NikAB- or NikB-Dependent Intracellular Recombination between Tandemly RepeatedoriTSequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors

Abstract

The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.Conjugative transfer of plasmid DNA is initiated and terminated at a specific site termed the origin of transfer, oriT. During conjugation, site-and strand-specific cleavage is introduced into oriT by a specific nicking enzyme called relaxase (for reviews, see references 4 and 17). After nicking, the relaxase protein remains covalently attached to the 5Ј phosphate of the nicked strand. The nicked strand is displaced from the complementary strand and transferred from the donor to the recipient cells with the 5Ј terminus leading. In the recipient cells, the transferred strand is recircularized by phosphotransfer from the 5Ј end to its 3Ј hydroxyl group. The complementary strand is synthesized to establish the double-stranded circular plasmid. In the donor cells, DNA transfer is accompanied by rolling-circle DNA synthesis of the replacement strand, which resembles the replication of single-stranded DNA phages, such as X174, and rolling-circle plasmids of grampositive bacteria (35).Various conjugative and mobilizable plasmids, such as F, R388, RP4, R1162 (RSF1010), and R64, carry their own specific oriT sites (17). Each oriT sequence is recognized by its cognate proteins, consisting of a specific relaxase and some auxiliary protein(s). Three major groups of oriT-relaxase systems have been identified (17). The P-type oriT of R64, RP4, and R751 carries the sequence YATCCTG/Y (the shill represents the nick site) at the nick site (6,11,26). The relaxases R64 NikB and RP4 and R751 TraI share conserved motifs I, II, and III at their N-terminal positions (18,29). The tyrosine residue in motif I and the histidine residue in motif III constitut...

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.…”

“…Although CPR may not be the primary conjugative pathway, plasmids with relaxases capable of resolving CPR intermediates would be expected to have a selective advantage. 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).…”

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

“…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.…”

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

“…Conversely, on single-stranded DNA (ssDNA) substrates NikB was sufficient to promote recombination, although NikA does accelerate the reaction. A hypothetical role for NikA in the generation of ssDNA for efficient recombination was proposed by those authors (14). Similarly, in TrwC-mediated recombination, we have observed a dependence on situations such as replication or transcription that favors ssDNA exposure at recombination locus oriT1 (4).…”

“…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). Conversely, on single-stranded DNA (ssDNA) substrates NikB was sufficient to promote recombination, although NikA does accelerate the reaction.…”