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 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...

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

“…Single-tyrosine relaxases could achieve resolution of CPR intermediates through relaxase multimerization (45) or cooperation with a second nonrelaxase protein (46). The ability to resolve undesirable replication products would also confer oriT specific recombinase activity between plasmids (47,48) or between tandem oriT repeats on the same plasmid (46,49). However, for our purposes, a key prediction that arose from this model is that multityrosine relaxases are capable of accommodating two phosphotyrosine intermediates simultaneously within their active sites.…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…Where characterized, relaxases covalently bound to the 5Ј end of T strands carry C-terminal signals specifying DNA substrate-channel interactions. Moreover, several relaxases including MobA RSF1010 , VirD2 At , TraA pATC58 , and TrwC R388 are bona fide substrates of T4SS even without any associated DNA, as demonstrated by the Cre reporter assay for translocation (84,214,239,269). With this assay, it was also shown that the C-terminal 50 residues of MobA RSF1010 are sufficient to mediate Cre translocation (269).…”

“…The relaxase also "pilots" the T strand through the translocation channel. In the recipient cell, the relaxase catalyzes the recircularization of the T strand and may also participate in second-strand synthesis or recombination into the chromosome (52,84,105).…”

Mobility of Plasmids

“…It is also thought to energize the process of DNA transport (135,136). The conjugative mating channel is basically a protein secretion channel, which transports the relaxase protein bound to the DNA to be transferred (43,58). According to the nomenclature of protein secretion mechanisms, it is a T4SS (54).…”