Integration site selection by the Bacteroides conjugative transposon CTnBST

Abstract

A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end.

FIG. 1.

In vivo integration assay for attBST derivatives. E. coli BW19851 with pattBST derivatives, containing various lengths of attB cloned in pEPE, was the donor B. thetaiotaomicron BT4001 contains pIntBST and was used as the recipient. The integration frequency was calculated by determining the number of transconjugants per recipient. The types of resistance in parentheses function in the Bacteroides recipient BT4001, and those outside parentheses function in the E. coli donor. Cm^R, chloramphenicol resistance; Em^R, erythromycin resistance; Tc^R, tetracycline resistance; Ap^R, ampicillin resistance.

FIG. 2.

Phylogenetic tree of members of the tyrosine recombinase family obtained using Clustal W. IntBST is underlined.

FIG. 3.

Minimal attBST site determined by the in vivo integration assay. The filled rectangle represents the 18-bp common core sequence. intBST is at the right end of the sequence. The positive control, shown at the top, was pDJE2.1 containing about 700 bp of attBST, including the 18-bp sequence. The negative control, shown at the bottom, was the pEPE vector. The pattBST vectors were constructed as described in Materials and Methods, and filter mating was performed as shown in Fig. 1. The filled rectangle indicates the 18-bp sequence where crossover occurs.

FIG. 4.

Southern blot analysis of mini-BST insertions in BT4001 by G9C mutants. Mini-BST was transferred from E. coli S17-1 carrying pattBSTG(9)C to BT4001 by conjugation. DNA was extracted from eight independent isolates, digested with HindIII, and run on an agarose gel. The Southern blot of the gel was probed with a labeled 423-bp region of intBST which detected right-end junctions. The HindIII fragments of lambda were in lane λ, and the sizes of the bands (in kilobase pairs) are indicated on the left. The arrow indicates the expected band for the wild type.

FIG. 5.

Southern blot and PCR results for CTnBST and mini-BST excision. Three strains containing CTnBST (CTnBST lanes) and three strains containing mini-BST (Mini-BST lanes) were tested to detect excision either by Southern blotting (A) or by PCR (B). The Southern blot of the gel was probed with a right-end fragment containing intBST. The location of the 2.4-kb PstI fragment from the excised CTnBST containing the joined ends is indicated on the left (BJE), and the location of the expected HindIII fragment of the excised mini-BST is indicated on the right (BJE?). The results for PCR amplification of BJE of the strains shown in panel A are shown immediately below the Southern blot in panel B. The size of the BJE amplicon is 410 bp.

FIG. 6.

Sequences of attB sites used by mutant mini-elements and consensus sequences of the attB sites relative to attBST. The panels show the 18-bp sequences of an integrating mini-element and the corresponding 18-bp sequence of the chromosomal attB site. The sequences are shown with spaces between bp 6 and bp 7 and between bp 13 and bp 14 to emphasize the putative crossover between the attBST and the attBs. The 18-bp attBST sequence of CTnBST is shown at the top. The underlined bases in the attBST sites are the mutated bases. Immediately below each attBST sequence is the sequence of the site in the chromosome (attB) into which the mini-element integrated. Below each pair of attBST and attB sequences are the left and right junctions of the integrated mini-element. The left junction of each insertion was cloned by plasmid rescue and sequenced. The right junction was obtained by PCR using a primer designed from the identified chromosomal site and the right end of the mini-element. An alignment of attBST and the attB sites is included. The consensus sequence is shown at the bottom of each panel; residues in uppercase letters are conserved in all the attBST and attB sites, and residues in lowercase letters are conserved in a majority of the sequences. Panels A, B, and C show the integration sequences of three mutants. The vertical arrows indicate the base pair that underwent mismatch correction.

FIG. 7.

Possible staggered cut sites for CTnBST integration. The 18-bp common core region for CTnBST and attB1 is shown. The vertical arrows indicate possible IntBST cleavage sites on the top and bottom strands. The underlined base pairs were important in target specificity. The italicized base pairs were shown to produce heterology which was resolved after integration. The inverted repeats are indicated by horizontal arrows.