Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase

Abstract

Background: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.

Methodology/principal findings: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.

Conclusions/significance: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.

Figure 1. Site-specific integration assays with different recipient plasmids.

A. Scheme of the assay and the cointegrate molecule obtained. The suicide plasmid is represented with a grey line and the recipient plasmid with a black line. The nic site is indicated by an arrowhead. P1 and P2, oligonucleotides used in the PCR reaction to detect the cointegrates. B and C. Restriction analysis with enzymes that cut only once in the recipient plasmid. NdeI was used for integrants obtained in pKK:oriT (in B, to the left of the MW marker), XcmI for integrants in pSU:oriT (in B, to the right of the MW marker), and BstEII for integrants in pLA58 and pLA59 (in C, nic▴ and nic▾, respectively). The cointegrate is indicated with an arrow. DP, donor plasmid; RP, recipient plasmid. I1-I2, DNA from two independent integrants obtained on each RP. Sizes in kb of the MW marker from top of the gel: 10-8.0-6.0-5.0-4.0-3.0-2.5.

Figure 2. Integration assays expressing trwC from the donor and/or the recipient cell.

A. Integration assay via TrwC from the donor. The suicide plasmid is mobilized by R388_TrwC relaxase from a II1 donor strain to a DH5α recipient which harbours plasmid pSU::oriTw. Integration mediated by TrwC takes place and the cointegrate is formed. B. Integration assay via TrwC from the recipient. The suicide plasmid is mobilized by RP4_TraI relaxase from an S17.1 λpir donor strain to a DH5α recipient which harbours pSU::oriTw and a plasmid coding for trwC. TrwC in the recipient is able to locate both oriT-containing plasmids and catalyze integration. C. Integration assay via TrwC from the donor and the recipient. The suicide plasmid is mobilized as in A. into a DH5α recipient which harbours plasmid pSU::oriTw plus a plasmid coding for trwC. R388 and RP4 non-mobilizable conjugative systems are represented with a dark and light grey square, respectively. TrwC is represented by an ellipse and RP4_TraI by a diamond. Arrowheads represent the nic site at the recipient oriTw. The T-strand of the suicide plasmid is represented by a wavy grey line containing the oriTw (black arrow) and the oriTp (grey arrow), and the recipient plasmid, with a thick black line. D. XcmI restriction pattern of integrant DNA. RP, recipient plasmid with R388 oriT; DP, suicide donor plasmid; HP, helper plasmid in the recipient. HL: Hyperladder MW marker. −, +A, +C, and +AC refer to the proteins produced by the helper plasmids in the recipient (none, TrwA, TrwC, or both). Top gel: integration assay via TrwC from the recipient (mobilization of donor plasmid with RP4-TraI). Bottom gel: integration assay via TrwC from the donor (mobilization of donor plasmid with R388-TrwC) with different helper plasmids in the recipient cell. The cointegrate molecular species is indicated with a black arrow.

Figure 3. DNA analysis of colonies obtained in the integration assay from the recipient cell expressing different TrwC derivatives.

A. TrwC catalytic mutants plus TrwA. B. TrwC wt or N600 with or without TrwA. Symbols as in Fig. 2.

Figure 4. Integration assays on different target sequences.

A. DNA sequence of the central R388 oriT region, coordinates 201 to 169 from . The arrows show inverted repeat IR2. The nic site is indicated by a slash. Horizontal bars indicate minimal sequence requirements for different TrwC activities. Below are shown the DNA sequences of the oriT mutations MutIR and Mut23-25, the minimal oriTw (oriT 14+3), and the human sequences resembling the R388 nic site, HuX and Hu5. B. DNA analysis of integrants obtained with recipient plasmids containing the indicated target sequences. Symbols as in Fig. 2. RP containing the minimal oriT is around 300 bp shorter than RP with full-length oriT.

Figure 5. Integration assay in the bacterial chromosome.

A. Scheme of the expected integrants in the chromosomal oriT copy of recipient strains CMS1 or CMS2. Symbols as in Fig. 1. B and C. PCR amplification with oligonucleotides P_A and P_B, flanking the lacZ gene (in B), or with P_A and P_C, annealing only to the cointegrate (in C). C1, C2 and HMS, strains CMS1, CMS2, and HMS174, used as a negative control. Int C1, integrants obtained with recipient strain CMS1. The black arrow indicates the amplification product of the suicide plasmid integrated in the chromosomal oriT copy. HL: MW marker.

Figure 6. A model for TrwC-mediated site-specific integration.

A. TrwC (blue oval) arrives to the recipient cell covalently bound to the suicide plasmid (dashed line) through the Y18 residue. The recipient contains the recipient plasmid (red lines). TrwA (green spheres) and IHF (yellow ovals) sit on the oriTw forming a relaxosome conformation which increase the exposure of ssDNA and the nic site (blue triangle). B. The incoming TrwC-DNA complex has a free Y26 residue. Y26 nicks (orange star) and binds covalently to the recipient nic site. C. Strand-transfer reactions are produced by the attack of the free –OH groups generated to Y18 covalently bound to the suicide plasmid and to Y26 attached to the recipient plasmid. As a result, the transferred DNA strand is integrated into the recipient plasmid. D. The host replication machinery duplicates the integrated DNA. E. Two molecules are obtained, the cointegrate and the recipient plasmid (nic sites indicated by triangles). The cointegrate can be resolved by TrwC-mediated site-specific recombination (enhanced by TrwA), producing the two initial molecules. The suicide plasmid is lost, since it cannot be replicated in the recipient cell.