Tn 5 to assess soil fate of genetically marked bacteria: screening for aminoglycoside-resistance advantage and labelling specificity

Abstract The reliability of Tn 5 as labelling tool was investigated in soil microcosm. The occurence of a selective in soil microcosm. The occurence of resistances encoded by Tn 5 nptII gene was assesed by kanamycin and neomycin amendment. The bioassay developed to monitor the persistence of the soil-added kanamycin did not detect the antibiotic activity in soil extract. A nptII -engineered Escherichia coli strain showed no enhanced survival in aminoglycoside amended soil. Tn 5-marker properties were investigated within indigenous bacteria to determine the specificity of labelling to follow the fate of recombinant DNA. Kanamycin and neomycin resistant population levels made Tn 5 aminoglycoside-resistance phenotype non-sensitive enough to select a soil dissemination of the labelled DNA. The unexpected occurrence of homologous sequences among soil organisms also prevented Tn 5 from being a specific DNA marker. By contrast, colony hybridization did not reveal homology to nptII suggesting its use as a reliable gene transfer indicator.     

Spread of recombinant DNA by roots and pollen of transgenic potato plants,identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.     

Intrachromosomal recombination in plants.

Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin selection did not increase the number of recombinant kanamycin-resistant (KanR) cell clones. DNA analysis of KanR clones derived from cells carrying multiple tandem recombination units suggested that these units have a tendency to undergo concerted recombination. Recovery and analysis of kanamycin-sensitive seedlings with patches of KanR cells provided direct evidence for mitotic recombination in plant tissue.     

In solium Selection for Arabidopsis Transformants Resistant to Kanamycin

The kanamycin resistance encoded by the neomycin phosphotransferase II gene (nptII) of transposon Tn5 is widely used in higher plant genetic transformation. The general process of plant transformation using nptII as a selectable marker gene, however, requires selecting kanamycin-resistant plants or tissues in culture. Even with the recently developed vacuum infiltration method for Arabidopsis transformation, the plant culture steps are not completely eliminated in selection for kanamycin-resistant transformants. The herbicide resistance genes, such as bar, which provides resistance to bialaphos, allow Arabidopsis transformation to become a true non-culture procedure. In this report, we assessed the feasibility of applying kanamycin as a spray in selecting for kanamycin-resistant Arabidopsis transformants grown in soil. We find that kanamycin-resistant transformants were effectively selected by spraying soil-grown Arabidopsis seedlings.     

Transient and stable expression of marker genes in cotransformedPetunia protoplasts in relation to X-ray and UV-irradiation

Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of foreign DNA inPetunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not stimulated by irradiation when measuring expression. Twenty-five kanamycin resistant but hygromycin sensitive colonies were examined with Southern or slot blotting and all were found to contain the coding sequence for the hygromycinphosphotransferase gene in their genomes. No obvious differences regarding copy number of integrated genes were observed when comparing transformed colonies derived from irradiated and non-irradiated protoplasts.     

Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper

Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 6-(,-dimethylallylamino)purine - Kn kinetin - nptII neomycin phosphotransferaseII - MS Murashige and Skoog (1962) medium     

Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

Agrobacterium tumefaciens–mediatated transformation of shoot-buds of chicory

Vegetative propagation of chicory via axillary shoot proliferation is one of the best ways to obtain an offspring with complete genetic stability. These shoot buds were used in transformation experiments using Agrobacterium tumefaciens strains containing binary plasmids carrying the neomycin phosphotransferase gene (nptII) and the β-glucuronidase gene (uidA). Selection was carried out on basal medium containing 100 mg l⁻¹ kanamycin. Transformed plantlets were recovered at a frequency of about 10% within four weeks after co-cultivation. The presence of the uidA gene was demonstrated by transient gene expression experiments using the histochemical GUS staining procedure. Evidence for stable transformation was shown by subculturing leaf discs on kanamycin selection medium, and Southern blot analysis confirmed the integration of the nptII and the uidA genes in the plant genome. Analysis of the progenies showed that kanamycin resistance was inherited as a single dominant trait. This method for obtaining transgenic chicory plants represents an alternative to leaf disc transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.     

Homologous sequences other than insertion elements can serve as recombination sites in plasmid drug resistance gene amplification.

A plasmid (pRR983) was constructed which has a gene coding for neomycin and kanamycin resistance flanked by direct repeats of regions of homology which contain no known insertion sequences. pRR983 does not have any homologous IS1 sequences. Growth of Proteus mirabilis harboring pRR983 in medium containing high concentration of neomycin resulted in cells which were highly resistant to both neomycin and kanamycin. Plasmid DNA was analyzed by using restriction endonucleases. In most cases the neomycin resistance gene had been tandemly duplicated by using the homologous DNA sequences flanking the resistance gene as recombination sites. This is analogous to tandem duplication of drug resistance genes on NR1 using the two direct repeats of IS1 as recombination sites. The amplified plasmid DNA returned to its original structure by the deletion of amplified neomycin resistance determinants when the host cells were cultured without selection for high resistance to neomycin.     

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.     

Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10(9) CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.     

FLP recombinase-mediated site-specific recombination in rice

The feasibility of using the FLP/ FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene ( neomycin phosphotransferase II , nptII ) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA . Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA . While the hybrid progeny from various crosses exhibited β-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/ FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/ FRT system in rice genetic improvement.     

Protein fusions with the kanamycin resistance gene from transposon Tn5.

The gene for the neomycin phosphotransferase II (NPT II) from transposon Tn5 was fused at the amino or carboxy terminus to foreign DNA sequences coding for 3-300 amino acids and the properties of the fused proteins were investigated. All amino-terminal fusions examined conferred kanamycin resistance to their host cell, but profound differences in their enzymatic activity and stability were detected. Short additions to the amino terminus of the NPT II resulted in highly enzymatically active fusion proteins whereas long amino-terminal fusions often had to be proteolytically degraded to release active proteins. Fusions at the carboxy-terminal end of the NPT II protein did not always induce kanamycin resistance and their enzymatic activity depended more stringently on the nature of the junction sequence.     

Aminoglycoside 3'-phosphotransferase gene: its cloning, characteristics and use as a molecular probe

Aminoglycoside resistance genes were cloned from transconjugates of aminoglycoside resistant clinical strains of gramnegative bacteria. The resistance determinant cloned from the strains of E. coli transferred kanamycin, monomycin and neomycin resistance to laboratory strains. It was shown that the cloned resistance gene encoded the type I 3'-aminoglycoside phosphotransferase. The results of spot and blot hybridization of the gramnegative bacteria clinical strains with the Bam HI-Pst I fragment of the cloned resistance determinant were indicative of wide-spread distribution of the APH 3' (I) and closely related genes in clinical microbial populations.     

Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml^-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.     

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.Abbreviations NPTII neomycin phosphotransferase - SDS sodium dodecyl sulphate