Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer

Abstract

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but terminationproficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3 terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.Intercellular transfer of plasmid DNA during bacterial conjugation is accomplished by the function of transfer genes borne on each conjugative plasmid (for reviews, see references 4 and 15). All conjugative and mobilizable plasmids, such as R64, F, RP4, and R1162, contain oriT sites as cis elements which function as the origin of transfer of plasmid DNA. At the initiation stage of DNA transfer, a site-and strand-specific nick is introduced into the oriT site with a covalent attachment of the cognate relaxase protein to the 5Ј terminus of the nicked strand. The nicked strand is transferred from donor to recipient cells with the 5Ј terminus leading through a putative channel. In the donor cells, replacement strand DNA synthesis reconstitutes the double-stranded plasmid DNA. After one round of DNA transfer, the relaxase-attached 5Ј terminus of the transferred-strand DNA is religated to its 3Ј terminus to reconstitute the circular structure, and complementary-strand synthesis establishes double-stranded plasmid DNA in the recipient cells. Among these steps, the mechanisms by which initiation and termination of conjugative DNA transfer occur are important issues which remain to be elucidated.Initiation and termination of conjugative DNA transfer at the oriT site were extensively studied using a small mobilizable plasmid, R1162 (RSF1010). R1162 oriT consists of a specific nick site and a 10-bp inverted repeat with one mismatch, which is situated 8 bp from the nick site (3, 27). Three R1162 proteins, MobA, MobB, and MobC, form a protein-DNA complex called the relaxosome at oriT (27). From ...

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

“…An additional 20-generation growth of E. coli cells increased the ratio of the recombinant plasmid up to 95% (data not shown). Such recombination was also previously found when pKK541 was mobilized by the mini-R64 plasmid pKK610a (10). However, the oriT-specific recombination in pKK561 appears to be independent of conjugation, since we incubated E. coli cells without recipient cells.…”

“…E. coli K-12 strain NF83 recA (10) was used for the oriT-specific recombination of the dual oriT plasmids. E. coli K-12 JM109 FЈ gyrA96 recA (36) and MV1184 FЈ recA rpsL (34) were used for the propagation of the dual oriT phages.…”

Identification of the Origin of Transfer (oriT) and a New Gene Required for Mobilization of the SXT/R391 Family of Integrating Conjugative Elements

“…These repeats might be involved in the specific recognition of the oriT locus by the relaxase and/or accessory proteins, allowing the single-strand, site-specific cleavage reaction to take place at the nic site (21,24,39).…”

Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system

“…Plasmid pSKU146 contains two identical 175 bp direct repeat units, designated Repeat Alpha (bases 377-551) and Repeat Beta (bases 1160-1334), in each of which is embedded the sequence ATCCTG, a sequence that is conserved at the oriT nick region among conjugative plasmids (Furuya and Komano, 2000;Herrera-Cervera et al, 1998). The ATCCTG in pSKU146 is part of an imperfect inverted repeat, that can potentially form a hairpin structure important for relaxosome formation and endonuclease protein binding that results in a single-stranded nick required for plasmid mobilization during conjugation.…”