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

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 termination-proficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3' terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.     

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.     

Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.     

Specific binding of the NikA protein to one arm of 17-base-pair inverted repeat sequences within the oriT region of plasmid R64.

Products of the nikA and nikB genes of plasmid R64 have been shown to form a relaxation complex with R64 oriT DNA and to function together as an oriT-specific nickase. We purified the protein product of the nikA gene. The purified NikA protein bound specifically to the oriT region of R64 DNA. Gel retardation assays and DNase I footprinting analyses indicated that the NikA protein bound only to the right arm of 17-bp inverted repeat sequences; the right arm differed from the left arm by a single nucleotide. The binding site is proximal to the nick site and within the 44-bp oriT core sequence. Binding of the NikA protein induced DNA bending within the R64 oriT sequence.     

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.     

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT.

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.     

Streptococcal plasmid pIP501 has a functional oriT site.

DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.     

Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer.

R1162 is efficiently comobilized during conjugative transfer of the self-transmissible plasmid R751. Bacteriophage M13 derivatives that contain two directly repeated copies of oriT, the site on R1162 DNA required in cis for mobilization, were constructed. Phage DNA molecules underwent recombination during infection of Escherichia coli, with the product retaining a single functional copy of oriT. Recombination was strand specific and depended on R1162 gene products involved in mobilization, but did not require the self-transmissible plasmid vector. Two genes were identified, one essential for recombination and the other affecting the frequency of recombination. Recombination of bacteriophage DNA could form the basis of a simple model for some of the events occurring during conjugation without the complexity of a true mating system.     

Deletion analysis of the F plasmid oriT locus.

Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis. DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans. Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval. Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site. Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function. These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.     

A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase.

The polypeptide encoded by a segment of a gene required for the conjugal mobilization of the broad host-range plasmid R1162 has been purified as a beta-galactosidase fusion protein. The hybrid protein binds specifically to a small, double-stranded DNA fragment containing the origin of transfer (oriT), and specifically cleaves oriT single-stranded DNA at the position cleaved during transfer. Only one of the two DNA strands is a substrate. A fraction of the digested DNA is resistant to lambda exonuclease digestion, indicating that some molecules have protein covalently attached at the 5' end. After prolonged incubation with fusion protein, some of the cleaved molecules are religated. In vivo, M13 phage DNA containing two, directly-repeated copies of oriT recombine in cells containing the fusion protein. The single-stranded viral DNA forms are the probable substrates for the protein, the cleaved DNA being subsequently religated to form recombinant molecules. Cleavage of the DNA might be the reverse reaction of the ligation that normally takes place after conjugative transfer of a single, linear plasmid DNA strand.     

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.     

DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA

The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer.     

Recombination between directly repeated origins of conjugative transfer cloned in M13 bacteriophage DNA models ligation of the transferred plasmid strand.

When two, directly-repeated copies of the origin of transfer (oriT) of the conjugatively mobilizable, broad host-range plasmid R1162 are cloned into bacteriophage M13mp9 DNA, they undergo recombination in the presence of one of the R1162-encoded proteins required for mobilization [Meyer, R. (1989) J. Bacteriol., 171, 799-806]. Mutations in the outer arm of the inverted repeat within oriT inhibit this recombination. These mutations also affect a late step in transfer. We propose that recombination on the phage DNA models the processing of single-stranded DNA after entry into a recipient cell. The two, directly-repeated oriTs are not equivalent during the recombination reaction, because they are differently affected by the outer-arm mutations. A mutation was also isolated that reduces the specificity of the cleavage site in one of the two oriTs. Together, the results with the mutations suggest that phage recombinants can form only when the first cleavage occurs at one of the two oriTs. This is followed by the resulting free 3' end joining to the 5' end at the cleavage site of the other oriT.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization. Evidence for genetically distinct events at oriT

A segment of R1162 DNA containing genes for conjugative mobilization (Mob) and the origin of transfer (oriT) was integrated into the Escherichia coli chromosome. Bacterial genes were transferred in a polar fashion during conjugative mobilization of the integrated segment by a self-transmissible plasmid vector. The direction of transfer, together with the properties of mutated derivatives of oriT, indicate that initial cleavage of oriT, and subsequent religation after transfer, entail different mechanisms that can be distinguished genetically.     

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.     

Specificity determinants of conjugative DNA processing in the Enterococcus faecalis plasmid pCF10 and the Lactococcus lactis plasmid pRS01

The DNA-processing region of the Enterococcus faecalis pheromone-responsive plasmid pCF10 is highly similar to that of the otherwise unrelated plasmid pRS01 from Lactococcus lactis. A transfer-proficient pRS01 derivative was unable to mobilize plasmids containing the pCF10 origin of transfer, oriT. In contrast, pRS01 oriT-containing plasmids could be mobilized by pCF10 at a low frequency. Relaxases PcfG and LtrB were both capable of binding to single-stranded oriT DNAs; LtrB was highly specific for its cognate oriT, whereas PcfG could recognize both pCF10 and pRS01 oriT. However, pcfG was unable to complement an ltrB insertion mutation. Genetic analysis showed that pcfF of pCF10 and ltrF of pRS01 are also essential for plasmid transfer. Purified PcfF and LtrF possess double-stranded DNA binding activities for the inverted repeat within either oriT sequence. PcfG and LtrB were recruited into their cognate F-oriT DNA complex through direct interactions with their cognate accessory protein. PcfG also could interact with LtrF when pCF10 oriT was present. In vivo cross-complementation analysis showed that ltrF partially restored the pCF10DeltapcfF mutant transfer ability when provided in trans, whereas pcfF failed to complement an ltrF mutation. Specificity of conjugative DNA processing in these plasmids involves both DNA-protein and protein-protein interactions.     

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.     

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.     

Cloning the origin of transfer region of the resistance plasmid R1

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.