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.     

The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101

The components for the mobilization function of a plasmid DNA during conjugation include a cis-acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins. By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain. Within a 2797 bse-pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6). Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010. Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101. Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites. By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1. An identical nick site, which is adjacent to a characteristic 10 base-pair inverted repeat sequence, is also found for plasmid RSF1010. A recombinant plasmid containing a 42 base-pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT-active.     

The primase of broad-host-range plasmid R1162 is active in conjugal transfer.

The broad-host-range plasmid R1162 is conjugally mobilized at high frequency by the IncP-1 plasmid R751 but is poorly mobilized by pOX38, a derivative of the F factor. In both cases, the origin of transfer (oriT) and the Mob proteins of R1162 are required, indicating that these plasmids are mobilized by similar mechanisms. R1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of MobA, one of the R1162 mobilization proteins (P. Scholz, V. Haring, B. Wittman-Liebold, K. Ashman, M. Bagdasarian, and E. Scherzinger, Gene 75:271-288, 1989). When R751 is the mobilizing vector, the primase is not required for mobilization of plasmids containing cloned mob-oriT R1162 DNA. However, detectable mobilization of such plasmids by pOX38 requires both the primase and its cognate initiation site, oriented for synthesis of the complement to the transferred strand. The long form of the primase is required for optimal transfer: R1162 replicons lacking this form also are not transferred detectably by pOX38 and are less well mobilized by R751. The distance between oriT and the primase initiation site affects the frequency of mobilization, and this effect is polar in the direction of transfer. Our results indicate that the R1162 primase is active in mobilization of R1162 and suggest that the MobA-linked form is an adaptation increasing its effectiveness during transfer.     

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.     

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 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.     

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.     

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.     

MobB protein stimulates nicking at the R1162 origin of transfer by increasing the proportion of complexed plasmid DNA.

An essential early step in conjugal mobilization of R1162, nicking of the DNA strand that is subsequently transferred, is carried out in the relaxosome, a complex of two plasmid-encoded proteins and DNA at the origin of transfer (oriT). A third protein, MobB, is also required for efficient mobilization. We show that in the cell this protein increases the proportion of molecules specifically nicked at oriT, resulting in lower yields of covalently closed molecules after alkaline extraction. These nicked molecules largely remain supercoiled, with unwinding presumably constrained by the relaxosome. MobB enhances the sensitivity of the oriT DNA to oxidation by permanganate, indicating that the protein acts by increasing the fraction of complexed molecules. Mutations that significantly reduce the amount of complexed DNA in the cell were isolated. However, plasmids with these mutations were mobilized at nearly the normal frequency, were nicked at a commensurate level, and still required MobB. Our results indicate that the frequency of transfer is determined both by the amount of time each molecule is in the nicked form and by the proportion of complexed molecules in the total population.     

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.     

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.     

The structure of the minimal relaxase domain of MobA at 2.1 A resolution

The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism.     

Genetic organization of plasmid R1162 DNA involved in conjugative mobilization.

DNA involved in the mobilization of broad-host-range plasmid R1162 was localized to a region of 2.7 kilobases within coordinates 3.4 to 6.1 kilobases on the R1162 map. By examining the transfer properties of plasmids containing cloned fragments of DNA from within this region, we showed that at least four trans-active products and a cis-active site (oriT) were involved in mobilization. A cloned DNA fragment of 155 base pairs was capable of providing full oriT activity. This fragment was located within 600 base pairs of DNA containing the origin of replication of R1162, and its nucleotide sequence and that of neighboring DNA were determined. Activation of oriT required R1162-encoded, trans-acting products. Deletions which resulted in the loss of one or more of these had a variable effect on transfer efficiency and indicated the presence of both essential and nonessential Mob products. Regions encoding these products flanked oriT and in one case appeared to overlap a gene essential for plasmid replication. The implications of these findings with respect to the broad host range of R1162 are discussed.     

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.     

Complete sequence of pSC101.

We have completed the pSC101 sequence. The coding capacities of the newly sequenced regions show the presence of two large open reading frames close to the oriT region. Their size and localization suggest that these polypeptide chains could be involved in the transfer process of pSC101.     

Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization. Evidence for genetically distinct events at oriT

A segment of R1162 DNA containing genes for conjugative mobilization (Mob) and the origin of transfer (oriT) was integrated into the Escherichia coli chromosome. Bacterial genes were transferred in a polar fashion during conjugative mobilization of the integrated segment by a self-transmissible plasmid vector. The direction of transfer, together with the properties of mutated derivatives of oriT, indicate that initial cleavage of oriT, and subsequent religation after transfer, entail different mechanisms that can be distinguished genetically.     

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.     

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.