Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC-oligonucleotide complexes could be separated from noncovalently bound protein by SDS-PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18-DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.     

The F-plasmid TraI protein contains three functional domains required for conjugative DNA strand transfer

The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site- and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5'-to-3' helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first approximately 310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraIDelta252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraIDelta252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraIDelta252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.     

The diversity of conjugative relaxases and its application in plasmid classification

Bacterial conjugation is an efficient and sophisticated mechanism of DNA transfer among bacteria. While mobilizable plasmids only encode a minimal MOB machinery that allows them to be transported by other plasmids, conjugative plasmids encode a complete set of transfer genes (MOB+T4SS). The only essential ingredient of the MOB machinery is the relaxase, the protein that initiates and terminates conjugative DNA processing. In this review we compared the sequences and properties of the relaxase proteins contained in gene sequence databases. Proteins were arranged in families and phylogenetic trees constructed from the family alignments. This allowed the classification of conjugative transfer systems in six MOB families: MOB_F, MOB_H, MOB_Q, MOB_C, MOB_P and MOB_V . The main characteristics of each family were reviewed. The phylogenetic relationships of the coupling proteins were also analysed and resulted in phylogenies congruent to those of the cognate relaxases. We propose that the sequences of plasmid relaxases can be used for plasmid classification. We hope our effort will provide researchers with a useful tool for further mining and analysing the plasmid universe both experimentally and in silico .     

An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage

Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.     

Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies

Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.     

Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that can transfer from one bacterial cell to another by conjugation. ICEBs1 is integrated into the trnS-leu2 gene of Bacillus subtilis and is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system. When B. subtilis senses DNA damage or high concentrations of potential mating partners that lack the element, ICEBs1 excises from the chromosome and can transfer to recipients. Bacterial conjugation usually requires a DNA relaxase that nicks an origin of transfer (oriT) on the conjugative element and initiates the 5'-to-3' transfer of one strand of the element into recipient cells. The ICEBs1 ydcR (nicK) gene product is homologous to the pT181 family of plasmid DNA relaxases. We found that transfer of ICEBs1 requires nicK and identified a cis-acting oriT that is also required for transfer. Expression of nicK leads to nicking of ICEBs1 between a GC-rich inverted repeat in oriT, and NicK was the only ICEBs1 gene product needed for nicking. NicK likely mediates conjugation of ICEBs1 by nicking at oriT and facilitating the translocation of a single strand of ICEBs1 DNA through a transmembrane conjugation pore.     

Two atypical mobilization proteins are involved in plasmid CloDF13 relaxation

The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT-mobB-mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5'-GGGTG/GTCGGG-3' by primer extension and sequencing reactions. Analysis of Mob- insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which nic cleavage results in a free nicked-DNA intermediate.     

Exploration of DNA processing features unravels novel properties of ICE conjugation in Gram-positive bacteria

Integrative and conjugative elements (ICEs) are important drivers of horizontal gene transfer in prokaryotes. They are responsible for antimicrobial resistance spread, a major current health concern. ICEs are initially processed by relaxases that recognize the binding site of oriT sequence and nick at a conserved nic site. The ICESt3/Tn916/ICEBs1 superfamily, which is widespread among Firmicutes, encodes uncanonical relaxases belonging to a recently identified family called MOB_T. This family is related to the rolling circle replication initiators of the Rep_trans family. The nic site of these MOB_T relaxases is conserved but their DNA binding site is still unknown. Here, we identified the bind site of RelSt3, the MOB_T relaxase from ICESt3. Unexpectedly, we found this bind site distantly located from the nic site. We revealed that the binding of the RelSt3 N-terminal HTH domain is required for efficient nicking activity. We also deciphered the role of RelSt3 in the initial and final stages of DNA processing during conjugation. Especially, we demonstrated a strand transfer activity, and the formation of covalent DNA-relaxase intermediate for a MOB_T relaxase.     

Stringent and relaxed recognition of oriT by related systems for plasmid mobilization: implications for horizontal gene transfer

The plasmids R1162 and pSC101 have origins of conjugative transfer (oriTs) and corresponding relaxases that are closely related. The oriTs are made up of a highly conserved core, where DNA is cleaved by the relaxase prior to transfer, and an inverted repeat that differs in size and sequence. We show that in each case the seven base pairs adjacent to the core and within one arm of the inverted repeat are sufficient to determine specificity. Within this DNA there are three AT base pairs located 4 bp from the core. Mutations in the AT base pairs suggest that the relaxase makes essential contacts at these locations to the minor groove of the DNA. The remaining four bases are different for each oriT and are both necessary and sufficient for stringent recognition of oriT by the pSC101 mobilization proteins. In contrast, the R1162 mobilization proteins have a much more relaxed requirement for the base sequence of this specificity region. As a result, the R1162 mobilization proteins can initiate transfer from a variety of sites, including those derived from the chromosome. The R1162 mobilization proteins could therefore contribute to the horizontal gene transfer of DNA from diverse sources.     

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 Å crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 Å out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid’s origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.     

The R1162 relaxase/primase contains two, type IV transport signals that require the small plasmid protein MobB

The relaxase of the plasmid R1162 is a large protein essential for conjugative transfer and containing two different and physically separate catalytic activities. The N-terminal half cleaves one of the DNA strands at the origin of transfer (oriT) and becomes covalently linked to the 5' terminal phosphate; the C-terminal half is a primase essential for initiation of plasmid vegetative replication. We show here that the two parts of the protein are independently transported by the type IV pathway. Part of the domain containing the catalytic activity, as well as an adjacent region, is required in each case, but the required regions do not physically overlap. Both transport systems contribute to the overall frequency of conjugative transfer. MobB is a small protein, encoded within mobA but in a different reading frame, that stabilizes the relaxase at oriT. MobB is required for efficient type IV transport of both the complete relaxase and its two, separate functional halves. MobB inserts into the membrane and could thus stabilize the association between the relaxase and the type IV transfer apparatus.     

Mobility of Plasmids

Summary: Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance. While many studies have decorticated the mechanisms of mobility in model plasmids, the identification and characterization of plasmid mobility from genome data are unexplored. By reviewing the available data and literature, we established a computational protocol to identify and classify conjugation and mobilization genetic modules in 1,730 plasmids. This allowed the accurate classification of proteobacterial conjugative or mobilizable systems in a combination of four mating pair formation and six relaxase families. The available evidence suggests that half of the plasmids are nonmobilizable and that half of the remaining plasmids are conjugative. Some conjugative systems are much more abundant than others and preferably associated with some clades or plasmid sizes. Most very large plasmids are nonmobilizable, with evidence of ongoing domestication into secondary chromosomes. The evolution of conjugation elements shows ancient divergence between mobility systems, with relaxases and type IV coupling proteins (T4CPs) often following separate paths from type IV secretion systems. Phylogenetic patterns of mobility proteins are consistent with the phylogeny of the host prokaryotes, suggesting that plasmid mobility is in general circumscribed within large clades. Our survey suggests the existence of unsuspected new relaxases in archaea and new conjugation systems in cyanobacteria and actinobacteria. Few genes, e.g., T4CPs, relaxases, and VirB4, are at the core of plasmid conjugation, and together with accessory genes, they have evolved into specific systems adapted to specific physiological and ecological contexts.     

The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions

Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer (oriT), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICEBs1 can transfer three different plasmids to recipient bacteria at high frequencies. Strikingly, these plasmids do not have dedicated mobilization-oriT functions. Plasmid mobilization required conjugation proteins of ICEBs1, including the putative coupling protein. In contrast, plasmid mobilization did not require the ICEBs1 conjugative relaxase or cotransfer of ICEBs1, indicating that the putative coupling protein likely interacts with the plasmid replicative relaxase and directly targets the plasmid DNA to the ICEBs1 conjugation apparatus. These results blur the current categorization of mobilizable and nonmobilizable plasmids and indicate that conjugative elements play a role in horizontal gene transfer even more significant than previously recognized.     

The F plasmid-encoded TraM protein stimulates relaxosome-mediated cleavage at oriT through an interaction with TraI

Conjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer - they introduce a site- and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5'-end of the cleaved DNA strand. The action of the initiator protein at the origin of transfer (oriT) is governed by auxiliary proteins that alter the architecture of the DNA molecule, allowing binding of the initiator protein. In the F plasmid system, two auxiliary proteins have roles in establishing the relaxosome: the host-encoded IHF and the plasmid-encoded TraY. Together, these proteins direct the loading of TraI which contains the catalytic centre for the transesterification. The F-oriT sequence includes a binding site for another plasmid-encoded protein, TraM, which is required for DNA transfer. Here the impact of TraM protein on the formation and activity of the F plasmid relaxosome has been examined. Purified TraM stimulates the formation of relaxed DNA in a reaction that requires the minimal components of the relaxosome, TraI, TraY and IHF. Unlike TraY and IHF, TraM is not essential for the formation of the relaxosome in vitro and TraM cannot substitute for either TraY or IHF in this process. The TraM binding site sbmC, along with both IHF binding sites, is essential for stimulation of the relaxase reaction. In addition, stimulation of transesterification appears to require the C-terminal domain of TraI suggesting that TraM and TraI may interact through this domain on TraI. Taken together, these results provide additional evidence of a role for TraM as a component of the relaxosome, suggest a previously unknown interaction between TraI and TraM, and allow us to propose a molecular role for the C-terminal domain of TraI.     

Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4.

Two essential transfer genes of the conjugative plasmid RP4 were altered by site-directed mutagenesis: traG of the primase operon and traI of the relaxase operon. To evaluate effects on the transfer phenotype of the point mutations, we have reconstituted the RP4 transfer system by fusion of the transfer regions Tra1 and Tra2 to the small multicopy replicon ColD. Deletions in traG or traI served to determine the Tra phenotype of mutant plasmids by trans complementation. Two motifs of TraG which are highly conserved among TraG-like proteins in several other conjugative DNA transfer systems were found to be essential for TraG function. One of the motifs resembles that of a nucleotide binding fold of type B. The relaxase (TraI) catalyzes the specific cleaving-joining reaction at the transfer origin needed to initiate and terminate conjugative DNA transfer (W. Pansegrau, W. Schröder, and E. Lanka, Proc. Natl. Acad. Sci. USA 90:2925-2929, 1993). Phenotypes of mutations in three motifs that belong to the active center of the relaxase confirmed previously obtained biochemical evidence for the contributions of the motifs to the catalytic activity of TraI. Expression of the relaxase operon is greatly increased in the absence of an intact TraI protein. This finding suggests that the relaxosome which assembles only in the presence of the TraI in addition to its enzymatic activity plays a role in gene regulation.     

Evolution of Plasmid Mobility: Origin and Fate of Conjugative and Nonconjugative Plasmids

Conjugation drives the horizontal transfer of adaptive traits across prokaryotes. One-fourth of the plasmids encode the functions necessary to conjugate autonomously, the others being eventually mobilizable by conjugation. To understand the evolution of plasmid mobility, we studied plasmid size, gene repertoires, and conjugation-related genes. Plasmid gene repertoires were found to vary rapidly in relation to the evolutionary rate of relaxases, for example, most pairs of plasmids with 95% identical relaxases have fewer than 50% of homologs. Among 249 recent transitions of mobility type, we observed a clear excess of plasmids losing the capacity to conjugate. These transitions are associated with even greater changes in gene repertoires, possibly mediated by transposable elements, including pseudogenization of the conjugation locus, exchange of replicases reducing the problem of incompatibility, and extensive loss of other genes. At the microevolutionary scale of plasmid taxonomy, transitions of mobility type sometimes result in the creation of novel taxonomic units. Interestingly, most transitions from conjugative to mobilizable plasmids seem to be lost in the long term. This suggests a source-sink dynamic, where conjugative plasmids generate nonconjugative plasmids that tend to be poorly adapted and are frequently lost. Still, in some cases, these relaxases seem to have evolved to become efficient at plasmid mobilization in trans, possibly by hijacking multiple conjugative systems. This resulted in specialized relaxases of mobilizable plasmids. In conclusion, the evolution of plasmid mobility is frequent, shapes the patterns of gene flow in bacteria, the dynamics of gene repertoires, and the ecology of plasmids.     

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.     

A novel relaxase homologue is involved in chromosomal DNA processing for type IV secretion in Neisseria gonorrhoeae

The Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA that acts in natural transformation. To examine the mechanism of DNA processing for secretion, we made mutations in the putative relaxase gene traI and used nucleases to characterize the secreted DNA. The nuclease experiments demonstrated that the secreted DNA is single-stranded and blocked at the 5' end. Mutation of traI identified Tyr93 as required for DNA secretion, while substitution of Tyr201 resulted in intermediate levels of DNA secretion. TraI exhibits features of relaxases, but also has features that are absent in previously characterized relaxases, including an HD phosphohydrolase domain and an N-terminal hydrophobic region. The HD domain residue Asp120 was required for wild-type levels of DNA secretion. Subcellular localization studies demonstrated that the TraI N-terminal region promotes membrane interaction. We propose that Tyr93 initiates DNA processing and Tyr201 is required for termination or acts in DNA binding. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion. The TraI domain architecture, although not previously described, is present in 53 uncharacterized proteins, suggesting that the mechanism of TraI function is a widespread process for DNA donation.     

Tracking F plasmid TraI relaxase processing reactions provides insight into F plasmid transfer

Early in F plasmid conjugative transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the origin of transfer called nic. The reaction covalently links TraI Tyr16 to the 5'-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3'-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is transferred to the recipient with a free 3'-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for transfer. Therefore, two TraI molecules are required for F plasmid transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3'-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization.     

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.