Tumefaciens Ti Plasmid Research Articles

A Survey of Genetically Modified Foods and Feed in Namibian Supermarkets

The current study was carried out to survey if genetically modified organisms (GMOs) are present in feeds and foods found in local supermarkets in Namibia. Little is known of the presence and prevalence of GMOs in the local supermarkets or information available on the labelling provided on some. The aim was to prove the absence or existence of GMOs in the foods and feeds by means of molecular extraction and screening procedures. Samples purposefully selected were nine (6 food and 3 chicken feed samples). DNA was extracted from them, and they were screened by means of polymerase chain reaction (PCR) for the presence of the Cauliflower mosaic virus (CaMV) 35S promoter and the terminator of Agrobacterium tumefaciens Ti plasmid nopaline synthase gene (NOS) that are commonly found in transgene constructs. Screening results indicated that GMOs were present in the 3 chicken feed samples, absent in 1 food sample, present in 3 food samples and the other 2 food samples’ results were inconclusive. These results and survey information reveal the extent of GMOs in the food chain in supermarkets in Namibia and highlight the need for regulatory bodies in the food and feed industry to protect consumers from being misled by absence of properly informative labels on genetic modifications.

Features of Expression of Foreign Genes in Complex Insertions in Transgenic Tobacco Plants with a Mosaic Pattern of nptII Gene Expression

The features of a mosaic pattern of the expression of the nptII selective gene, which provides plant resistance to kanamycin antibiotic, and the target gene of Serratia marcescens secretory endonuclease under control of the bidirectional MAS promoter of the mannopine synthase gene of Agrobacterium tumefaciens Ti plasmid in epiallelic lines of transgenic tobacco plants (Nu5 and Nu6) were studied. Both genes are part of a complex insertion represented by two full-size T-DNA copies and one truncated copy located between them in the reverse orientation. Transgenic tobacco lines contrastingly differ in the phenotypic expression of the nptII gene (low frequency of mosaics in the Nu5 line and high in Nu6). It was established that a decrease in the level of expression of the selective gene occurs when transgenic plants pass from hemi- to homozygous state, and it is most pronounced for the Nu6 epiallele. It was demonstrated that synthesis of aberrant sense and antisense transcripts in the region of a truncated T-DNA copy occurs in the transgenic lines. Namely these transcripts can act as triggers and trigger the inactivation of the selective nptII gene expression.

Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population

ABSTRACTAccumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations.

The plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.

In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified plants and bacteria, we evaluated the impact of plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type tumors relative to uninfected plant tissues. Moreover, comparisons of tumors induced on Col-0 and her1 plants showed that the increase in the plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in plant tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights plant GABA content as an important trait in the struggle against pathogenic bacteria.

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes.

BackgroundRecently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies.ResultsThe 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome.ConclusionsThis study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).

RepC protein of the octopine‐type Ti plasmid binds to the probable origin of replication within repC and functions only in cis

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.

Identification of amino acid residues of the pheromone‐binding domain of the transcription factor TraR that are required for positive control

Genes required for replication and for conjugal transfer of the Agrobacterium tumefaciens Ti plasmid are regulated by the quorum sensing transcription factor TraR, whose N-terminal domain binds to the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and whose C-terminal domain binds to specific DNA sequences called tra boxes. Here, we constructed 117 mutants, altering 103 surface-exposed amino acid residues of the TraR N-terminal domain. Each mutant was tested for activation of the traI promoter, where TraR binds to a site centred 45 nucleotides upstream of the transcription start site, and of the traM promoter, where TraR binds a site centred 66 nucleotides upstream. Alteration of 18 residues blocked activity at the traI promoter. Of these, alteration at three positions impaired TraR abundance or DNA binding, leaving 15 residues that are specifically needed for positive control. Of these 15 residues, nine also blocked or reduced activity at the traM promoter, while six had no effect. Amino acid residues required for activation of both promoters probably contact the C-terminal domain of the RNA polymerase alpha subunit, while residues required only for traI promoter activation may contact another RNA polymerase component.

Molecular biology and plant breeding

The use of modern molecular biological techniques has played a significant role in elucidating various aspects of the molecular organization of plant genes. Most of the information gained concerns characters and phenomena that are of relevance to plant breeding. It has been shown at the molecular level that cereal and legume seed storage proteins are controlled by complex multigenic nuclear loci. The analysis of movable nuclear genes, exemplified by the Ds-Ac transposable elements in maize, has given insight into the nature of these elements and the genetic changes that occur at their insertion and deletion sites. The characterization of the heterogeneous mitochondrial DNA and the uniform chloroplast DNA has helped to elucidate the molecular basis of cytoplasmic male sterility, RuBP carboxylase enzyme synthesis and herbicide resistance, In N2 fixation, the emphasis has been put on the molecular dissection of N2-fixing genes in bacteria and the interactions between symbiotic and host DNAs. It has been possible to develop vectors of Agrobacterium tumefaciens Ti-plasmids which lack oncogenes for DNA transfer. Cauliflower and gemini viruses as well as liposomes are also potential systems for DNA transport to protoplasts which can be regenerated into plants. Furthermore, it has been shown that certain molecular biological techniques are useful for screening purposes in genetic analysis and plant breeding work.

Localization of the plasmid-encoded proteins TraI and MobA in eukaryotic cells

Conjugation mediates gene transfer not only between bacterial species but also from bacteria to yeast, plant, and animal cells. DNA transferred by conjugative plasmids from bacteria to eukaryotes must traverse subcellular membranes in the recipient before the transferred genes can be expressed and inherited. This process is most likely facilitated by putative DNA pilot proteins such as VirD2 of the Agrobacterium tumefaciens Ti plasmid. Here, we test this model as a general feature of trans-kingdom conjugation using the DNA-relaxases TraI and MobA of the IncP and IncQ groups. TraI localized unambiguously and uniformly to the nuclei of both yeast and human cells, whereas MobA displayed a range of subcellular localization patterns. The tendency to localize to the nucleus was not correlated with predicted nuclear localization sequence motifs in either protein, suggesting a lack of stringent requirements for nuclear localizing potential in pilot proteins mediating conjugative DNA transfer to eukaryotes. Further, our results indicate that nuclear localization ability may be more commonly associated with conjugative pilot proteins than previously recognized.

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells.

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

Crystallization and preliminary X-ray diffraction studies of the quorum-sensing regulator TraM from Agrobacterium tumefaciens.

TraM is a 11.4 kDa protein involved in the control of the conjugal transfer of Agrobacterium tumefaciens Ti plasmids by quorum-sensing. TraM was overexpressed and purified from Escherichia coli. This protein binds to the transcriptional regulator TraR, abolishing its function. Size-exclusion chromatography and dynamic light scattering show that the recombinant protein has an apparent molecular weight of 30 kDa in solution. Crystals have been obtained of both native and selenomethionine-substituted TraM by the vapour-diffusion method. Crystals diffract to 1.67 A and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 76.43, b = 47.09, c = 47.46 A and two molecules in the asymmetric unit. A two-wavelength MAD data set for the selenomethionine-substituted form has been collected to a resolution of 2.0 A. The selenium substructure (five out of six possible sites) has been solved using direct methods.

Developing the Agrobacterium Ti plasmid as a vector for plant genetic engineering: the 2002 Benjamin Franklin Medal in life sciences presented to Mary-Dell Chilton

The Franklin Institute, Philadelphia, Pennsylvania, awarded the 2002 Benjamin Franklin Medal in Life Sciences to Mary-Dell Chilton for her key discoveries and wide-ranging contributions in the development of the Agrobacterium tumefaciens Ti plasmid as a major vector system for plant genetic engineering.

Mutational Analysis of TraR: CORRELATING FUNCTION WITH MOLECULAR STRUCTURE OF A QUORUM-SENSING TRANSCRIPTIONAL ACTIVATOR

TraR, the quorum-sensing activator of the Agrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone, N-(3-oxooctanoyl)-L-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to lambda cI' confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in alpha-helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.

Heterologous expression of a bacterial homospermidine synthase gene in transgenic tobacco: effects on the polyamine pathway.

Homospermidine synthase (HSS) is a branch-point enzyme that links the secondary pathway (pyrrolizidine alkaloids) to primary metabolism (polyamines). Since the diamine putrescine is a precursor of homospermidine and nicotine in tobacco, we performed heterologous expression of a bacterial homospermidine synthase gene (hss)in Nicotiana tabacum and determined the effect on free and conjugated polyamine levels. The hss gene from Rhodopseudomonas viridis was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Agrobacterium tumefaciens Ti-plasmid in sense and antisense orientation and both hss constructs were transformed into tobacco plants. Expression of the hss gene was verified by "Northern" and "Southern Blot" analysis. 2 transgenic sense lines were generated from 1000 calli which showed weak expression of homospermidine synthase, i.e. 50 pktal/mg protein and 45 pktal/mg protein. These transgenic sense plants showed a significantly decreased content of free spermidine while the pool of conjugated spermidine was not affected. The 2 sense plants exhibited a range of abnormal phenotypes such as dwarfness and stunted growth. Homospermidine was sporadically detectable in wild type tobacco. To our knowledge, this is the first biotechnological approach to express a prokaryotic homospermidine synthase gene in tobacco plants.

Transgenic tobacco (Nicotiana tabacum SR1) plants expressing the gene coding for Serratia marcescens nuclease

The gene coding for the secreted Serratia marcescens endonuclease was fused with the mannopine synthase promoter of Agrobacterium tumefaciens Ti plasmid and transferred to Nicotiana tabacum SR1 plants. The promoter is leaf- and root-specific. The resulting transgenic plants demonstrated elevated nuclease activity. The level of the transgene product was determined in the transgenic lines.

Co-evolution of the agrocinopine opines and the agrocinopine-mediated control of TraR, the quorum-sensing activator of the Ti plasmid conjugation system.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is controlled by a hierarchical system in which opines, substrates produced by crown gall tumours, induce a quorum-sensing system. The cascade results from the control of expression of traR, the quorum-sensing activator, by a regulator responsive to the opine. In the two cases studied to date, the gene arrangements responsible for the cascade differ remarkably, suggesting that considerable diversity exists among the many Ti-like plasmids in the agrobacteria. In this study, we demonstrated that the novel Ti plasmid pTiChry5 is induced to transfer at high frequency by extracts from tumours initiated by strain Chry5. The purified inducer had the chemical and biological properties of agrocinopines C and D, a set of sugar phosphodiester opines known to induce transfer of another Ti plasmid, pTiBo542. The T-region of pTiChry5 contained a gene whose product, called Acs(Chry5), is virtually identical to the agrocinopine C+D synthase from the T-region of pTiBo542. The two genes are less closely related to acs of pTiC58, which is responsible for the production of agrocinopines A+B, a similar but not identical set of phosphodiester opines by tumours induced by strain C58. Agrocinopines A+B induce transfer of pTiC58 but did not induce transfer of pTi(Chry5). A single copy of traR was identified at the 11 o'clock region of pTi(Chry5), where it is part of a two-gene operon called arc(Chry5). Although altered by deletions, arc(Chry5) is related to the five-gene arc operon that controls the expression of traR on pTiC58. Expression of traR(Chry5) was induced by agrocinopines C+D and the opines isolated from Chry5 tumours but not by agrocinopines A+B. A mutation in traR(Chry5) abolished transfer, and transfer was restored by complementation in trans. We conclude that the agrocinopine opines and the corresponding opine-meditated conjugal regulatory regions of pTiChry5 and pTiC58 share a common origin, but that the opine signals for the two Ti plasmids have evolved divergently through changes in the opine synthase enzymes. The alterations in the opines, in turn, necessitated a co-evolutionary change in the opine recognition systems responsible for controlling expression of the traR genes on these two types of Ti plasmids.

The Antiactivator TraM Interferes with the Autoinducer-dependent Binding of TraR to DNA by Interacting with the C-terminal Region of the Quorum-sensing Activator

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via the transcriptional activator TraR and the acyl-homoserine lactone Agrobacterium autoinducer (AAI). Unique to this system, the activity of TraR is negatively modulated by an antiactivator called TraM. Analyses from yeast two-hybrid studies suggest that TraM directly interacts with the activator, but the conditions under which these components interact and the region of TraR responsible for this interaction are not known. Induction of traM in a strain in which TraR was activating transcription of a reporter system led to rapid cessation of gene expression. As assessed by a genetic assay that measures AAI-dependent DNA binding, TraM inhibited TraR function before and after the transcription factor had bound to its DNA recognition site. Consistent with this observation, in gel retardation assays, purified TraM abolished the DNA binding activity of TraR in a concentration-dependent manner. Such inhibition occurred independent of the order of addition of the reactants. As assessed by far Western analyses TraM interacts with TraR by directly binding the activator. TraM in its native form interacted with native TraR and also with heat-treated TraR but only when SDS was included with the denatured protein. TraM interacted with TraR on blots prepared with total lysates of cells grown in the presence and absence of AAI. Far Western analysis of N- and C-terminal deletion mutants localized a domain of TraR contributing to TraM binding to the C-terminal portion of the activator protein. Random mutagenesis by hydroxylamine treatment and error-prone polymerase chain reaction identified several residues in this region of TraR important for interacting with TraM as well as for transcriptional activation or/and DNA binding. We conclude that TraM inhibits TraR by binding to the activator at a domain within or close to the helix-turn-helix motif located at the C terminus of the protein.

Erratum

Erratum

The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid.

Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450-type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon. We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2. Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH-proficient host. For all tested compounds, VirH2 appeared to be solely responsible for this effect. One such compound, ferulic acid, was chosen for biochemical analysis. Ferulic acid was degraded by a VirH-proficient host but not by a VirH mutant. The wild-type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid. This indicates that wild-type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O-demethylation reaction. Ferulic acid was far more toxic than caffeic acid to the wild-type strain, although the wild-type strain was more resistant to ferulic acid than was the virH mutant. Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.