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.     

Recognition of oriT for DNA processing at termination of a round of conjugal transfer

Conjugal transfer of plasmid DNA is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (oriT), is recircularized. For the plasmid R1162, it is the protein MobA, covalently linked to the linear strand, that rejoins the ends by a reversible transesterification reaction. We have identified from those oligonucleotides with a partially degenerate oriT base sequence, subpopulations bound by MobA that undergo transesterification, and support efficient termination of conjugal transfer. Two domains of oriT, a ten base-pair inverted repeat and an adjacent TAA, are required for tight binding by the protein, whereas the location of the dinucleotide YG determines the site of strand cleavage. The results indicate that capture of MobA by oriT, and subsequent processing of the DNA for termination, are determined by different sequence motifs within this locus.     

Location of the nick at oriT of the F plasmid

The oriT locus of the Escherichia coli K12 F plasmid contains a site at which one of the DNA strands is cleaved as a prelude to conjugal transmission to recipient bacteria. We have remapped this site biochemically by using oriT-containing plasmids that were purified from bacteria expressing the F transfer (tra) functions. The strand interruption was found on the transferred strand 137 base-pairs clockwise of the center of the BglII site at 66.7 on the F map. This location is consistent with the locations anticipated from studies of delta traF' plasmids, but it differs from previous results by other investigators. The strand interruption produced a 3'-OH, but the nature of the 5' terminus of the strand on the other side of the nick was not determined. Some DNA sequence motifs in the vicinity of the oriT nick site of F resemble the chromosomal site involved in formation of delta traF'purE plasmids.     

Characterizing the DNA contacts and cooperative binding of F plasmid TraM to its cognate sites at oriT

TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT: two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase I, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.     

Intrinsic bends and integration host factor binding at F plasmid oriT.

F plasmid oriT DNA extending from the F kilobase coordinate 66.7 (base pair [bp] 1 on the oriT sequence map) rightward to bp 527 was analyzed for intrinsic bends (by permutation assays) and for binding of integration host factor (IHF) (by gel retardation and DNase footprinting). Intrinsic bending of the 527-bp fragment (bend center approximately at bp 240) was represented as a composite of at least two components located near bp 170 and near bp 260. IHF bound primarily to a site extending from bp 165 to 195 and with lower affinity to a site extending from bp 287 to 319. The intrinsic curvature and sequences to which IHF binds (IHF is known to bend DNA) may play a structural role in oriT function.     

Boundaries of the nicking region for the F plasmid transfer origin, oriT

The extent of the F plasmid oriT nicking region was determined from the properties of successive substitution mutations in the region from base pair 121 to base pair 174 and from KMnO₄ probing of DNA structural distortions induced in vivo by tra gene products. Nicking and transfer assays indicated that the left margin of oriT Wes predominantly at the nick site, and that the nicking domain primarily lies within 17bp to the right of the nick. Some mutants that were proficient for nicking showed reduced frequencies of termination, indicating that oriT nicking does not guarantee efficient termination. DNA in the vicinity of the nick (G₁₃₇, T₁₃₈, G₁₄₀, and T₁₄₁ on the nicked strand) showed elevated sensitivity to KMnO₄ when tra gene products were present in the donor. Bases C₁₄₅, C₁₄₆, C₁₄₇, C₁₄₉, and G₁₅₀ on the un-nicked strand also became more sensitive to oxidation under tra⁺ conditions. The bases preferentially oxidized by KMnO₄ lie within the nicking domain, as defined by the substitution mutants, and they include dinucleotides that can produce kinks in the DNA. Base pairs in the nicking region are calculated to be more thermodynamically stable than base pairs in the flanking regions.     

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.     

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.     

Conjugal mobilization of plasmid DNA: termination frequency at the origin of transfer of plasmid R1162.

Conjugal transfer of plasmid R1162 is initiated and terminated at a 38-bp origin of transfer (oriT). Plasmids containing two directly repeated copies of oriT were used to determine the actual frequency of termination at this site. This frequency was calculated both for oriTnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated oriT where transfer had been initiated. In both cases, the termination frequency decreased as the distance between the initiation and termination sites became greater and was significantly less than one for plasmids the size of R1162. A substantial proportion of recipient cells received more than one plasmid copy during transfer. Our results indicate that termination is inefficient but that this is partly compensated for by the transmission of multiple plasmid copies.     

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

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.     

Mutational and physical analysis of F plasmid traY protein binding to oriT

F plasmid traY protein binding to wild-type or deleted regions containing the TraY-binding site, sbyA, was studied in vitro. The principal DNA-protein complex was formed with DNA segments including the sbyA site defined by footprinting and (with lesser affinity) with truncated segments that retained the leftward two-thirds of sbyA. This located the major sequence determinants for TraY binding between bp 204 and 227 on the oriT map. For all sequences tested, bound TraY induced bending of approximateiy 50 to 55°, and centred between bp 214 and 221. Thermodynamic and mobility analyses indicated that two TraY protomers bind to sbyA. At higher TraY concentrations, additional TraY bound to the left of the sbyA in a region previously shown to bind IHF (site IHF A). TraY binding to this additional site (sbyC) was inhibited by IHF. Sequence similarities shared by sbyA, sbyB, and SbyC may include the critical base pairs for TraY binding.     

Cloning of F DNA fragments carrying the origin of transfer oriT and the fertility inhibition gene finP

The two SalI fragments derived from the F transfer region that are bounded at one end by the SalI cleavage site in traM were cloned into pBR322. From these, smaller (540 bases) SalI-BglII fragments were subcloned to give plasmids containing the origin of transfer oriT (pED806) and finP (pED812), respectively, but no entire tra genes. All four plasmids were characterized by genetic tests and by restriction endonuclease analysis. pED806 could not be used to search for an F oriT-related “relaxation complex” because of its unexpected instability in the presence of Flac, and extensive efforts to prepare such a complex using other oriT⁺ plasmids were unsuccessful. We therefore suggest that a cell-free F relaxation complex does not exist. Protein synthesis directed by pED812 in minicells allowed the finP product to be tentatively identified as a 4000 M_r, protein.     

F plasmid conjugative DNA transfer: the TraI helicase activity is essential for DNA strand transfer

The product of the Escherichia coli F plasmid traI gene is required for DNA transfer via bacterial conjugation. This bifunctional protein catalyzes the unwinding of duplex DNA and is a sequence-specific DNA transesterase. The latter activity provides the site- and strand-specific nick required to initiate DNA transfer. To address the role of the TraI helicase activity in conjugative DNA transfer traI mutants were constructed and their function in DNA transfer was evaluated using genetic and biochemical methods. A traI deletion/insertion mutant was transfer-defective as expected. A traI C-terminal deletion that removed the helicase-associated motifs was also transfer-defective despite the fact that the region of traI encoding the transesterase activity was intact. Biochemical studies demonstrated that the N-terminal domain was sufficient to catalyze oriT-dependent transesterase activity. Thus, a functional transesterase was not sufficient to support DNA transfer. Finally, a point mutant, TraI-K998M, that lacked detectable helicase activity was characterized. This protein catalyzed oriT-dependent transesterase activity in vitro and in vivo but failed to complement a traI deletion strain in conjugative DNA transfer assays. Thus, both the transesterase and helicase activities of TraI are essential for DNA strand transfer.     

Specific binding of MobA, a plasmid-encoded protein involved in the initiation and termination of conjugal DNA transfer, to single-stranded oriT DNA.

MobA protein, encoded by the broad host-range plasmid R1162, is required for conjugal mobilization of this plasmid. The protein is an essential part of the relaxosome, and is also necessary for the termination of strand transfer. In vitro, MobA is a nuclease specific for one of the two DNA strands of the origin of transfer (oriT). The protein can cleave this strand at the same site that is nicked in the relaxosome, and can also ligate the DNA. We show here that purified MobA protein forms a complex that is specific for this single oriT strand. The complex is unusually stable, with a half-life of approximately 95 min, is not disrupted by hybridization with the complementary strand, and reforms rapidly after boiling. Both the inverted repeat within oriT, and the eight bases between this repeat and the site cleaved by MobA, are required for binding by the protein. Mutations reducing base complementarity between the arms of the inverted repeat also decrease binding. This effect is partially suppressed by second-site mutations restoring complementarity. These results parallel the effects of these mutations on termination. Footprinting experiments with P1 nuclease indicate that the DNA between the inverted repeat and the nick site is protected by MobA, but that pairing between the arms of the repeat, which occurs in the absence of protein, is partially disrupted. Our results suggest that termination of strand transfer during conjugation involves tight binding of the MobA protein to the inverted repeat and adjacent oriT DNA. This complex positions the protein for ligation of the ends of the transferred strand, to reform a circular plasmid molecule.     

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing

Summary The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.     

Cloning and polypeptide analysis of the leading region in F plasmid DNA transfer

A segment of the F plasmid DNA, located between the origin of transfer and the primary F replication region, is the first to enter the recipient cell during conjugation.PstI,SalI, andSmaI restriction endonuclease sites have been mapped within this leading region in conjugational DNA transfer and chimeric plasmids carrying overlapping fragments of the region have been constructed. Analyses of polypeptides synthesized by maxicells carrying these chimeric plasmids have shown four new polypeptides ofM_r 27,800, 23,100, 14,400, and 11,000 to be encoded by sequences within the leading region.     

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.