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.     

Covalent association of the traI gene product of plasmid RP4 with the 5'-terminal nucleotide at the relaxation nick site

Formation of relaxosomes is the first step in the initiation of transfer DNA replication during bacterial conjugation. This nucleoprotein complex contains all components capable of introducing a site- and strand-specific nick at a cognate transfer origin (oriT) on supercoiled plasmid DNA, thus providing the substrate for generation of the strand to be transferred. Characterization of the terminal nucleotides at the oriT nick site revealed that relaxation occurs by hydrolysis of a single phosphodiester bond between a 2'-deoxyguanosyl and a 2'-deoxycytidyl residue. The relaxation nick site and a 19-base pair invert repeat sequence that is recognized by asymmetric binding of the RP4 TraJ protein are interspaced by 8 base pairs. The nicking reaction results in covalent attachment of the RP4 TraI protein to the 5'-terminal 2'-deoxycytidyl residue of the cleaved strand. The arrangement of the TraJ binding site and the relaxation nick site on the same side of the DNA double helix suggests that protein-protein interactions between TraJ and TraI are a prerequisite for oriT specific nicking. In accordance with the current model of transfer DNA replication, the 3' end remains accessible for primer extension by DNA polymerase I, enabling replacement strand synthesis in the donor cell by a rolling circle-type mechanism.     

Effect of divalent ions on the minimal relaxase domain of MobA

The MobA protein encoded by plasmid R1162 plays an important role in conjugative mobilization between bacterial cells. It has two functional domains, the N-terminal relaxase domain and C-terminal primase domain. The N-terminal 186 residues (minMobA) is the minimal domain required for relaxase activity. We investigated the effects of different divalent metallic cations on minMobA activity measuring DNA binding, DNA nicking, and protein denaturation experiments. The results show that divalent cations are not required for DNA binding but are required for DNA nicking. The range of metals that function in minMobA suggests the cation role is largely structural. The most tightly binding cation is Mn²⁺, but the expressed protein shows roughly equal amounts of Mg²⁺ and Ca²⁺, both of which facilitate substrate binding and catalysis. Surprisingly, Zn²⁺ does not facilitate DNA binding nor allow nicking activity.     

Investigating the basis of substrate recognition in the pC221 relaxosome

The nicking of the origin of transfer (oriT) is an essential initial step in the conjugative mobilization of plasmid DNA. In the case of staphylococcal plasmid pC221, nicking by the plasmid-specific MobA relaxase is facilitated by the DNA-binding accessory protein MobC; however, the role of MobC in this process is currently unknown. In this study, the site of MobC binding was determined by DNase I footprinting. MobC interacts with oriT DNA at two directly repeated 9 bp sequences, mcb1 and mcb2, upstream of the oriT nic site, and additionally at a third, degenerate repeat within the mobC gene, mcb3. The binding activity of the conserved sequences was confirmed indirectly by competitive electrophoretic mobility shift assays and directly by Surface Plasmon Resonance studies. Mutation at mcb2 abolished detectable nicking activity, suggesting that binding of this site by MobC is a prerequisite for nicking by MobA. Sequential site-directed mutagenesis of each binding site in pC221 has demonstrated that all three are required for mobilization. The MobA relaxase, while unable to bind to oriT DNA alone, was found to associate with a MobC-oriT complex and alter the MobC binding profile in a region between mcb2 and the nic site. Mutagenesis of oriT in this region defines a 7 bp sequence, sra, which was essential for nicking by MobA. Exchange of four divergent bases between the sra of pC221 and the related plasmid pC223 was sufficient to swap their substrate identity in a MobA-specific nicking assay. Based on these observations we propose a model of layered specificity in the assembly of pC221-family relaxosomes, whereby a common MobC:mcb complex presents the oriT substrate, which is then nicked only by the cognate MobA.     

Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein

Site-specific and strand-specific nicking at the origin of transfer (oriT) of the F sex factor is the initial step in conjugal DNA metabolism. Then, DNA helicase I, the product of the traI gene, processively unwinds the plasmid from the nick site to generate the single strand of DNA that is transferred to the recipient. The nick at oriT is produced by the combined action of two Tra proteins, TraY and TraZ. The traZ gene was never precisely mapped, as no available point mutation uniquely affected TraZ-dependent oriT nicking. With several new mutations, we have demonstrated that TraZ activity is dependent upon traI DNA sequences. The simplest interpretation of this finding is that the F TraI protein is bifunctional, with DNA unwinding and site-specific DNA nicking activities.     

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer

The frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50-fold if donor cells lack MobC, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer ( oriT  ). The absence of MobC has several different effects on oriT DNA. Site- and strand-specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome-induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single-stranded substrate required for cleavage by MobA.     

Localized denaturation of oriT DNA within relaxosomes of the broad-host-range plasmid R1162

The broad-host-range, multicopy plasmid R1162 is efficiently mobilized during conjugation by the self-transmissible plasmid R751. The relaxosome, a complex of plasmid DNA and R1162-encoded proteins, forms at the origin of transfer ( oriT ) and is required for mobilization. Transfer is initiated by strand- and site-specific nicking of the DNA within this structure. We show by probing with potassium permanganate that oriT DNA is locally melted within the relaxosome, in the region from the inverted repeat to the site that is nicked. Mutations in this region of oriT , and in genes encoding the protein components of the relaxosome, affect both nicking and melting of the DNA. The nicking protein in the relaxosome is MobA, which also ligates the transferred linear, single strand at the termination of a round of transfer. We propose that there is an underlying similarity in the substrates for these two MobA-dependent, DNA-processing reactions. We also show that MobA has an additional role in transfer, beyond the nicking and resealing of oriT DNA.     

TraY and integration host factor oriT binding sites and F conjugal transfer: sequence variations, but not altered spacing, are tolerated

Bacterial conjugation is the process by which a single strand of a conjugative plasmid is transferred from donor to recipient. For F plasmid, TraI, a relaxase or nickase, binds a single plasmid DNA strand at its specific origin of transfer (oriT) binding site, sbi, and cleaves at a site called nic. In vitro studies suggest TraI is recruited to sbi by its accessory proteins, TraY and integration host factor (IHF). TraY and IHF bind conserved oriT sites sbyA and ihfA, respectively, and bend DNA. The resulting conformational changes may propagate to nic, generating the single-stranded region that TraI can bind. Previous deletion studies performed by others showed transfer efficiency of a plasmid containing F oriT decreased progressively as increasingly longer segments, ultimately containing both sbyA and ihfA, were deleted. Here we describe our efforts to more precisely define the role of sbyA and ihfA by examining the effects of multiple base substitutions at sbyA and ihfA on binding and plasmid mobilization. While we observed significant decreases in in vitro DNA-binding affinities, we saw little effect on plasmid mobilization even when sbyA and ihfA variants were combined. In contrast, when half or full helical turns were inserted between the relaxosome protein-binding sites, mobilization was dramatically reduced, in some cases below the detectable limit of the assay. These results are consistent with TraY and IHF recognizing sbyA and ihfA with limited sequence specificity and with relaxosome proteins requiring proper spacing and orientation with respect to each other.     

Characterization of the functional sites in the oriT region involved in DNA transfer promoted by sex factor plasmid R100.

We have previously identified three sites, named sbi, ihfA, and sbyA, specifically recognized or bound by the TraI, IHF, and TraY proteins, respectively; these sites are involved in nicking at the origin of transfer, oriT, of plasmid R100. In the region next to these sites, there exists the sbm region, which consists of four sites, sbmA, sbmB, sbmC, and sbmD; this region is specifically bound by the TraM protein, which is required for DNA transfer. Between sbmB and sbmC in this region, there exists another IHF-binding site, ihfB. The region containing all of these sites is located in the proximity of the tra region and is referred to as the oriT region. To determine whether these sites are important for DNA transfer in vivo, we constructed plasmids with various mutations in the oriT region and tested their mobilization in the presence of R100-1, a transfer-proficient mutant of R100. Plasmids with either deletions in the sbi-ihfA-sbyA region or substitution mutations introduced into each specific site in this region were mobilized at a greatly reduced frequency, showing that all of these sites are essential for DNA transfer. By binding to ihfA, IHF, which is known to bend DNA, may be involved in the formation of a complex (which may be called oriT-some) consisting of TraI, IHF, and TraY that efficiently introduces a nick at oriT. Plasmids with either deletions in the sbm-ihfB region or substitution mutations introduced into each specific site in this region were mobilized at a reduced frequency, showing that this region is also important for DNA transfer. By binding to ihfB, IHF may also be involved in the formation of another complex (which may be called the TraM-IHF complex) consisting of TraM and IHF that ensures DNA transfer with a high level of efficiency. Several-base-pair insertions into the positions between sbyA and sbmA affected the frequency of transfer in a manner dependent upon the number of base pairs, indicating that the phasing between sbyA and sbmA is important. This in turn suggests that both oriT-some and the TraM-IHF complex should be in an appropriate position spatially to facilitate DNA transfer.     

The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette.

Bacteroides conjugative transposons can act in trans to excise, circularize, and transfer unlinked integrated elements called NBUs (for nonreplicating Bacteroides units). Previously, we localized and sequenced the mobilization region of one NBU, NBU1, and showed that this mobilization region was recognized by the IncP plasmids RP4 and R751, as well as by the Bacteroides conjugative transposons. We report here that the single mobilization protein carried by NBU1 appears to be a bifunctional protein that binds to the oriT region and catalyzes the nicking reaction that initiates the transfer process. We have also localized and sequenced the mobilization region of a second NBU, NBU2. The NBU2 mobilization region was 86 to 90% identical at the DNA sequence to the oriT-mob region of NBU1. The high sequence similarity between NBU1 and NBU2 ended abruptly after the stop codon of the mob gene and about 1 kbp upstream of the oriT region, indicating that the oriT-mob regions of NBU1 and NBU2 may be on some sort of cassette. A region on NBU1 and NBU2 which lies immediately upstream of the oriT region had 66% sequence identity to a region upstream of the oriT region on a mobilizable transposon, Tn4399, an element that had previously appeared to be completely unrelated to the NBUs.     

Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC

Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer alpha/beta sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a beta-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.     

Concomitant reconstitution of TraI-catalyzed DNA transesterase and DNA helicase activity in vitro

TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing < or =30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic.     

Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis.

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.     

Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100.

We developed an in vitro system to reproduce a site- and strand-specific nicking at the oriT region of plasmid R100. The nicking reaction was dependent on the purified TraY protein and on the lysate, which was prepared from cells overproducing the TraI protein. This supports the idea that the protein products of two genes, traY and traI, constitute an endonuclease that introduces a specific nick in vivo in the oriT region of the conjugative plasmids related to R100. The products were the "complex" DNA molecules with a protein covalently linked with the 5'-end of the nick. The nick was introduced in the strand, which is supposed to be transferred to recipient cells during conjugation, and was located at the site 59 base pairs upstream of the TraY protein binding site, sbyA.     

In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.     

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.     

Formation of delta tra F' plasmids: specific recombination at oriT

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.     

Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase

Background

Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.

Methodology/Principal Findings

We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.

Conclusions/Significance

The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.