Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer.

Transfer genes of the IncP plasmid RP4 are grouped in two separate regions, designated Tra1 and Tra2. Tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells. To provide information relevant to understanding the function of Tra2 gene products, the nucleotide sequence of the entire RP4 Tra2 region is presented here. Twelve open reading frames were identified in the Tra2 core region, being essential for intraspecific Escherichia coli matings. Predicted sizes of 11 of the 12 Tra2 polypeptides could be verified by expression in E. coli. Based on hydropathy plot analysis, most of the Tra2 open reading frames encode proteins that may interact with membranes. Interestingly, six of the predicted Tra2 gene products exhibited significant sequence similarities to gene products encoded by the VirB operon of the Agrobacterium Ti plasmid. VirB proteins are thought to function in the formation of a transmembrane structure that mediates the passage of T-DNA molecules from bacteria into plant cells. Because of this analogy and the hydropathy of Tra2 gene products, we assume that the DNA transfer machineries acting in bacterial conjugation and T-DNA transfer are structurally and functionally similar. Therefore, the data presented here, support the hypothesis that Ti vir and IncP tra genes evolved from a common ancestor. This suggestion is favored by previous findings of sequence similarities between the IncP and Ti DNA transfer system.     

Introduction of bacteriophage Mu into bacteria of various genera and intergeneric gene transfer by RP4::Mu.

The host range of coliphage Mu was greatly expanded to various genera of gram-negative bacteria by using the hybrid plasmic RP4::Mu cts, which is temperature sensitive and which confers resistance to ampicillin, kanamycin, and tetracycline. These drug resistance genes were transferred from Escherichia coli to members of the general Klebsiella, Enterobacter, Citrobacter, Salmonella, Proteus, Erwinia, Serratia, Alcaligenes, Agrobacterium, Rhizobium, Pseudomonas, Acetobacter, and Bacillus. Mu phage was produced by thermal induction from the lysogens of all these drug-resistant bacteria except Bacillus. Mu phage and RP4 or the RP4::Mu plasmid were used to create intergeneric recombinant strains by transfer of some genes, including the arylsulfatase gene, between Klebsiella aerogenes and E. coli. Thus, genetic analysis and intergeneric gene transfer are possible in these RP4::Mu-sensitive bacteria.     

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.     

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.     

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.     

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.     

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.     

Transfer of Drug Resistance Factors to the Dimorphic Bacterium CAULOBACTER CRESCENTUS

The P-type drug resistance factors RP4, RK2, R702, R68.45, and the N-type drug resistance factor R46 are transferred to Caulobacter crescentus at high frequencies. They are stably maintained and their antibiotic resistances are expressed. Experiments with RP4 have shown that intergeneric transfer of RP4 occur at a frequency of 10(-1). C. crescentus strains maintain RP4 as a plasmid, are sensitive to RP4-specific phage, and segregate phage-resistant cells at a frequency of 10(-4) to 10(-5). The RP4 plasmid can be used in several ways: (1) the RP4 plasmid will promote chromosomal exchange between C. crescentus strains at frequencies ranging from 10(-6) to 10(-8); (2) RP4 will promote the transfer of nonconjugative colE1 plasmids from E. coli to C. crescentus; once transferred, the colE1 plasmid is stably maintained under nonselective conditions, can be transferred serially, and segregates independently from RP4; and (3) RP4 can be used to introduce transposons into the C. crescentus chromosome, providing the basis for additional genetic techniques.     

IncP Plasmids Are Unusually Effective in Mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System

Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10⁻⁵ transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of oriT in cis and the provision in trans of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.     

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.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

Mutational analysis of the tra locus of the broad-host-range Streptomyces plasmid pIJ101

The tra gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the tra gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211-6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.     

Transgenosis with participation of plasmid RP1; indications of the presence of a "composit plasmid" in an interspecies hybrid of Escherichia coli]

One of the transconjugants (1-7) obtained by the authors earlier in the conjugation of Escherichia coli J-62 with Pseudomonas aeruginosa 1822, besides the plasmic RP1 has acquired the ability to grow without proline and tryptophan. The detailed analysis has shown that in the conjugation of the transconjugant 1-7 with different strains of E. coli the plasmic RP1 and chromosomal genes were transmitted together, but in transduction--by means of bacteriophage P1, independently of each other. The fertility was found only in the transductants carrying the plasmid RP1. This suggests that in the intergeneric conjugations the transmission of chromosomal genes may occur without any firm link with the plasmid (as in the case of "aggregated plasmids"). In E. coli cells these chromosomal fragments of Ps. aeruginosa apparently formed small nontransmissible replicons.     

委内瑞拉链霉菌秦岭变种属间接合转移系统的构建及透明颤菌血红蛋白的表达

【目的】构建委内瑞拉链霉菌秦岭变种属间接合转移系统及透明颤菌血红蛋白的表达。【方法】以链霉菌广泛使用的整合型质粒pSET152和复制型pHZ1358为出发质粒,通过供体大肠杆菌(Escherichia coli)ET12567(pUZ8002)进行属间接合转移委内瑞拉链霉菌秦岭变种。【结果】确定了该变种的最佳接合转移条件;通过SOE-PCR(Splicing by overlap extension PCR)技术构建含PermE和vhb结构基因融合片段的整合型表达载体pJD100,转化ET12567(pUZ8002)后属间接合转移委内瑞拉链霉菌秦岭变种。通过PCR和CO结合差光谱验证了vhb基因在委内瑞拉链霉菌秦岭变种中的整合表达。【结论】本文首次探索了委内瑞拉链霉菌秦岭变种接合转移系统,确定了委内瑞拉链霉菌秦岭变种的最佳接合转移条件,并采用基因工程手段使vhb基因在委内瑞拉链霉菌秦岭变种中获得表达。     

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.     

Fostriecin产生菌Streptomyces pulveraceus遗传转化体系的建立与优化

【目的】建立并优化链霉菌Fostriecin产生菌Streptomyces pulveraceus的遗传转化系统。【方法】以整合型质粒pSET152为出发质粒,通过供体菌E.coli ET12567/pUZ8002与受体菌Streptomyces pulveraceus进行接合转移。【结果】确定了链霉菌Streptomyces pulveraceus的最佳接合转移条件:培养基为终浓度含15%甘氨酸的MS培养基;孢子热激条件为50°C 10 min;阿伯拉霉素覆盖的时间为18 h,终浓度为20 mg/L。同时,把组成型启动子ermE+与绿色荧光蛋白基因(gfp)克隆到pSET152载体上,通过接合转移整合到该链霉菌中,gfp获得表达。【结论】建立Fostriecin产生菌的遗传转化系统,并发现甘氨酸能显著提高链霉菌的接合转移效率。     

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.     

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

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