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Bacterial antibiotic resistance genes are still frequently used as markers in transgenic plants. Due to the problems caused by antibiotic-resistant pathogens, the use of antibiotic resistance genes in transgenic plants is subject to debate. It is hypothesized that the introduction of bacterial genes into the plant genome leads to a higher probability of gene transfer from plants to bacteria due to the presence of homologous sequences. However, until now, there has been a lack of clear experimental evidence that successful gene transfer from plants to bacteria can occur at all. Natural transformation, the ability of bacteria to actively take up free DNA, is a way plant DNA can be transferred to bacteria. At present, around 40 species, some of which are soil- or water-borne bacteria, are known to develop the ability (called competence) for natural transformation (13). Prerequisites for natural transformation under soil conditions are the availability of free DNA, the development of competence, and the stable integration of the captured DNA into the bacterial genome. In microcosm experiments, bacterial DNA adsorbed to soil particles was able to transform competent bacteria and to persist in soil (7, 12, 13, 17). DNA adsorbed to soil particles is, to some degree, protected against attacks of nucleases (12, 13, 19, 20). In agricultural soils, the persistence of transgenic plant DNA released into the environment, e.g., from senescent or rotting plant material, has been observed by several researchers (2, 19, 24, 26). Several groups have tried to transform bacteria with transgenic plant DNA from different plants and naturally competent bacteria (2, 3, 16, 23). However, transformation of bacteria with transgenic plant DNA has not yet been demonstrated. In this study, experiments were done under optimized laboratory conditions to examine the ability of Acinetobacter sp. strain BD413 to capture and integrate transgenic sugar beet DNA based on homologous recombination. All transformation experiments were done with a rifampin-resistant mutant of Acinetobacter sp. strain BD413 (obtained from J. D. van Elsas, Institute for Plant Protection [IPO-DLO], Wageningen, The Netherlands) which is known to be naturally transformable (11). Plasmid pGSFR160, which was provided by Plant Genetic Systems, Ghent, Belgium, harbored a gene construct consisting of the phosphinothricin acetyltransferase gene (bar), the neomycin phosphotransferase gene (npt) (both under control of the TR1/TR2 promoter from Agrobacterium tumefaciens [31]), the 3'ocs terminator, and nonspecific parts (Fig. 1). The same construct was present in the transgenic sugar beet plants obtained from Planta GmbH, Einbeck, Germany. Due to low-level expression of nptII controlled by TR1, the Acinetobacter sp. remained kanamycin sensitive even after the introduction of pGSFR160.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Map of the gene construct. The gene construct is part of pGSFR160 and is chromosomally integrated into the genome of the sugar beet. It consisted of the gene bar, the bidirectional promoter TR1/TR2, nptII, the 3'ocs terminator sequence, and nonspecified parts.

Construction of pFG4nptII. To facilitate the stable integration of transgenic plant DNA in Acinetobacter sp. strain BD413 and the selection of transformants, plasmid pFG4nptII, carrying a part of Tn5 with an incomplete nptII (a deletion of 317 bp), was constructed and introduced in Acinetobacter sp. strain BD413. A 1,693-bp fragment of transposon Tn5 (nucleotide 1236 to nucleotide 2929) consisting of the nptII promoter, the nptII gene (kanamycin resistance), and the bleomycin resistance gene was amplified by PCR with P1 (5' TGC TAA AGG AAG CGG AAC 3') and P2 (5' AGG TCA ACA GGC GGT AAC 3') as forward and reverse primers, respectively. Primers were designed on the basis of the published Tn5 sequence (accession no. L19385 and U00004) with the Oligo 4.0 program (21). PCR mixtures contained 0.1 µmol of both the forward and reverse primer, 3.75 mM MgCl₂, 0.2 mM deoxyribonucleoside triphosphates, 1× Stoffel buffer, and 2.5 U of Taq polymerase Stoffel fragment (Applied Biosystems). Amplification involved a 7-min step at 92°C; subsequently, 35 cycles consisting of 1 min at 92°C (denaturation), 1 min at 58°C (primer annealing), and 1.5 min at 72°C (primer extension) were performed, followed by a 10-min final extension step at 72°C. The 1,693-bp PCR product was cloned into PCR vector pT7-blue (Novagen, Madison, Wis.). Digestion with NcoI and Tth111I introduced a 317-bp deletion into the central part of nptII. After the ends were blunted by integrating deoxyribonucleoside triphosphates with the Klenow fragment, the plasmid was recircularized. A HindIII-BamHI fragment of the resulting plasmid containing the insert was introduced into the multiple cloning site of pMMB190 (15), resulting in pFG4nptII (Fig. 2). The IncQ plasmid pMMB190 was provided by M. Bagdasarian, Michigan State University, East Lansing, Mich. All digestions were carried out with restriction enzymes from Boehringer, Mannheim, Germany, or New England Biolabs, Beverly, Mass., according to the manufacturers' instructions. All ligations were carried out with Fast-Link ligase (Epicentre Technologies, Madison, Wis.). For blunting of digested fragments, Klenow fragment (Boehringer) was used. Buffer and media were prepared according to Sambrook et al. (22) unless indicated otherwise. Both pMMB190 and pFG4nptII contained an ampicillin resistance gene which could be used for selection. Transformation of Escherichia coli XL-blue cells was carried out by electroporation according to the protocol of Bio-Rad (Hercules, Calif.). Plasmid pFG4nptII was introduced into Acinetobacter sp. strain BD413 by natural transformation as described previously (17).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Map of pFG4nptII. The insert of the plasmid consisted of the promoter region, the deleted gene nptII (nptII [deletion of 317 bp]), and the bleomycin resistance gene (bleo). Primer binding sites and PvuII restriction sites are indicated.

Transformation experiments. The resulting Acinetobacter sp. strain BD413(pFG4nptII), which was rifampin and ampicillin resistant but kanamycin sensitive due to the 317-bp deletion in nptII, was used as a recipient in transformation experiments. Transformation experiments were done with either DNA of plasmid pGSFR160, linearized pGSFR160 without an origin of replication (oriV), or DNA extracted from transgenic sugar beet plants harboring the gene construct. In addition, transformations were attempted with leaf homogenates of transgenic sugar beet carrying the gene construct. DNA of plasmid pGSFR160 was isolated with the plasmid extraction kit from Qiagen (Hilden, Germany). To obtain linearized plasmid DNA without oriV, pGSFR160 was treated with SacI and HindIII. DNA from transgenic and nontransgenic sugar beets was extracted by the hexadecyltrimethyl ammonium bromide method (30). Extracted DNA was dissolved in sterile deionized water. To produce plant homogenate, leaves of sugar beet were frozen at 70°C and homogenized afterwards. The liquid phase obtained was suitable for transformation without being treated further. Transformation experiments were carried out basically as described by Nielsen et al. (17) on Mueller-Hinton agar (MHA; Merck, Darmstadt, Germany) containing 50 µg of rifampin per ml and 100 µg of ampicillin per ml. Natural competence of Acinetobacter sp. strain BD413 was developed by growing it in 10 ml of Luria-Bertani medium at 28°C until early stationary phase. Cells were washed once in 10 ml of saline and stored at 70°C in 10 ml of saline containing 15% glycerol until use. After the addition of DNA, competent cells (100 µl = approximately 5 × 10⁷ CFU) were placed on a nylon membrane filter (Millipore, Bedford, Mass.) on top of the agar and grown at 28°C for 24 h. The bacterial layer was resuspended in 4 ml of saline, concentrated by centrifugation for 2 min at 5,500 × g or diluted 10-fold, and plated on MHA supplemented with 50 µg of kanamycin per ml, 50 µg of rifampin per ml, and 100 µg of ampicillin per ml to detect transformants. The number of CFU of recipient Acinetobacter sp. strain BD413(pFG4nptII) was determined by plating on MHA containing 50 µg of rifampin per ml and 100 µg of ampicillin per ml. The colonies were counted after incubation at 28°C for 2 days. The transformation frequency was determined as the ratio of the number of transformants to the number of Acinetobacter sp. strain BD413(pFG4nptII) CFU. All experiments were conducted twice in two (plasmid transformation) or three (plant DNA or homogenate) replicates. Controls were made with water and nontransgenic plant DNA. Transformations with all sources of DNA listed above resulted in kanamycin-resistant Acinetobacter sp. strain BD413(pFG4nptII), indicating the restoration and expression of nptII located on pFG4nptII. Transformation frequencies are shown in Table 1. Compared to transformation with plasmid DNA, transformation frequency with plant DNA was drastically reduced. With plant homogenate of sugar beet leaves, the transformation of Acinetobacter sp. strain BD413(pFG4nptII) occurred at a frequency of 1.5 × 10¹⁰. Controls made with water and nontransgenic plant DNA were negative, indicating that no kanamycin-resistant spontaneous mutants appeared. In contrast, Nielsen et al. (16) failed to demonstrate the transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA due to the absence of homologous sequences.

View this table: [in this window] [in a new window]   TABLE 1.   Transformation frequencies of Acinetobacter sp. strain BD413(pFG4nptII) transformed with transgenic DNA and plant homogenate

Characterization of transformants. Representative numbers of kanamycin-resistant colonies (a total of 20 transformants) obtained from transformations with the purified DNA samples were screened by PCR (P1/P2 system) for the presence of the restored gene nptII. The appearance of specific PCR products of 1.7 kb for all transformants tested demonstrated the restoration of the deleted nptII (data not shown). The size of the control PCR product obtained with Acinetobacter sp. strain BD413(pFG4nptII) was approximately 1.4 kb. The restoration of the gene nptII was confirmed by Southern blot hybridization of the PCR products with a probe specific for the deletion (nptII). The 1,693-bp PCR product obtained with Tn5 template was digested with Tth111I-NcoI, generating a 317-bp fragment. After separation on a 0.7% agarose gel with Tris-acetate-EDTA buffer, the 317-bp fragment was recovered by the QiaQuick gel extraction kit (Qiagen) and labelled with digoxigenin-11dUTP (Boehringer).

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Southern blot hybridization of PvuII-digested plasmid DNA extracted from kanamycin-resistant Acinetobacter sp. obtained after transformation with plant homogenates. Hybridization was carried out with the nptII probe.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   (A) Sequence comparison of pFG4nptII and the gene construct. Sequences were compared in 8-base segments, and homologous segments are indicated by dots. (B) Map of the insert of pNT2 based on restriction analysis and sequencing. As a result of homologous recombination, the insert of pNT2 consisted of the promoter region, the restored nptII, and the 3'ocs terminator integrated into the bleomycin resistance gene (bleo). Primer sites and restriction sites for PvuII are indicated.