Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

Abstract

Conjugative relaxases are the proteins that initiate bacterial conjugation by a site-specific cleavage of the transferred DNA strand. In vitro, they show strand-transferase activity on single-stranded DNA, which suggests they may also be responsible for recircularization of the transferred DNA. In this work, we show that TrwC, the relaxase of plasmid R388, is fully functional in the recipient cell, as shown by complementation of an R388 trwC mutant in the recipient. TrwC transport to the recipient is also observed in the absence of DNA transfer, although it still requires the conjugative coupling protein. In addition to its role in conjugation, TrwC is able to catalyze site-specific recombination between two origin of transfer (oriT) copies. Mutations that abolish TrwC DNA strand-transferase activity also abolish oriT-specific recombination. A plasmid containing two oriT copies resident in the recipient cell undergoes recombination when a TrwC-piloted DNA is conjugatively transferred into it. Finally, we show TrwC-dependent integration of the transferred DNA into a resident oriT copy in the recipient cell. Our results indicate that a conjugative relaxase is active once in the recipient cell, where it performs the nicking and strand-transfer reactions that would be required to recircularize the transferred DNA. This TrwC site-specific integration activity in recipient cells may lead to future biotechnological applications.

Fig. 1.

Scheme of triparental matings. Bacteria are represented by ovals containing the respective plasmids. Antibiotic resistance of strains and mobilizable plasmids is indicated. Plasmid pSU1371, containing R388-oriT, is mobilized from strain 1 by nonmobilizable helper plasmids pET29:trwABC (providing R388 trwA, trwB, and trwC genes) and pKM101Δmob (providing the T4SS). TrwC is represented by a diamond, leading or not the transferred DNA (wavy line) into a recipient cell. TrwC in strain 2 is able or not to complement the R388 mutants (strain 2, left and right, respectively), leading to transconjugants or lack thereof in strain 3 (strain 3 left and right, respectively). Nx, nalidixic acid; Sm, streptomycin; Rf, rifampicin.

Fig. 2.

Assays for TrwC-mediated oriT-specific recombination. (a) Structure of the recombination cassette and the resulting product after recombination between the two oriT copies (black circles). Arrows point in the direction of transcription. Plac, lactose promoter. npt II, neomycin phosphotransferase gene conferring Km resistance. (b) DH5α (pRec2oriT-Cm) colonies transformed with pET3::trwAC and plated on CmAp X-Gal plates. Each blue sector originated from a single recombination event. (c) Assay for TrwC transfer into recipient cells carrying the recombination cassette. Helper nonmobilizable plasmids in the donor are indicated in brackets. Relaxases are shown as diamonds leading the DNA (wavy line) into recipient cells.

Fig. 3.

Analysis of integrants. (a) Structure of the oriT-containing plasmids present in donor (pR6K::oriT_W) and recipient (pKK::oriT) cells and of the resulting cointegrant upon integration. Arrows point in the direction of transcription or DNA transfer. Small inner arrows indicate the annealing sites for the primers used in PCR reactions. (b) DNA analysis of integrants. Lanes 1-6, NdeI restriction digestion. Lane 7, 1-kb ladder (sizes in kb from the bottom of the gel: 1.0-1.5-2.0-2.5-3.0-4.0-5.0-6.0-8.0-10). Lanes 8-14, products of PCR reactions with oligonucleotides M13R (-48) and BamHindnptIIR (see Table 6, which is published as supporting information on the PNAS web site), which anneal 5′ to the vector Ptac promoter and 3′ to the npt II gene, respectively. Lanes 1-4 and 8-11, four different colonies obtained in the integration assays. Lanes 5 and 12, pKK::oriT. Lanes 6 and 13, pR6K::oriT_W. Lane 14, positive control (plasmid pKK::oriT-Km). Other plasmids present in the matings (pKM101Δmob, pET29::trwABC, pKK223-3) were also PCR-negative (not shown).