Protein apparatus for horizontal transfer of agrobacterial T-DNA to eukaryotic cells

This review analyzes agrobacterial virulence proteins and recipient cell proteins involved in horizontal transfer of a T-DNA-protein complex. Specifically, it considers the early stages of the interactions of partners (signal exchange, attachment, close contact); T-DNA release from bacterial cells; channel formation for the transfer of ssDNA between the partners; transfer of agrobacterial T-DNA through the membrane, cytoplasm, and nuclear membrane of the recipient cell and its incorporation into the recipient cell genome. It further discusses possible pathways of agrobacterial ssDNA transfer to the recipient cells. In particular, the possible role of T-pili and VirE2 protein during conjugative transfer of agrobacterial ssDNA between donor and recipient cells is discussed.     

Transfer of genetic information from agrobacteria to bacterial and plant cells: membrane and supramembrane structures involved in transfer

The review deals with the supramembrane and membrane structures involved in the initial contact (attachment) of an agrobacterial cell with a bacterial or plant cell during the transfer of the agrobacterial genetic information. The relationships between the donor cell attachment to the recipient cell surface and the infection and conjugation processes are discussed. Experimental data on the recently found agrobacterial pili and surface protein rhicadhesin, which are involved in the conjugative transfer of the plasmid between agrobacteria, are considered. The role of adhesive and conjugative pili of E. coli in the initial and tight contacts is analyzed in the context of the recently proved similarity between the mechanisms of agrobacterial transformation in plants and conjugative transfer in bacteria. Possible involvement of the pilus in the conjugative transfer of agrobacterial DNA across the membranes of donor and recipient (bacterial and plant) cells is discussed.     

Formation of extracellular structures in conjugated cultures of agrobacteria

Electron microscopy of noncentrifugated agrobacterial cells on a nitrocellulose membrane labeled with colloid gold-conjugated antibodies to VirB1 showed that the labeled complex bound to acetosyringone (AS)-induced cells but failed to form red-colored stains during incubation with Ti aplasmid cells. Supramembrane structures of AS-treated A. tumefaciens cells were for the first time visualized by transmission electron microscopy. Colloid gold labeling of VirB2-specific antibodies showed that VirB2 proteins produce long thin pilus structures emerging at the poles of AS-induced agrobacterial cells but never on the surface untreated with AS and Ti-plasmid-free agrobacterial cells. As a rule, one (or rarely two) thread-like connections and bridges were observed between the cells at the primary contact stage. The bridges were not destroyed by SDS, did not react with VirB2-specific antibodies, and remained visible at 30 degrees C. Visible close contacts between mating bacteria did not cease after SDS treatment. SDS pretreatment of donor cells or a mating cell suspension significantly modified the efficiency of pTd33 plasmid transfer from donor to recipient agrobacterial cells. In the presence of AS the optimal temperature for transfer was 25 degrees C. The frequency of plasmid pTd33 transfer from A. tumefaciens via vir-dependent pathway decreased 2-4-fold due to increase of temperature from 19.25 to 31 degrees C.     

Formation of TRA-dependent surface structures in Agrobacterium tumefaciens and their absence in R1 (deltatraR) mutant

Agrobacteria have Ti plasmid DNA delivering systems for the transfer to recipient cells by the conjugation mechanism. This transfer is absolutely dependent on induction tra genes. It is not clear which tra-dependent surface (extracellular) proteins (structures) are involved in the transport mechanism and whether these proteins also play a role in the contact formation. SDS-PAGE electrophoresis of proteins released from the cell showed disappearance of 63 and 67 kD proteins in R1(delta traR) strain, which were found in the growth medium and triton extract from the outer membrane of Ti plasmid-harboring A. tumefaciens R10 strains. The traR defective mutant did not express these proteins and had a higher hemagglutination and flocculation capacity than the wild strain. On the other hand, the wild strain showed D-galactose and N-acetyl-galactosamine specific hemagglutination which was not shown by traR mutant. Motility and chemotactic behavior of traR mutant in semisolid medium were defective. As a rule, one (or rarely two) thread-like connections in vir(-) and tra(+) conditions were observed on the agrobacterial cell surface. SDS pretreatment of agrobacterial cells had a significant effect on the expression of tra-dependent surface structures.     

Transfer of cytoplasmic and nuclear proteins between cells in culture

Evidence is presented for transfer of proteins between cells in culture, using techniques which previously have shown RNA transfer and the lack of DNA transfer between cells in culture. These techniques involved making donor cells heavier than recipient cells by having them ingest tantalum particles. After coculture of donor and recipient cells the two cell types were separated by centri- fugation on Ficoll gradients and the recipient cells analyzed for radioactively labeled proteins that may have passed from the prelabeled donor cells.These techniques also provided evidence for passage of donor cell proteins to recipient cell nuclei. Examination of the nuclear proteins in the recipient cells revealed that histones were transferred intercellularly to a greater extent than other nuclear proteins. The histone subfractions in the recipient cell nuclei were studied by acrylamide gel electrophoresis. No major differences were found in the proportion of each histone subfraction that was transferred to the recipient cell nuclei.     

Cut and move: protein machinery for DNA processing in bacterial conjugation

Conjugation is a paradigmatic example of horizontal or lateral gene transfer, whereby DNA is translocated between bacterial cells. It provides a route for the rapid acquisition of new genetic information. Increased antibiotic resistance among pathogens is a troubling consequence of this microbial capacity. DNA transfer across cell membranes requires a sophisticated molecular machinery that involves the participation of several proteins in DNA processing and replication, cell recruitment, and the transport of DNA and proteins from donor to recipient cells. Although bacterial conjugation was first reported in the 1940s, only now are we beginning to unravel the molecular mechanisms behind this process. In particular, structural biology is revealing the detailed molecular architecture of several of the pieces involved.     

Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool

Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT^P) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT^P loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.     

Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.     

The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer

pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer. A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer. Initiation (i.e. excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain. pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome. Transfer generally involves single-stranded DNA. In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer. Using the differential sensitivity to the SalI restriction-modification system of transfers involving single- and double-stranded DNA, we found that pSAM2 was probably transferred to the recipient as double-stranded DNA. This provides the first experimental evidence for the transfer of double-stranded DNA during bacterial conjugation. Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double-stranded DNA actively, either from donor to recipient or from mother cell to prespore.     

VirE2: a unique ssDNA-compacting molecular machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.     

Genetic functions and cell–cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis

Pheromone-inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1-5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell-cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI II. Properties of Host and Plasmid Mutations

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.     

A Fast and Practical Yeast Transformation Method Mediated by Escherichia coli Based on a Trans-Kingdom Conjugal Transfer System: Just Mix Two Cultures and Wait One Hour

Trans-kingdom conjugation is a phenomenon by which DNA is transferred into a eukaryotic cell by a bacterial conjugal transfer system. Improvement in this method to facilitate the rapid co-cultivation of donor bacterial and recipient eukaryotic cell cultures could make it the simplest transformation method, requiring neither isolation of vector DNA nor preparation of competent recipient cells. To evaluate this potential advantage of trans-kingdom conjugation, we examined this simple transformation method using vector combinations, helper plasmids, and recipient Saccharomyces cerevisiae strains. Mixing donor Escherichia coli and recipient S. cerevisiae overnight cultures (50 μL each) consistently yielded on the order of 10¹ transformants using the popular experimental strain BY4742 derived from S288c and a shuttle vector for trans-kingdom conjugation. Transformation efficiency increased to the order of 10² using a high receptivity trans-kingdom conjugation strain. In addition, either increasing the amount of donor cells or pretreating the recipient cells with thiols such as dithiothreitol improved the transformation efficiency by one order of magnitude. This simple trans-kingdom conjugation-mediated transformation method could be used as a practical yeast transformation method upon enrichment of available vectors and donor E. coli strains.     

Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter

The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to lambda phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4'312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.     

Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli

We used the LacO/GFP-LacI system to label and visualize the IncP beta plasmid R751 fluorescently during conjugative transfer between live donor and recipient bacteria. Comparisons of R751 in conjugative and non-conjugative conditions have allowed us to identify key localizations and movements associated with the initiation of conjugative transfer in the donor and the establishment of R751 in the recipient. A survey of successful mating pairs demonstrates that close physical contact between donor and recipient bacteria is required for DNA transfer and that regions of intimate contact can occur at any location on the donor or recipient cell membrane. The transferred DNA is positioned at the characteristic centre or quarter-cell position after conversion to a double-stranded molecule in the recipient cell. Initial duplication of plasmids often results in an asymmetric distribution of plasmid foci. Symmetric localization (either at centre or at 1/4 and 3/4 cell lengths) occurs only after a significant lag, presumably reflecting the time required to synthesize the plasmid-encoded partitioning proteins.     

Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.