Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27

The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed.     

Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes.

The conjugation system of the IncP alpha plasmid RK2/RP4 is encoded by transfer regions designated Tra1, Tra2, and Tra3. The Tra1 core region, cloned on plasmid pDG4 delta 22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncP alpha plasmid-specific functions that allow pDG4 delta 22 to be mobilized by the heterologous IncP beta plasmid R751. Tn5 insertions in pDG4 delta 22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tra1 core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traJ. The traJ and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

Assembly of a functional phage PRD1 receptor depends on 11 genes of the IncP plasmid mating pair formation complex.

PRD1, a lipid-containing double-stranded DNA bacteriophage, uses the mating pair formation (Mpf) complex encoded by conjugative IncP plasmids as a receptor. Functions responsible for conjugative transfer of IncP plasmids are encoded by two distinct regions, Tra1 and Tra2. Ten Tra2 region gene products (TrbB to TrbL) and one from the Tra1 region (TraF) form the Mpf complex. We carried out a mutational analysis of the PRD1 receptor complex proteins by isolating spontaneous PRD1-resistant mutants. The mutations were distributed among the trb genes in the Tra2 region and accumulated predominantly in three genes, trbC, trbE, and trbL. Three of 307 phage-resistant mutants were weakly transfer proficient. Mutations causing a phage adsorption-deficient, transfer-positive phenotype were analyzed by sequencing.     

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.     

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.     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.     

Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.     

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.     

Interaction of Bacteroides fragilis pLV22a relaxase and transfer DNA with Escherichia coli RP4-TraG coupling protein

Many Bacteroides transfer factors are mobilizable in Escherichia coli when coresident with the IncP conjugative plasmid RP4, but not F. To begin characterization and potential interaction between Bacteroides mobilizable transfer factors and the RP4 mating channel, both mutants and deletions of the DNA processing (dtr), mating pair formation (mpf) and traG coupling genes of RP4 were tested for mobilization of Bacteroides plasmid pLV22a. All 10 mpf but none of the four dtr genes were required for mobilization of pLV22a. The RP4 TraG coupling protein (CP) was also required for mobilization of pLV22a, but could be substituted by a C-terminal deletion mutant of the F TraD CP. Potential interactions of the TraG CP with relaxase protein(s) and transfer DNA of both RP4 and pLV22a were assessed. Overlay assays identified productive interactions between TraG and the relaxase proteins of both MbpB and TraI from pLV22a and RP4 respectively. The Agrobacterium Transfer-ImmunoPrecipitation (TrIP) assay also identified an interaction between TraG and both RP4 and pLV22a transfer DNA. Thus, mobilization of the Bacteroides pLV22a in E. coli utilizes both RP4 Mpf and CP functions including an interaction between the relaxosome and the RP4 CP similar to that of cognate RP4 plasmid.     

Initiation of DNA synthesis in the transfer origin region of RK2 by the plasmid-encoded primase: detection using defective M13 phage

The broad host range IncP (IncP1) plasmids of gram-negative bacteria encode DNA primases that are involved in conjugal DNA synthesis. The primase of RK2/RP4 is required for efficient DNA transfer to certain gram-negative bacteria, indicating that the enzyme primes complementary strand synthesis in the recipient. In vitro, the primase initiates synthesis of oligoribonucleotides at 3'-dGdT-5' dinucleotides on the template strand. In this report, replication-defective M13 phage are used to assay the ability of the RK2-encoded primase to initiate complementary strand synthesis in vivo on single-strand templates containing the RK2 origin of conjugal transfer (oriT) or the RK2 origin of vegetative replication (oriV). The results show that sequences from either strand of the oriT region serve as efficient substrates for the RK2 primase and can enhance the growth of the defective M13 vectors delta E101 and delta Elac to levels approaching wild-type. The primise-oriT interaction appeared specific, since neither the oriV sequence nor another RK2 region, trfB, significantly enhanced growth of the defective phage, either in the presence or in the absence of the primase. In contrast to ColEl and F, this study also shows that the oriV region of RK2 lacks sites that are recognized by the host-specified DNA priming systems. The results suggest that the oriT region contains sites on both DNA strands that are efficient substrates for the plasmid-encoded primase, facilitating initiation of complementary strand DNA synthesis in both donor and recipient during conjugation.     

Characterization of the Tra2 Region of the IncHI1 Plasmid R27

In this study, the DNA sequence of one of the transfer regions of the IncHI1 plasmid R27 was determined. This region, which corresponds to coordinates 0-40 on the R27 map has been called the Tra2 region, and is believed to be involved in mating pair formation. DNA sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems. The R27 transfer genes appear to most closely resemble the genes from the F plasmid and Sphingomonas aromaticivorans plasmid pNL1, both within the individual genes and in the overall gene order. The Tra2 region is also distinct in that replication, partitioning, and stability genes are found in the middle of the transfer region. The R27 Tra2 region also contains a gene, trhF, which appears to be related to the TraF genes of Agrobacterium and Rhizobium species. This, along with the temperature-sensitive transfer system found in both H plasmids and Agrobacterium, leads to the speculation that the R27 transfer region evolved from both ancestral F-like and P-like plasmids.     

Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation

F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.     

Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system

A cryptic plasmid of the wall-less plant pathogenic mollicute, Spiroplasma kunkelii CR2-3X, was cloned and its sequence analyzed. The 14,615 bp plasmid, designated pSKU146, has a nucleotide content of 28 mol% G + C, and contains 18 potential protein-coding regions (open reading frames, ORFs), of which six encode proteins that exhibit similarity to virulence-associated proteins involved in cell-to-cell adhesion or conjugal DNA transfer. One ORF encodes a 96 kDa protein, SkARP1, that is highly similar to SARP1 adhesin involved in attachment of Spiroplasma citri to insect vector gut membrane. Five ORFs encode proteins similar to TraE and Mob in walled bacteria, and to ORFs found in the integrative, conjugative element (ICEF) of Mycoplasma fermentans, respectively. Presence of domains similar to proteins of the Type IV secretion system in pathogenic bacteria suggests that spiroplasma possesses a related translocation system. Plasmid pSKU146 also contains two identical oriT regions each containing a nick sequence characteristic of the IncP conjugative plasmid family, as well as a 58 bp palindromic sequence, palSK1. Features in pSKU146 suggest that the plasmid functions as a mobile genetic element in conjugative transmission of spiroplasma pathogenicity-related genes.     

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.     

Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.