Purified Escherichia coli F-factor TraY protein binds oriT.

The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation. In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein. The plasmid-encoded TraY protein was specifically labeled with [35S]methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label. Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene. The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T. Fowler, L. Taylor, and R. Thompson, Gene 26:79-89, 1983). This start sequence confirmed the premise of Inamoto et al. that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S. Inamoto, Y. Yoshioka, and E. Ohtsubo, J. Bacteriol. 170:2749-2757, 1988). The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid. However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.     

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.     

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.     

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 TraY protein to oriT and the promoter region for the traY gene of plasmid R100

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.     

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.     

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.     

TraY DNA recognition of its two F factor binding sites

F factor TraY, a ribbon-helix-helix DNA-binding protein, performs two roles in bacterial conjugation. TraY binds the F origin of transfer (oriT) to promote nicking of plasmid DNA prior to conjugative transfer. TraY also binds the P(Y) promoter to up-regulate tra gene expression. The two plasmid regions bound by TraY share limited sequence identity, yet TraY binds them with similar affinities. TraY recognition of the two sites was first probed using in vitro footprinting methods. Hydroxyl radical footprinting at both oriT and P(Y) sites indicated that bound TraY protected the DNA backbone bordering three adjacent DNA subsites. Analytical ultracentrifugation results for TraY:oligonucleotide complexes were consistent with two of these subsites being bound cooperatively, and the third being occupied at higher TraY concentrations. Methylation protection and interference footprinting identified several guanine bases contacted by or proximal to bound TraY, most located within these subsites. TraY affinity for variant oriT sequences with base substitutions at or near these guanine bases suggested that two of the three subsites correspond to high-affinity, cooperatively bound imperfect inverted GA(G/T)A repeats. Altering the spacing or orientation of these sites reduced binding. TraY mutant R73A failed to protect two symmetry-related oriT guanine bases in these repeats from methylation, identifying possible direct TraY-DNA contacts. The third subsite appears to be oriented as an imperfect direct repeat with its adjacent subsite, although base substitutions at this subsite did not reduce binding. Although unusual for ribbon-helix-helix proteins, this binding site arrangement occurs at both F TraY sites, consistent with it being functionally relevant.     

Mobilization of chimeric oriT plasmids by F and R100-1: role of relaxosome formation in defining plasmid specificity

Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).     

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.     

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.     

The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .     

Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT.

A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction.     

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.     

Transfer protein TraY of plasmid R1 stimulates TraI-catalyzed oriT cleavage in vivo

The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated. As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at oriT in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations, traY was dispensable for mobilization in the presence of traM, but mobilization did not occur in the absence of both traM and traY. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.     

Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1.

The complete nucleotide sequences of the ColB4-K98 (ColB4) plasmid transfer genes oriT, traM, and traY as well as the traY gene of R100-1 are presented and compared with the corresponding regions from the conjugative plasmids F, R1, and R100. The sequence encoding the oriT nick sites and surrounding inverted repeats identified in F was conserved in ColB4. The adenine-thymine-rich sequence following these nick sites was conserved in R1 and ColB4 but differed in F and R100, indicating that this region may serve as the recognition site for the traY protein. A series of direct repeats unique to the ColB4 plasmid was found in the region of dyad symmetry following this AT-rich region. This area also encodes 21-base-pair direct repeats which are homologous to those in F and R100. The traM gene product may bind in this region. Overlapping and following these repeats is the promoter(s) for the traM protein. The traM protein from ColB4 is similar to the equivalent products from F, R1, and R100. The traY protein from ColB4 is highly homologous to the R1 traY gene product, while the predicted R100-1 traY product differs at several positions. These differences presumably define the different alleles of traM and traY previously identified for IncF plasmids by genetic criteria. The translational start codons of the ColB4 and R100-1 traY genes are GUG and UUG, respectively, two examples of rare initiator codon usage.     

Location of the relaxation complex nick site within the minimal origin of transfer region of RK2

Transfer of plasmid DNA during bacterial conjugation begins at a specific site: the origin of transfer (oriT). The oriT region of the broad host range plasmid RK2 is located on a 250 bp fragment. Deletions involving either end of this region reduce transfer function, indicating that an extended sequence is required for optimal oriT activity. The single-strand nick induced by the RK2 DNA-protein relaxation complex is located adjacent to the 19 bp inverted repeat within the minimal oriT sequence. These results provide strong evidence that the plasmid relaxation event induced in vitro represents the nicking reaction that initiates DNA transfer at oriT during conjugation.     

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.     

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.