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.     

Fertility inhibition of RP1 by IncN plasmid pKM101.

IncN plasmids, including pKM101, strongly inhibit the conjugal transfer of cohabiting IncP plasmids. We localized the pKM101 DNA sufficient for this phenomenon to a 1.1-kilobase region (denoted fip). Two fip-deficient Tn5 insertion derivatives of pKM101 were isolated; neither affected other pKM101-mediated functions. fip did not inhibit either the synthesis of the IncP plasmid's sex pilus or its ability to mediate entry exclusion against other IncP plasmids.     

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.     

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.     

A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis.

TraF, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4 (IncP), which is located at the periplasmic side of the cytoplasmic membrane, encodes a specific protease. The traF gene products of IncP and Ti plasmids show extensive similarities to prokaryotic and eukaryotic signal peptidases. Mutational analysis of RP4 TraF revealed that the mechanism of the proteolytic cleavage reaction resembles that of signal and LexA-like peptidases. Among the RP4 transfer functions, the product of the Tra2 gene, trbC, was identified as a target for the TraF protease activity. TrbC is homologous to VirB2 of Ti plasmids and thought to encode the RP4 prepilin. The maturation of TrbC involves three processing reactions: (i) the removal of the N-terminal signal peptide by Escherichia coli signal peptidase I (Lep), (ii) a proteolytic cleavage at the C terminus by an as yet unidentified host cell enzyme, and (iii) C-terminal processing by TraF. The third reaction of the maturation process is critical for conjugative transfer, pilus synthesis, and the propagation of the donor-specific bacteriophage PRD1. Thus, cleavage of TrbC by TraF appears to be one of the initial steps in a cascade of processes involved in export of the RP4 pilus subunit and pilus assembly mediated by the RP4 mating pair formation function.     

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.     

Prevotella intermedia native plasmid can be mobilized by an Escherichia coli conjugal IncP plasmid

We have determined the nucleotide sequence of a small Prevotella intermedia cryptic plasmid, pYHBi1, which consisted of sequences that were highly homologous to the amino acid sequence of the replication and mobilization proteins found in related organisms. We have also demonstrated that chimeric plasmids derived from this P. intermedia native plasmid can be mobilized between Escherichia coli strains by using a broad-host-range E. coli conjugative plasmid, IncP plasmid RP4. The results suggest that pYHBi1 possesses gene(s) responsible for conjugal transfer.     

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.     

Requirements for mobilization of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB and RP4 traG are interchangeable.

Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB. traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388. This result suggests a dual specificity of TrwB-like proteins in conjugation. The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1.     

Retrotransfer of IncP piasmid R751 from Escherichia coli maxicells: evidence for the genetic sufficiency of self-transferable plasmids for bacterial conjugation

Gene transfer between organisms is a prime contributor to evolution. Bacterial conjugation is probably the most important mechanism by which genes are spread among prokaryotes and perhaps also contributes to eukaryotic evolution. Conjugation is mediated by plasmids. The mechanism of conjugation remains ill-understood despite progress in the identification, mapping and sequencing of genes required for plasmid transmission. All conjugation-specific genes (those required only for DNA transfer and establishment) identified to date map to plasmids. We found that IncP plasmids could enter and subsequently convert maxicells, which are trapped in a metabolic state that prevents de novo expression of chromosomal genes, into conjugative donors. This suggests that IncP plasmids encode not only necessary functions but indeed all functions specific to DNA transmission. Thus, like viruses, plasmids can convert non-viable cells into gene vectors.     

Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC.

Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.     

Intergeneric conjugation between Escherichia coli and Streptomyces species.

We have constructed Escherichia coli-Streptomyces shuttle plasmids which are capable of conjugal transfer from E. coli to Streptomyces spp. These plasmids contained the pBR322 and pIJ101 origins of replication and the RK2 (IncP) origin of transfer. The transfer of plasmid was specifically dependent the presence of a 760-base-pair, cis-acting, oriT-containing fragment and on RP4 (IncP) functions supplied in trans. Conditions of mating and selection of exconjugants were analyzed with Streptomyces lividans as recipient. Plasmid transfer to other Streptomyces species was also demonstrated.     

Conjugal transfer system of the IncN plasmid pKM101.

The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically. Its organization differed significantly from that of the F plasmid. The tra genes are located in three regions, each between 3 and 4 kilobases in length. All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus. The plasmid's origin of transfer was localized to a 1.2-kilobase region at an extreme end of the transfer region. Using two different methods, we have identified 11 complementation groups required for transfer. One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids. One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused.     

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker.     

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.     

Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli.

The first demonstration of conjugal plasmid transfer from Escherichia coli to Bartonella henselae is reported. Transconjugants bearing plasmids of incompatibility groups P (IncP) and Q (IncQ), expressing various resistance markers, were generated. Tn5 transposons delivered on suicide plasmids by conjugation showed transpositional insertion into random chromosomal sites.     

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.     

Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid.

A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K. In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication. This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria. Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability.