Amplification of the Tetracycline Resistance Determinant of pAMα1 in
            <i>Enterococcus faecalis</i>
            Requires a Site-Specific Recombination Event Involving Relaxase

Francia, María Victoria; Clewell, Don B.

doi:10.1128/jb.184.18.5187-5193.2002

Cited by 43 publications

(36 citation statements)

References 42 publications

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“…There have been scarce reports that relate conjugative relaxases to site-specific recombination reactions (21,(35)(36)(37). How these proteins accomplish a reaction characteristic of the family of site-specific recombinases is still to be determined.…”

Section: Discussionmentioning

confidence: 99%

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

Draper

César

Machón

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

139

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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 strandtransferase 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.bacterial conjugation ͉ plasmid R388 ͉ site-specific integration ͉ strand transferase ͉ TrwC B acterial conjugation is a widespread mechanism for horizontal DNA transfer among prokaryotes. Under laboratory conditions, conjugation has also been reported between bacteria and eukaryotic cells (1-3). Any DNA molecule containing a short segment called the origin of transfer (oriT) can be conjugatively transferred to a recipient cell if the rest of the conjugation machinery is provided, either in cis or in trans. The transfer apparatus can be divided into three functional modules (4 -6). (i) A nucleoprotein complex known as relaxosome consists of oriT-binding proteins and their cognate DNA. These proteins include one relaxase plus one or more accessory nicking proteins; the relaxase introduces a site-specific nick at the oriT and remains covalently bound to the 5Ј end of the strand that is to be transferred. (ii) A type IV secretion system (T4SS) is a multiprotein complex spanning the inner and outer membranes through which the substrate is secreted. (iii) The conjugative coupling protein (T4CP) is responsible for connecting the two other functional modules. Current models for DNA transfer through T4SS, in both conjugation and the Agrobacterium tumefaciens T-DNA transfer system (7,8), postulate that a T4CP recruits the relaxosome to the T4SS via protein-protein interactions.For Ͼ20 years, models for conjugative DNA transport have postulated that the relaxase catalyzes the final recircularization step of the transferred DNA because of its strand-transfer activity (8, 9). Nevertheless, it was not until r...

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Section: Discussionmentioning

confidence: 99%

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

Draper

César

Machón

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

139

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“…Within the putative replication module (Fig. 3A, positions 1919 to 3208), one predicted product (351 amino acids) shares 38% identity to the putative replication initiation proteins of Enterococcus faecalis plasmids pS68 (GenBank accession number CAA11136) (34), pEF47 (GenBank accession number AAX44237) (42), and pAM␣1 (GenBank accession number NP863355) (16). All of the closest matches shared homology to the first 220 N-terminal amino acids of several known or putative replication initiation proteins of gram-positive and gramnegative plasmids.…”

Section: Resultsmentioning

confidence: 99%

Development of Experimental Genetic Tools for Campylobacter fetus

Kienesberger

Gorkiewicz

Joainig

et al. 2007

Appl Environ Microbiol

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Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.

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“…As for TrwC (4,9,20), a similar oriT-oriT recombination ability has been described for three additional relaxases, NikB of the IncI1 plasmid R64 (14) and the relaxases of Enterococcus faecalis plasmids pAD1 (12) and pAM␣1 (11). In the case of the oriT recombination catalyzed by NikB, the reaction on dsDNA substrates was strictly dependent on the presence of protein NikA, which is homologous to TrwA (14).…”

mentioning

confidence: 93%

TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

César

Llosa

2007

J Bacteriol

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R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a "closed" conformation. In an IHF-deficient (IHF ؊ ) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF ؊ background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an "open" form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

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Amplification of the Tetracycline Resistance Determinant of pAMα1 in Enterococcus faecalis Requires a Site-Specific Recombination Event Involving Relaxase

Cited by 43 publications

References 42 publications

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

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

Development of Experimental Genetic Tools for Campylobacter fetus

TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

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