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.     

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.     

Reconstitution of a staphylococcal plasmid-protein relaxation complex in vitro

The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nicking-closing equilibrium in vivo that serves to ready the plasmids for conjugal transfer. In pC221 and pC223, the components required for in vivo site- and strand-specific nicking at oriT are MobC and MobA. In order to investigate the minimal requirements for nicking in the absence of host-encoded factors, the reactions were reconstituted in vitro. Purified MobA and MobC, in the presence of Mg2+ or Mn2+, were found to nick at oriT with a concomitant phosphorylation-resistant modification at the 5' end of nic. The position of nic is consistent with that determined in vivo. MobA, MobC, and Mg2+ or Mn2+ therefore represent the minimal requirements for nicking activity. Cross-complementation analyses showed that the MobC proteins possess binding specificity for oriT DNA of either plasmid and are able to complement each other in the nicking reaction. Conversely, nicking by the MobA proteins is plasmid specific. This suggests the MobA proteins may encode the nicking specificity determinant.     

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.     

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.     

Functional analysis of ori1 and repA of the R-plasmid pSJ5.6 from Neisseria gonorrhoeae

The functional ori1 of the 5.6kb gonococcal R-plasmid pSJ5.6 contains an A-T rich region followed by four 22bp direct repeats and one 19bp inverted repeat. The replication region of the plasmid also contains a gene encoding for a 39kD RepA protein. We have further assessed the functionality of the replication region in pSJ5.6, an-iteron type plasmid, using in vivo complementation assays in Escherichia coli. A 2.1kb PstI-RsaI fragment containing the ori1 and repA gene of pSJ5.6 was cloned into vector pZErO -2 to obtain pZA-MRR. The pUC origin in pZA-MRR was deleted to render the plasmid dependable on the cis-acting ori1 for replication. The resulting plasmid, pMRR, was capable of replication and maintenance in E. coli. We also cloned the ori1 and repA gene separately to obtain pA-Ori and pZG-Rep, respectively. Using in vivo complementation assays, we demonstrated that the ori1(+) plasmid (pA-Ori) was maintained only when the RepA protein was supplied in trans by the high copy number plasmid pZG-Rep.     

Mobilization of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 into Escherichia coli by cointegration with several gram-conjugative plasmids

We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.     

Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids

The primary DNA processing protein for conjugative mobilization of the plasmid R1162 is the transesterase MobA, which acts at a unique site on the plasmid, the origin of transfer (oriT). Both MobA and oriT are members of a large family of related elements that are widely distributed among bacteria. Each oriT consists of a highly conserved core and an adjacent region that is required for binding by its cognate MobA. The sequence of the adjacent region is important in determining the specificity of the interaction between the Mob protein and the oriT DNA. However, the R1162 MobA is active on the oriT of pSC101, another naturally occurring plasmid. We show here that MobA can recognize oriTs having different sequences in the adjacent region and, with varying frequencies, can cleave these oriTs at the correct position within the core. Along with the structure of the oriTs themselves, these characteristics suggest a model for the evolution of this group of transfer systems.     

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.     

An accessory protein is required for relaxosome formation by small staphylococcal plasmids

Mobilization of the staphylococcal plasmid pC221 requires at least one plasmid-encoded protein, MobA, in order to form a relaxosome. pC221 and closely related plasmids also possess an overlapping reading frame encoding a protein of 15 kDa, termed MobC. By completing the nucleotide sequence of plasmid pC223, we have found a further example of this small protein, and gene knockouts have shown that MobC is essential for relaxosome formation and plasmid mobilization in both pC221 and pC223. Primer extension analysis has been used to identify the nic site in both of these plasmids, located upstream of the mobC gene in the sense strand. Although the sequence surrounding the nic site is highly conserved between pC221 and pC223, exchange of the oriT sequence between plasmids significantly reduces the extent of relaxation complex formation, suggesting that the Mob proteins are selective for their cognate plasmids in vivo.     

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.     

Region of the streptococcal plasmid pMV158 required for conjugative mobilization.

The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUB110. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.     

MobA, the DNA strand transferase of plasmid R1162: the minimal domain required for DNA processing at the origin of transfer

MobA is a DNA strand transferase encoded by the plasmid R1162 and required for plasmid DNA processing during conjugal transfer. The smallest active fragment was identified using phage display and partial enzymatic digestion of the purified protein. This fragment, consisting of approximately the first 184 amino acids, is able to bind and cleave its normal DNA substrate, the origin of transfer (oriT). Smaller fragments having one of these activities were not obtained. An active intermediate consisting of MobA linked to DNA was isolated and used to show that a single molecule of MobA is sufficient to carry out all of the DNA processing steps during transfer. These results, along with those obtained earlier, point to a single large, active site in MobA that makes several different contacts along the oriT DNA strand.     

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.     

Requirements for strand- and site-specific cleavage within the oriT region of Tn4399, a mobilizing transposon from Bacteroides fragilis.

Replicons that contain Tn4399, a conjugal mobilizing transposon isolated from Bacteroides fragilis, can be mobilized in the presence of broad-host-range IncP plasmids RP4 and R751 in Escherichia coli to B. fragilis or E. coli recipients (C. G. Murphy and M. H. Malamy, J. Bacteriol. 175:5814-5823, 1993). To identify the initial DNA processing events involved in Tn4399-mediated mobilization in E. coli, plasmid DNA from pCGM328 (a pUC7 vector that contains the mobilization region of Tn4399) was isolated from donor cells following the release of plasmid DNA from the relaxation complex. Site- and strand-specific cleavage within the oriT region of Tn4399 was detected by denaturing gel electrophoresis and Southern hybridization analysis of this DNA in the presence or absence of IncP plasmids. Mutations in either mocA or mocB, two genes which are encoded by Tn4399 and are required for mobilization, significantly decrease the amount of specifically nicked DNA detected. These results suggest roles for the MocA and MocB gene products in specific processing of Tn4399-containing plasmid DNA prior to mobilization. By isolation of the nicked strand and primer extension of this template, we mapped the precise 5' end of the single-stranded cleavage reaction. The nucleotide position of nicTn4399 is adjacent to two sets of inverted repeats, a genetic arrangement similar to those of previously characterized oriT regions. Two site-directed mutations which remove nicTn4399 (oriT delta 1 and oriT delta 2) cannot be mobilized to recipients when they are present in trans along with functional MocA and MocB proteins and an IncP mobilizing plasmid; they are cis-dominant loss-of-function mutations.     

oriT-directed cloning of defined large regions from bacterial genomes: identification of the Sinorhizobium meliloti pExo megaplasmid replicator region

We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia coli but not in the target organism. Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E. coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. meliloti pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.