Integration and excision of a Bacteroides conjugative transposon, CTnDOT

Abstract

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.

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.

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 ΩCTnDOT in BT4107N1. The Bacteroides transconjugant was selected as Cm^r. 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 Cm^r Ap^r 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.

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 oriT_RK2 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.

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.

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 (IR_Right and IR_Left), 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.