ to 10⁴ transconjugants per recipient were obtained in each of several independent matings. Since this is comparable to the frequency of transfer of a plasmid that replicates in Bacteroides species, the integration efficiency of the minielement may be close to 100% once the element enters the recipient. We also constructed a DJE vector that contained the joined ends of CTnDOT but only a truncated integrase gene, pDJE1.1 (Fig. 3). When pDJE1.1 was mobilized into BT4001 under the same conditions, no transconjugants (<10⁹ per recipient) were obtained. These results prove that the joined ends alone were not sufficient for integration but that including the intDOT ORF allowed the plasmid to integrate. The fact that integration occurred independently of tetracycline stimulation and independently of the rte genes on CTnDOT or CTnERL, which mediate tetracycline-inducible activities of the CTns, demonstrates that integration is not the tetracycline-regulated step in CTnDOT transfer.

Coupling sequences of CTnDOT in BT4107N1. Sequence analysis of the right and left ends of the element, as defined by comparison of junction regions with the sequence of the joined ends, revealed that when CTnDOT excises from chromosome, it brings 5 bp of chromosomal sequence with it. We call these sequences "coupling sequences" following the model for the conjugative transposon Tn916 (21, 22). When the excised CTn forms a circular intermediate, the coupling sequence is located between the joined ends (Fig. 5A). According to the model for Tn916 excision, the initial form of the excised element has coupling sequences from the two ends of the element, which form a short region of heterology. In Enterococcus faecalis, as Manganelli et al. (12) have shown, the heterology is rapidly eliminated in the donor in favor of one coupling sequence or the other. Thus, prior to conjugal transfer, either coupling sequence could be found in the excised circular form. To determine if this was the case for CTnDOT, PCRs were performed to amplify the 1.1-kbp DOT joined ends segment from DNA isolated from 10 independent tetracycline-induced BT4107N1 cultures as template. The amplicons were sequenced and compared to detect the coupling sequences. In 4 of the 10 excision events, the excised CTnDOT joined ends contained the GCAAT chromosomal sequence from the left side, and 6 contained AATTC from the right side. This is the outcome expected if excision of CTnDOT occurred similarly to that of Tn916. The PCR approach used here would not distinguish between a region of heterology and a region resolved in favor of one coupling sequence or the other, but the fact that both coupling sequences were seen indicates that excision involved an intermediate that contained both coupling sequences at one time.

Importance of the integrase for excision. Results presented above show that the int gene was needed for integration. To determine whether the integrase was essential for excision, we constructed a single-crossover disruption in the integrase gene of CTnERL, BT4004-int. Excision of wild-type CTnERL was easily detectable by PCR amplification of the joined ends, but no excision was detected in the disruption strain (Table 3). Thus the int gene appears to be essential for excision. Excision of the wild-type CTnERL element occurred only after exposure of cells to tetracycline. This was also true for CTnDOT. These results demonstrate that excision is a tetracycline-regulated step in transposition. To determine whether the downstream orf2 played a role in excision, we made a single-crossover insertion into it. The disruption mutant, BT4004-orf2, was still able to excise. This shows not only that orf2 is not essential for excision but that loss of the ability to excise caused by the disruption in integrase was not due to a polar effect on orf2 or to insertion of a large DNA segment into this region of the CTn.

	DISCUSSION

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Although the Bacteroides CTns of the CTnDOT family share no discernible DNA sequence similarity with Tn916 in any of the sequences within 3 kbp of either end of the element, these two types of elements seem to integrate and excise by similar methods. That is, excision brings along a coupling sequence from chromosomal DNA adjacent to the excision site and introduces a coupling sequence into a target site when it integrates after having been transferred by conjugation. In earlier studies of the sequences of the ends of CTnXBU4422, which had been accidentally trapped on a plasmid in B. uniformis, we had concluded that CTnXBU4422 integrated by a blunt-ended integration mechanism. At that time, however, we were unable to detect the joined ends of CTnXBU4422 after excision and did not know if there was a bias for the left-end or right-end coupling sequences to be excised with the element. We have since sequenced the PCR products of the joined ends and found that the excised form of CTnXBU4422 detected in B. uniformis 0061 contained the 4-bp left-end sequence (CCCG) about half the time and the 5-bp right-end sequence (GAAAA) about half the time. However, insertions into the plasmid targets on a plasmid replicating in B. uniformis contained the CCCG coupling sequence at the right end of the integrated element in 9 out of 10 cases tested and GAAAA was at the right end in one (2). The reason for the observed bias is not understood. We were also unable to observe conjugal transfer and integration of CTnXBU4422 into B. thetaiotaomicron from B. uniformis, either from the chromosomal insertion or from a plasmid containing CTnXBU4422 (25).

Whereas Tn916 integrates randomly (21, 22), CTnDOT and CTnERL exhibit some site selectivity. This site selectivity may be due to a 10-bp consensus sequence at the right end of the CTn, which has sequence identity to a 10-bp sequence adjacent to the integration site. IntDOT and IntTn916 are both members of the lambda family of site-specific integrases, but the amino acid sequence identity is below 30% even within the C-terminal region of the proteins. The integrase most closely related to the CTnDOT integrase was that of Tn5520 (49% identity and 69% similarity), a 5.5-kbp Bacteroides fragilis integrated element (33), which is probably mobilized by a CTnDOT-type element. There was also limited amino acid sequence similarity to the mobilizable transposons Tn4555, NBU1, and NBU2. The sequence of the integrase of another integrated mobilizable Bacteroides element, Tn4399 (8), is not yet available.

A major difference between the Bacteroides CTns and Tn916 is that the excision reaction catalyzed by the Bacteroides CTns is dependent on tetracycline stimulation and appears to be more complex than that of Tn916. The excision of CTnDOT and related CTns requires more than just the int gene and an xis-like ORF downstream of int. By contrast, the Tn916 int and xis genes seem to be sufficient for excision (15). In the case of the Bacteroides CTns, the int gene was essential for excision as well as for integration, but the downstream orf2 was neither essential for nor sufficient for excision. The as yet unidentified excision genes may well be regulatory genes, although adding known CTn regulatory genes (rteA, rteB, and rteC) in trans with the integrated pDJE2.6 did not induce pDJE2.6 to excise. Future studies will involve identifying genes and DNA sequences that are required for CTnDOT excision.