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Journal of Bacteriology, July 2003, p. 3871-3877, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3871-3877.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Nobuhisa Furuya and Teruya Komano*

Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan

Received 2 December 2002/ Accepted 23 April 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 5' 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 5' terminus leading. In the recipient cells, the transferred strand is recircularized by phosphotransfer from the 5' end to its 3' 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 {phi}X174, and rolling-circle plasmids of gram-positive 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 constitute an active center for nicking and rejoining (18, 28, 29). The Q-type oriT of R1162 and pSC101 carries the sequence TAANWGCG/CCCT at the nick site (2). R1162 MobA and pSC101 Mob belong to the MobA-MobL relaxase family (2). The F-type oriT of F, R100, and R388 carries the sequence TGCGNNNNGTNT/RNNNC at the nick site (5, 19). F and R100 TraI and R388 TrwC carry a conserved relaxase domain at their N-terminal positions (19). The nick site sequences described above are thought to be recognized by the cognate relaxases. Each oriT contains additional specific sequences of variable lengths which are recognized by its auxiliary protein(s) (17).

The relaxase and auxiliary proteins bind to the oriT sequence to form a unique nucleoprotein complex called the relaxosome (17). Within relaxosomes, relaxases are thought to be in equilibrium between cleavage and rejoining of the specific nick site on supercoiled plasmid DNAs (29). The relaxosomes of F, R100, RP4, R388, and R1162 have been reconstructed in vitro using supercoiled oriT plasmid DNAs and purified relaxosome proteins (12, 13, 24, 25, 29, 32). In vitro experiments have shown that the purified relaxase proteins of R100 (TraI), RP4 (TraI), R388 (TrwC), and R1162 (MobA) have DNA strand transferase activities that specifically cleave and rejoin the single-stranded oligonucleotide containing their cognate nick region sequence (3, 5, 21, 28).

R64 is a conjugative plasmid belonging to the incompatibility group I1. Twenty-three genes, encoded within a 54-kb R64 transfer region, have been shown elsewhere to be essential for R64 conjugation in liquid and on surfaces (11, 14, 16, 37). An additional 12 pil genes are required only for liquid matings (37). R64 oriT is located at one terminus of the 54-kb transfer region (11). The fully active R64 oriT sequence (oriT92) consists of three subsequences (see Fig. 4), (i) the P-type nick region sequence CATCCTG/T at the nick site, (ii) the 17-bp inverted repeat sequence (repeats A and B), and (iii) the 8-bp GC-rich inverted repeat sequence (6). The oriT core sequence (oriT42) encompassing the nick region sequence and one arm (repeat A) of the 17-bp repeat sequence was shown previously to be required for relaxosome formation and to exhibit 1/25 of the oriT activity (9). Two genes, nikA and nikB, encoded in an operon adjacent to oriT were found to be essential for R64 relaxosome formation (11). NikA is a DNA-binding protein that specifically binds to the repeat A sequence (8). NikB, belonging to the RP4 TraI relaxase family (29), bears a DNA strand transferase activity that cleaves and rejoins R64 oriT DNA at the nick site.



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  		FIG. 4. oriT deletion alleles used in this study. The DNA sequences deleted from the oriT92 sequence are indicated by hyphens. The mobilization frequencies (Mob) of plasmids carrying a single copy of each oriT sequence are shown as the ratio of the transfer frequency of each oriT plasmid to that of the mini-R64 plasmid pKK607 (taken from reference 10). The bracket above repeat A shows the NikA-binding sequence from a footprint analysis (8). The nikAB-dependent deletion frequencies of the oriT-intervening sequence from the dual oriT plasmids carrying mutant oriT alleles at site 1 or site 2 were estimated from the gels shown in Fig. 2B and 3B and are expressed as the ratio (percentage) of the plasmid DNA that was subjected to oriT-specific deletion. NT, not tested.

 
In a previous study (10) using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for the initiation and termination of R64 transfer were determined by examining conjugation-mediated deletion and duplication between the repeated oriT sequences. The oriT core sequence was found to be required for the initiation of DNA transfer, indicating that the relaxosome formation is essential for initiation. In contrast, the entire oriT sequence was required for the termination of DNA transfer. Mutations in the repeat A sequence affecting the oriT initiation ability were found not to affect the termination ability, suggesting that the inverted repeat structures are important for the termination process.

Conjugation-independent site-specific recombination between two oriT sequences in Escherichia coli cells expressing the R388 trwC gene has been found for the IncW plasmid R388 (20). In the case of the broad-host-range mobilizable plasmid R1162, site-specific recombination between the two oriT sequences tandemly cloned into the M13 bacteriophage vector occurred during the phage propagation in E. coli cells, resulting in the deletion of the segment between the two oriT sequences (23). In the present study, we found a conjugation-independent site-specific recombination between tandemly repeated oriT sequences of R64 in both plasmid and M13 phage vectors. When two R64 oriT sequences were tandemly cloned in the plasmid vector pUC18, oriT-mediated site-specific recombination was observed within E. coli cells expressing the nikA and nikB genes. On the other hand, the oriT-specific recombination in M13 vectors was dependent only on nikB, provided that the nicked strand was on the viral strand of the vector phage.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, phages, and plasmids. 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 F' gyrA96 recA (36) and MV1184 F' recA rpsL (34) were used for the propagation of the dual oriT phages. The plasmid vectors pTK219 (15), pUC18, and pUC19 and the phage vectors M13mp18 and M13mp19 (36) were used.

Media. Luria-Bertani (LB) broth was prepared as previously described (31). The solid medium contained 1.5% agar. Antibiotics were added to the liquid or solid medium at the concentrations indicated: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; nalidixic acid, 25 µg/ml; streptomycin, 200 µg/ml; and tetracycline, 12 µg/ml.

Recombinant DNA techniques. The recombinant DNA techniques were performed as previously described (31). The dual oriT plasmids, pKK561 through pKK565 and pKK565r (see Fig. 3A), were constructed by inserting various oriT sequences and the tetracycline resistance (Tcr) gene cassette into the multicloning sites of pUC18 and pUC19, respectively. The dual oriT phages, {phi}KK18 through {phi}KK23 (see Fig. 5A), were constructed by inserting various oriT sequences into the multicloning sites of M13mp18 and M13mp19.



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  		FIG. 3. Effects of oriT deletion on the intracellular recombination at R64 oriT. (A) Structure of the dual oriT plasmids. The arrangement of the two oriT sequences with or without deletion mutations in each plasmid is listed. The frequency of oriT-specific deletion of each plasmid was estimated from the gel shown in panel B and is expressed as the ratio (percentage) of the deletion plasmid. In pKK561 to pKK565, the oriT nick strand is located on the leading strand of the DNA replication as shown in Fig. 1A, while in pKK561r, it is located on the lagging strand. (B) Each dual oriT plasmid was introduced into E. coli NF83 cells harboring the nikAB+ plasmid pKK518b. Plasmid DNA was extracted from the E. coli cells grown in LB medium, linearized by digestion with ScaI and BstPI, and subjected to agarose gel electrophoresis. DNA bands from pKK518b, dual oriT plasmids, and recombinant plasmids are indicated. The molecular lengths (in kilobases) are indicated on the right.

 


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  		FIG. 5. The oriT-specific deletion in a single-stranded phage. (A) Structure of the dual oriT phages. Two R64 oriT sequences with or without deletion mutations (Fig. 4) were tandemly cloned into the multicloning sites of M13mp18 ({phi}KK18, {phi}KK21, {phi}KK22, and {phi}KK23) or M13mp19 ({phi}KK19). In {phi}KK18, {phi}KK21, {phi}KK22, and {phi}KK23, the oriT nick strand is located on the viral strand (+) as shown in Fig. 1B, while in {phi}KK19 it is located on the nonviral strand (-). The frequencies of nikB- or nikAB-dependent deletions of the oriT-intervening sequence from each phage were estimated from the gel shown in panel B and are expressed as the ratio (percentage) of the deletion phage. (B) The indicated phages were used to infect E. coli JM109 cells harboring pTK219 (lanes 1, 7, 10, and 13), pKK518b (lanes 3, 5, 9, 12, and 15), pKK519b (lanes 2, 4, 8, 11, and 14), or pKK520b (lane 6). The resultant plaques were used to infect E. coli MV1184 cells. RF DNAs were extracted, digested with EcoRI and HindIII, and analyzed by 4% polyacrylamide gel electrophoresis. *, deletion of the NikB C-terminal region (pKK520b). Original, the EcoRI-HindIII fragments from the dual oriT phages; deletion, smaller EcoRI-HindIII fragments from the phages which underwent oriT-specific recombination.

 
oriT-specific recombination in a plasmid vector. Each dual oriT plasmid was transformed into E. coli NF83 cells harboring pKK607, pKK617, pKK518b, pKK519b, or pKK520b. The Apr and Kmr colonies were picked and cultured overnight in 2 ml of LB medium containing both ampicillin and kanamycin. Plasmid DNAs were extracted from the overnight cultures by the alkaline lysis method (31) and examined by restriction enzyme analysis.

oriT-specific recombination in a single-stranded phage vector. An overnight culture (0.1 ml) of E. coli JM109 cells harboring pTK219, pKK518b, pKK519b, or pKK520b was infected with diluted dual oriT phage solutions, plated, and incubated overnight at 37°C. Each single plaque was picked and transferred into 5 ml of LB medium containing streptomycin (200 µg/ml) and 10 µl of an overnight culture of E. coli MV1184. After overnight growth of the inoculated culture, the cells were recovered by centrifugation, and replicative-form (RF) DNA was extracted by the alkaline lysis method and examined by restriction enzyme analysis.

Estimation of recombination efficiency. The recombination efficiencies of dual oriT plasmids and dual oriT phages were estimated from the intensities of the ethidium bromide-stained DNA bands from the original and recombinant plasmids. The intensities of the DNA bands were estimated with NIH Image software.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intracellular recombination between two tandemly repeated R64 oriT sequences. In a previous study (10), we investigated the subregions of R64 oriT required for the initiation and termination of DNA transfer, using dual oriT plasmids, such as pKK541, based on pHSG398 (33). In the present study, we tandemly cloned two R64 oriT92 sequences into another vector, pUC18 (Fig. 1A). A tetracycline resistance gene (Tcr) was inserted between the two oriT sequences. The directions of the repeated oriT sequences in comparison to the replication of the vector plasmid were arranged so that the oriT nick strand corresponded to the leading strand of the DNA replication (Fig. 1A).



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  		FIG. 1. (A) Deletion of the Tcr segment from the dual oriT plasmid by oriT-mediated recombination occurs within E. coli cells expressing the nikA and nikB genes. The arrowheads indicate the 5' ends of the nicked strand at oriT. The arrows at the oriV site indicate the direction of DNA replication of the pMB1 replicon. Apr, ampicillin resistance gene; Tcr, tetracycline resistance gene. (B) Deletion of the DNA segment from the dual oriT single-stranded phage by oriT-specific recombination occurs within E. coli cells expressing the nikB gene. The arrowheads indicate the 5' ends of the nicked strand at oriT. (C) Recombination at oriT is illustrated. The fully active oriT92 sequences are depicted by boxes. The arrows and arrowheads within the boxes represent the 17- and 8-bp inverted repeats, respectively. The downward arrowheads above the boxes represent the nick sites introduced into the top strand.

 
The resultant dual oriT plasmid pKK561 was introduced into E. coli NF83 recA cells harboring the self-transmissible mini-R64 plasmid pKK607. Plasmid DNA was extracted from an overnight culture of E. coli cells, linearized at the unique ScaI site, and subjected to agarose gel electrophoresis. A plasmid smaller than pKK561 was seen in addition to the original one in E. coli cells harboring pKK561 and pKK607 (Fig. 2B, lane 1). Restriction mapping and DNA sequence analysis indicated that the small plasmid lacked the Tcr segment and carried only one oriT92 sequence, suggesting that the small plasmid was formed by recombination between the tandemly repeated oriT sequences (Fig. 1A and C). It was estimated that 65% of the plasmids carried the deletion. 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. It should be noted that conjugation between donor cells does not occur because of surface exclusion (7). When pKK561 was introduced into E. coli cells harboring pKK617, which lacked the nikAB genes in comparison to pKK607 and was transfer deficient, no deletion from pKK561 was observed (Fig. 2B, lane 2), indicating that the nikA and/or nikB gene is required for the oriT-specific recombination.



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  		FIG. 2. The oriT-specific intracellular recombination of the dual oriT plasmid is dependent on both the nikA and nikB genes. (A) Structure of the plasmids carrying the nikA and/or nikB gene. The oriT92 sequence is indicated by a hatched bar. The short and long wedges indicate the coding sequences of the nikA and nikB genes, respectively. B, BglII; E, EcoRI; X, XhoI; A and F, only the relevant restriction sites for AluI and FnuDII, respectively, are shown. (B) The dual oriT plasmid pKK561 was introduced into E. coli cells harboring the indicated plasmids. After overnight growth of the E. coli cells in LB medium, plasmid DNA was extracted, digested with BstPI and ScaI, and analyzed by agarose gel electrophoresis followed by ethidium bromide staining. DNA bands from nikAB+ plasmids are indicated by a bracket. The molecular lengths (in kilobases) are indicated on the right.

 
Requirement of both the nikA and nikB genes for the intracellular recombination of the dual oriT plasmid. To examine the role of the nikAB gene for the oriT-specific recombination, pKK561 was introduced into E. coli cells harboring pKK518b which contained the R64 nikA and nikB genes (Fig. 2A). After overnight growth of the transformed cells, a similar level of the small plasmid with the deletion was produced from pKK561 (Fig. 2B, lane 3). This result confirmed that the oriT-specific recombination in pKK561 was conjugation independent, since many other tra genes are strictly required for R64 conjugation (16). To examine whether both the nikA and nikB genes were required for the recombination, pKK519b and pKK520b were constructed, containing either the nikB or the nikA gene, respectively (Fig. 2A). When pKK561 was introduced into E. coli cells harboring pKK519b or pKK520b, no DNA deletion in pKK561 was detected (Fig. 2B, lanes 4 and 5), indicating that both the nikA and nikB genes are required for the intracellular recombination of the dual oriT plasmid.

Effects of oriT mutations on the intracellular recombination of the dual oriT plasmid. We previously showed that, in the conjugation-dependent recombination, the oriT44 core sequence was sufficient for the initiation of conjugation at site 1, while the fully active oriT92 sequence was required for the termination of conjugation at site 2 (10). To investigate the requirements of the oriT subregions for the oriT-specific recombination, we introduced oriT deletion mutations into sites 1 and 2 of the dual oriT plasmids (Fig. 3A). The DNA sequences of the various oriT deletion alleles are shown in Fig. 4. To construct pKK562, oriT64 lacking a 28-bp upstream sequence (Fig. 4) of oriT92 was inserted into site 2 of the dual oriT plasmid. The effect of the oriT deletion in pKK562 was prominent, since only 17% deletion was observed in E. coli cells harboring pKK562 and pKK518b (Fig. 3B, lane 2). pKK563, carrying oriT52 at site 2, exhibited no recombination (Fig. 3B, lane 3). These results strongly suggest that the oriT92 sequence with the 8- and 17-bp inverted repeats is required at site 2 for the intracellular recombination of the dual oriT plasmid.

Next, the oriT deletion alleles were inserted into site 1 of the dual oriT plasmid. pKK564, carrying oriT44 at site 1, exhibited the same recombination efficiency as did pKK561 (Fig. 3B, lane 4). However, introduction of a further deletion into the repeat A sequence (oriT32 in pKK565) completely diminished the recombination activity (lane 5). This finding is consistent with the result that oriT32 did not exhibit any mobilization activity (Fig. 4). Thus, the sequence requirement at the site 1 oriT is not equal to that of the site 2 oriT, indicating that the two oriT sequences may play different roles during recombination. The requirement of the site 1 and site 2 oriT sequences for conjugation-independent recombination of the dual oriT plasmids is similar to that for conjugation-dependent oriT recombination (10).

oriT-specific recombination did not occur when the orientations of the two oriT sequences were reversed in comparison to the vector sequence. We tested the effects of the orientations of the repeated oriT sequences in comparison to the vector plasmid on the oriT-specific recombination. With the use of the vector plasmid pUC19, pKK561r was constructed so that the directions of the two oriT92 sequences and the Tcr segment were reversed in comparison to those of pKK561. In E. coli NF83 cells harboring pKK561r and pKK518b, no deletion of the Tcr segment was observed in pKK561r (Fig. 3B, lane 6), suggesting that the oriT-specific recombination is dependent on the orientations of the two oriT sequences in comparison to the vector sequence, and most likely on the direction of replication of the vector plasmid.

oriT-specific recombination in a single-stranded phage vector: requirement for nikB. Intracellular recombination between two tandemly repeated oriT sequences of the mobilizable plasmid R1162 cloned into the M13 single-stranded phage vector has been reported elsewhere (1, 23). To examine whether R64 oriT-specific recombination occurs in a single-stranded phage vector, two tandemly repeated oriT92 sequences were cloned into the multicloning sites of M13mp18 and M13mp19 so that the nick strand was located on the plus and minus strand, respectively (Fig. 1B and 5A). The resultant phage DNAs, {phi}KK18 and {phi}KK19, were introduced into E. coli JM109 cells harboring pTK219, pKK518b nikAB+, pTK519b nikB+, or pKK520b nikA+. From the infected cells, RF DNAs were extracted, digested with EcoRI and HindIII, and analyzed by polyacrylamide gel electrophoresis (Fig. 5B). In E. coli JM109 cells harboring the pTK219 vector, no recombination was observed for either {phi}KK18 or {phi}KK19 (Fig. 5B, lane 1; data not shown for {phi}KK19). When propagated in E. coli JM109 cells expressing the nikB gene, almost all of the original 280-bp EcoRI-HindIII fragment of {phi}KK18 DNA was converted into the 147-bp recombinant one, whereas 41% of the original fragment of {phi}KK19 DNA was converted into the recombinant one (Fig. 5B, lanes 2 and 4). DNA sequence analysis of the recombinant phages produced from {phi}KK18 and {phi}KK19 revealed that DNA recombination occurred between the two oriT sequences of {phi}KK18 and {phi}KK19, respectively (Fig. 1B and C). When the recombinant phages were propagated in E. coli cells expressing both nikA and nikB, almost all of both {phi}KK18 and {phi}KK19 underwent recombination (Fig. 5B, lanes 3 and 5). pKK520b encodes nikA and truncated nikB lacking the C-terminal segment but retaining the relaxase domain (Fig. 2A). Propagation of {phi}KK18 in E. coli cells harboring pKK520b produced no recombinant phages (Fig. 5B, lane 6). These results indicate that the oriT-specific recombination of the dual oriT phages depends on the entire nikB, while nikA accelerates the recombination.

Effects of oriT mutations on the recombination of the dual oriT phages. To determine whether the 17- and 8-bp inverted repeat sequences within R64 oriT were required for the recombination in the phage vector, {phi}KK21, {phi}KK22, and {phi}KK23 were constructed. {phi}KK21 and {phi}KK22 carried the oriT44 allele at sites 2 and 1, and the oriT92 allele at sites 1 and 2, respectively (Fig. 5A). The propagation of {phi}KK21 and {phi}KK22 phages in E. coli cells harboring pKK519b nikB+ resulted in 70 and 72% of deletion phages by oriT-specific recombination, respectively (Fig. 5B, lanes 8 and 11). When {phi}KK21 and {phi}KK22 phages were propagated in E. coli harboring pKK518b nikAB+, almost all of the resultant phages were recombinant phages (Fig. 5B, lanes 9 and 12). {phi}KK23 contained oriT31 and oriT44 at sites 1 and 2, respectively. oriT31 lacked all the inverted repeat structures but retained the sequences sufficient for relaxosome formation (Fig. 4). No oriT-specific recombination was observed when {phi}KK23 was propagated in E. coli cells carrying pKK519b nikB+, while 70% recombination was detected in E. coli cells carrying pKK518b nikAB+ (Fig. 5B, lanes 14 and 15). These results suggest that the intracellular oriT-specific recombination in single-stranded phage vectors occurs at a frequency higher than that in plasmid vectors.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, intracellular recombination between two tandemly repeated R64 oriT sequences in plasmid and single-stranded phage vectors was examined. Recombination in the plasmid vectors was dependent on both nikA and nikB. Recombination in the phage vectors was dependent on nikB, while nikA facilitated the recombination. In general, oriT-specific recombination in phage vectors was more frequent than that in plasmid vectors (compare Fig. 3 and 5).

Intracellular recombination between two R64 oriT sequences in a single-stranded phage is easy to understand, since relaxase-dependent cleavage and rejoining of single-stranded oriT DNA have been demonstrated in vitro (3, 5, 21, 28). Each relaxase cleaves the transfer strand of cognate oriT DNA at the nick site, producing a relaxase-DNA intermediate linked by a phosphodiester bond between the 5' end of the recombining DNA and the tyrosine residue of the relaxase protein. The relaxase-DNA intermediate can react with oriT DNA with a 3' free hydroxyl group at the nick site, yielding a recombinant oriT DNA. Only a limited length of the oriT sequence is required for the in vitro cleavage and rejoining reaction, while a much longer oriT sequence is usually required for efficient conjugation. Our preliminary experiments indicated that purified R64 NikB protein also carries such activity (N. Furuya, unpublished results). Therefore, NikB-dependent cleavage and rejoining between tandemly repeated oriT sequences in single-stranded phages in E. coli cells may result in the deletion of the intervening segment. The present results revealed that longer oriT sequences facilitated the recombination, suggesting that the nick site-distal sequences are also involved in the recognition of the oriT sequence by NikB. It is likely that the oriT inverted repeat structures are recognized by NikB at the stage of ligation between the 5' and 3' ends of the cleaved oriT DNA. Although recombination occurred in the E. coli cells expressing nikB, the presence of nikA facilitated the reaction. NikA protein may enhance the recombination in the RF of the dual oriT phages. oriT-specific recombination on the plus strand was more frequent than on the minus strand (compare {phi}KK18 and {phi}KK19 in Fig. 5). It is likely that the plus strand exists in a single-stranded state more often than the minus strand does during phage replication in E. coli cells (30).

In R1162, intracellular recombination between the two oriT sequences tandemly cloned into the M13 vector was reported elsewhere (1, 23). Only a 204-amino-acid N-terminal region of the 708-amino-acid MobA protein was sufficient for recombination between the two oriT sequences in single-stranded phages (23), whereas the entire R64 NikB protein was necessary for the oriT-specific recombination. The in vitro cleavage and rejoining of single-stranded R1162 oriT DNA by use of a LacZ fusion protein containing the N-terminal half of R1162 MobA were reported previously (3). In addition, deletion of an oriT-intervening segment from a single-stranded DNA substrate containing two tandem oriT sequences was also demonstrated in vitro.

Intracellular recombination between two R64 oriT sequences in a plasmid vector is highly related to the conjugal DNA processing. Both the nikA and nikB genes were required for this recombination. The requirements of the site 1 and site 2 oriT sequences for intracellular recombination were similar to those of the oriT sequences required for the initiation and termination of conjugative DNA transfer (10), respectively. It is likely that the relaxosome formed at the site 1 oriT may provide a NikB-oriT (site 1) intermediate. To form the relaxosome at oriT, NikA protein is also required, while the oriT core sequence (oriT44) is sufficient. Subsequently, the NikB-oriT (site 1) intermediate may react with the free 3' hydroxyl group produced by the second cleavage with NikB at the site 2 oriT, resulting in recombination between the site 1 and site 2 oriT sequences. Our observation that the entire 92-bp oriT sequence at site 2 oriT is required for efficient recombination suggests that the inverted repeat structures within the oriT92 sequence are required for the recombination. The two inverted repeat sequences were also shown elsewhere to be required for efficient termination during R64 DNA transfer (10). The inverted repeat sequences may form a transient hairpin-loop structure during a DNA processing reaction such as conjugative DNA transfer or DNA replication. For the reaction of the free 3' hydroxyl group at the nick site of site 2 oriT with the NikB-oriT complex (site 1), a transient formation of hairpin-loop structures within site 2 oriT may be required. It is noteworthy that the directions of the tandemly repeated oriT sequences in comparison to the direction of replication of the vector plasmid were crucial for recombination. Since the mode of replication of pUC18 derived from pMB1 is unidirectional (22), the transfer strand is subjected to lagging-strand DNA synthesis. This may increase the frequency of a single-stranded state of the transfer strand in DNA replication.

In a previous study (10), conjugation-dependent deletion of the oriT-intervening sequence from a similar dual oriT plasmid, pKK541, based on pHSG398, another pMB1-derived vector plasmid carrying the chloramphenicol resistance gene (cat) (33), was observed. However, no intracellular oriT-specific recombination of pKK541 was observed within E. coli donor cells expressing the nikAB genes (Furuya, unpublished). In addition, intracellular recombination of pKK541r, with dual oriT insertion in the opposite direction, was also not observed. On the other hand, nikAB-dependent intracellular recombination was observed in the dual oriT plasmids, based on other Apr pMB1-derived plasmids, pBluescript SK(+) and pUC118 (31), equivalent to pKK561, whereas no recombination was observed in the counterpart dual oriT plasmids, equivalent to pKK561r. The reason for these findings is currently unknown. One possible explanation is that the direction of replication is the same as the direction of bla transcription in pUC-derived vectors, while it is the reverse in comparison to the direction of cat transcription in pHSG-series plasmids.

In conjugation-independent site-specific recombination between the two oriT sequences of plasmid R388, deletion of the oriT-intervening sequence was observed in E. coli cells expressing trwC which encoded the R388 relaxase-helicase protein (20). It was also found that not only intramolecular recombination but also intermolecular recombination between two oriT sequences separately cloned into two plasmids could occur (20). R388 trwA, encoding a homologue of R64 NikA protein, was not required for the intracellular R388 oriT recombination, while it was required for R388 conjugation (19, 20). Only a 272-amino-acid N-terminal segment of the 966-amino-acid TrwC protein, which corresponds to the domain for the relaxase activity, was sufficient for the oriT-mediated recombination (21). In contrast, the entire R64 NikB protein was necessary for oriT-specific recombination. We have previously shown that a 318-amino-acid N-terminal segment of the 899-amino-acid NikB protein was sufficient for in vitro relaxation of the oriT plasmid (11). This segment contains the motif I, II, and III sequences conserved in the RP4 TraI family of relaxases (29). The function of the NikB C-terminal domain is currently unknown. Its possible function is the dimerization of NikB monomers, since it was proposed elsewhere that RP4 TraI relaxase acts in a dimeric form at the rejoining step of the cleaved oriT sequences during the termination of conjugative DNA (27). Alternatively, it is possible that the C-terminal segment of R64 NikB relaxase may be directly involved in the termination of DNA transfer.

For further clarification of the DNA processing during R64 conjugal transfer, in vitro experiments using purified NikA and NikB proteins and oriT DNA are required. Purification of NikB protein and in vitro analyses are under way in our laboratory.


   ACKNOWLEDGMENTS
 
This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan. Phone: 81-426-77-2568. Fax: 81-426-77-2559. E-mail: komano-teruya{at}c.metro-u.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, July 2003, p. 3871-3877, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3871-3877.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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