Sandrine Demane
email: sandrine.demaneche@ec-lyon.fr

` Che

Jean-Michel Monier

Eric Dugat-Bony

Pascal Simonet

Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria

Keywords: GMO, HGT, antibiotic resistance gene, transplastomic tobacco, soil, microbial ecology

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.

Introduction

The use of transgenic plants in the field raises questions about the potential transfer of transgenes from these plants to soil bacteria that could lead to the dissemination of unwanted traits among bacterial communities in the environment. The main concern is about the transfer and dissemination of antibiotic resistance genes, usually cloned concomitant with the transgene to allow the selection of transformed plant cells [START_REF] Dröge | Horizontal gene transfer as a biosafety issue: a natural phenomenon of public concern[END_REF]. This is due to a higher possibility of recombination-mediated integration of these new plant DNA regions in bacterial genomes facilitated by sequence similarity between the transgene and the DNA of the recipient bacteria [START_REF] De Vries | Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination[END_REF][START_REF] Meier | Mechanisms of homologyfacilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri[END_REF]. In addition, studies indicate that several ecosystems in which physiologically active bacteria develop in close physical contact with DNA released by plant tissues constitute 'hot-spots' for horizontal gene transfer (HGT) between transgenic plants and bacteria [START_REF] Nielsen | Horizontal gene transfer from transgenic plants to terrestrial bacteria -a rare event?[END_REF][START_REF] Pontiroli | Visual evidence of horizontal gene transfer between plants and bacteria in the phytosphere of transplastomic tobacco[END_REF]. The likelihood of HGT is increased with transplastomic plants in which the transgene is cloned in the chloroplast genome composed of genes with a typical prokaryotic structure [START_REF] Mcfadden | Chloroplast origin and integration[END_REF][START_REF] Chu | Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes[END_REF]. The prokaryotic origin of the chloroplast genome suggests that transgene recombination in bacterial genomes could also be initiated based on the chloroplast DNA regions flanking the transgene, including the accD and rbcL genes routinely used for cloning purposes [START_REF] Svab | High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene[END_REF][START_REF] Birch-Machin | Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability[END_REF][START_REF] Zhou | High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes[END_REF]. The prokaryotic accD gene encodes the b-carboxyl transferase subunit of acetyl-CoA carboxylase (ACCase) required for fatty acid biosynthesis. Southern hybridization analysis demonstrated that most plants contain this accD gene in their genome and have the prokaryotic ACCase in their plastids [START_REF] Konishi | Acetyl-CoA carboxylase in higher plants: most plants other than gramineae have both the prokaryotic and the eukaryotic forms of this enzyme[END_REF]. The chloroplast rbcL gene encodes the large subunit of the CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and is present among Cyanobacteria and certain Proteobacteria (purple bacteria) [START_REF] Delwiche | Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids[END_REF]. Another factor increasing the likelihood of gene transfer from transplastomic plant to bacteria is the presence of the aadA gene. This gene encodes the aminoglycoside adenyl transferase conferring resistance to both streptomycin and spectinomycin antibiotics and is used to select for chloroplast transformants due to the effective inhibition of plastid protein biosynthesis in wild plants by spectinomycin [START_REF] Bock | Transgenic plastids in basic research and plant biotechnology[END_REF]. Moreover, the copy number of a chloroplastic transgenes in a plant cell is several orders of magnitude higher than that of a nuclear located DNA insert. A single leaf cell may contain dozens or even hundreds of chloroplasts. Chloroplastic DNA is organized in nucleoids harboring several copies of the plastid genome as typical of a prokaryotic system and several nucleoids are present in each chloroplast. Up to 10 000 (identical) copies of the plastid DNA can be found in a single pea leaf cell and even up to 50 000 copies in a wheat cell [START_REF] Bendich | Why do chloroplasts and mitochondria contain so many copies of their genome?[END_REF].

Studies already performed under conditions favoring HGT included the use of transplastomic plants concomitantly infected by pathogens (Kay et al., 2002a), transgenic plant residusphere (plant decaying material) [START_REF] Rizzi | Strategy for in situ detection of natural transformation-based horizontal gene transfer events[END_REF] and naturally transformable recipient bacteria specifically constructed to recombine and express transgenes [START_REF] Nielsen | Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants[END_REF]Kay et al., 2002b;[START_REF] Tepfer | Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer[END_REF][START_REF] Pontiroli | Visual evidence of horizontal gene transfer between plants and bacteria in the phytosphere of transplastomic tobacco[END_REF]. These conditions simulated those encountered in nature with the use of naturally transformable recipient bacteria commonly found in soil (e.g. Acinetobacter baylyi), and molecular constructions specifically engineered to simulate similarity between donor DNA and recipient bacterial genomes. Our study differs from previous ones because we used natural bacterial isolates as recipients.

The objective of this study was to determine the natural occurrence of DNA sequences among bacterial isolates on which recombination with transgene sequences or with the chloroplast DNA regions flanking the transgene could occur. Bacterial isolates able to grow on decaying tobacco plants [START_REF] Monier | Detection of potential transgenic plant DNA recipients among soil bacteria[END_REF] were screened for sequences that share a significant similarity level to three chloroplast-borne genes of transplastomic tobacco plants including accD, rbcL genes and the aadA marker gene. In order to assess the probability of HGT between transplastomic plants and indigenous bacteria, bacterial isolates were tested for their ability to take up extracellular DNA by natural or electrotransformation.

Materials and methods

Bacterial isolates, plants and plasmids

Sixteen environmental bacterial isolates able to grow on decaying plant tissues and presenting sequence similarities to the chloroplastic genes accD and/or rbcL [START_REF] Monier | Detection of potential transgenic plant DNA recipients among soil bacteria[END_REF] were used in this study. All bacterial strains were grown at 28 1C on a modified Luria-Bertani (LBm) medium (5 g L À1 NaCl). In order to test the resistance of the isolates to streptomycin and spectinomycin (antibiotic resistances encoded by the aadA gene), 100 mL of an overnight culture from the 16 isolates was spread on LBm plates supplemented with both antibiotics (50 mg mL À1 each).

Transplastomic tobacco plants (Nicotiana tabacum L. cv. PBD6) were grown in compost potting soil in a greenhouse at 23 1C ( AE 2 1C), with a daily regimen of 16 h of light and 8 h of darkness. Transplastomic plants harbored the transgenic aadA gene cloned between the chloroplastic genes rbcL and accD [START_REF] Svab | High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene[END_REF]Kay et al., 2002b) and contained c. 7000 copies of the transgene per plant cell.

Escherichia coli strain DH5a(pBHCrec) harbored the pBHCrec plasmid, a pBBR1MCS-3 derivative resistant to tetracycline, which contained the aadA gene flanked by part of the rbcL and accD tobacco plastid sequences [START_REF] Kovach | Four new derivatives of the broadhost-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes[END_REF][START_REF] Cérémonie | Isolation of lightning-competent soil bacteria[END_REF]. Escherichia coli strain DH5a(pLEP01) harbored the pLEP01 plasmid, an ampicillin -resistant cloning vector that contained the aadA gene, conferring resistance to spectinomycin and streptomycin, framed with plastid sequences corresponding to the rbcL and accD regions [START_REF] Svab | High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene[END_REF]Kay et al., 2002b). These strains were grown at 37 1C on LBm medium supplemented with spectinomycin (50 mg mL À1 ) and streptomycin (50 mg mL À1 ). Pseudomonas N3, an electrocompetent strain isolated from soil [START_REF] Cérémonie | Isolation of lightning-competent soil bacteria[END_REF], was grown at 29 1C on LBm medium and used as a positive control for electroporation experiments. Total transplastomic tobacco DNA, PCR products of the genes accD-aadA-rbcL generated from plasmid pLEP01 and plasmid pBHCrec were used for transformation and electroporation assays as described in the following sections.

DNA extraction

Genomic and plasmid DNA were isolated from bacterial strains using the NucleoSpin s Tissue and NucleoSpin s Plasmid Kits, respectively (Macherey-Nagel, Düren, Germany), following the manufacturer's instructions. Plant genomic DNA was extracted from plant leaves ground in liquid nitrogen using the DNeasy s Plant kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Following extraction, nucleic acid purity and concentration were determined by measuring the absorbance of DNA solutions at 260 nm using the NanoPhotometer (Implen GmbH, München, Germany) and by deposing 100 ng on agarose gel for comparison with the 1 kb1DNA ladder (Fermentas, Burlington, Canada).

Genetic amplification by PCR

For isolate identification, the 16S rRNA gene was amplified in a thermocycler (Applied Biosystems Inc., Foster City, CA) by PCR with the primers pA and pH (Table 1) and Titanium Taq 1 Â final (Clontech, St-Germain-en-Laye, France) on 70 ng of extracted genomic DNA (Table 1) according to the manufacturer's instructions. The PCR program consisted of a hot start at 94 1C for 5 min, followed by 30 cycles consisting of 94 1C for 30 s, annealing at 55 1C for 30 s and elongation at 68 1C for 1 min 30 s. A final elongation step at 72 1C for 7 min preceded cooling at 10 1C. Amplified DNA was purified on a 1% agarose gel using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK), according to the manufacturer's instructions. Purified PCR products were sequenced with pA and pH primers (GATC, Konstanz, Germany), assembled with SEQMAN version 7.2.1 (DNASTAR, Madison) and published in GenBank (accession numbers HQ670701-HQ670716).

The screening for the presence of aadA gene was performed by PCR with the primers p415 and p416 (Table 1) on 70 ng of purified genomic DNA. Amplifications were performed using the Invitrogen Taq polymerase (Invitrogen, Cergy Pontoise, France) according to the manufacturer's instructions. Amplified DNA (5 mL) was then loaded on a 2% w/v agarose gel.

For transformation and electroporation assays, the three genes were amplified by PCR with the primers accDF and rbcLR (Table 1) on 50 ng of plasmid pLEP01. PCR was performed with the Illustra HotStart Mix RTG (GE Healthcare) according to the manufacturer's instructions.

To analyze transformants, integration of the transgene into bacterial isolate's genome either by plasmidic replication or by genomic recombination was monitored by PCR with the primers rbcL455r and p416 (Table 1) on 70 ng of genomic DNA. PCR was performed with the Invitrogen Taq polymerase according to the manufacturer's instructions. Amplified DNA (5 mL) was then loaded on a 1% w/v agarose gel.

Transformation assays

Natural transformation

Natural transformation assays were performed as described previously (Demane `che et al., 2001b), except that overnight cultures were not 100-fold concentrated. Briefly, 100 mL of an overnight culture of the isolates was mixed with 0.1 pmol of plasmid pBHCrec, 0.1 pmol of PCR product or 500 ng of plant DNA directly on LBm Petri dishes. After drying of the droplets, the plates were incubated at 29 1C for 24 h and bacterial cells were resuspended in 500 mL of LBm medium with a sterile loop. Appropriate dilutions were then plated out on LBm medium supplemented or not with spectinomycin and streptomycin, to enumerate transformant and total cells, respectively. Transformations with plasmid pBHCrec were also plated on LBm medium supplemented with tetracycline (10 mg mL À1 ) to overcome problems linked to the natural resistance to streptomycin and spectinomycin of soil isolates. Negative controls were conducted without adding DNA. Experiments were conducted in triplicate.

Electroporation

The entry of DNA in bacterial cells due to electric discharges was tested by in vitro electroporations using the gene pulser TM apparatus (Bio-Rad Laboratories, Richmond, CA) by mixing 50 mL of electrocompetent cells [START_REF] Drury | Transformation of bacteria by electroporation[END_REF] with 0.1 pmol of plasmid pBHCrec, 0.1 pmol of PCR product or 500 ng of plant DNA. Mixes were incubated for 1 min on ice and electroporated in 0.2-cm cuvette at 12.5 kV cm À1 , 200 O, 25 mF, for 5 ms and immediately diluted in 450 mL of LBm medium [START_REF] Drury | Transformation of bacteria by electroporation[END_REF]. After 2 h at 29 1C, dilutions were plated out on LBm medium supplemented with appropriate antibiotics to determine transformation frequencies. Electroporations with plasmid pBHCrec were also plated on LBm medium supplemented with tetracycline (10 mg mL À1 ) to overcome problems linked to the natural resistance to streptomycin and spectinomycin of soil isolates. Negative controls were conducted without adding DNA. Positive controls were conducted on Pseudomonas N3 electroporated with 0.1 pmol of plasmid pBHCrec. Experiments were conducted in triplicate.

Results and discussion

Similarities of transplastomic genes with sequenced bacterial chromosomes

The transgene aadA and both flanking genes accD and rbcL of the transplastomic tobacco were BLASTed (BLASTN) using [START_REF] Altschul | Basic local alignment search tool[END_REF] against the NCBI chromosome database excluding eukaryotic sequences to detect which prokaryotic organism could be a potential transgene recipient. Seventy-three sequenced chromosomes possessed a high identity (ID) percentage (ID 4 60%) to the aadA gene, 108 to the accD gene and 79 to the rbcL gene (Fig. 1). Among BLAST results having an e-value 4 10 À5 , 66 sequences presenting 65-100% ID with the aadA gene were detected for 52% in enterobacteria, whereas 131 sequences with 65-87% ID with the accD gene were retrieved for 48% in Firmicutes and 66 sequences with 63-87% ID with the rbcL gene were rather of cyanobacterial origin for 55% (Fig. 1).

The alignment lengths of the BLASTs ranged from 58 to 1675 nucleotides, thus potentially allowing recombination [START_REF] Watt | Homology requirements for recombination in Escherichia coli[END_REF][START_REF] Prudhomme | Homologous recombination at the border: insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae[END_REF][START_REF] Monier | Detection of potential transgenic plant DNA recipients among soil bacteria[END_REF][START_REF] Brigulla | Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues[END_REF]. However, the presence of only one anchor sequence was shown to reduce the transformation frequency up to 10 6 -fold (de Vries & [START_REF] De Vries | Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination[END_REF][START_REF] Meier | Mechanisms of homologyfacilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri[END_REF][START_REF] Monier | Detection of potential transgenic plant DNA recipients among soil bacteria[END_REF]. For this reason, the presence of two of the three studied genes was monitored in these strains (Table 2). The presence of similarities to the aadA gene concomitant with similarities to the chloroplastic gene accD was shown in two strains of Acinetobacter baumannii, an aquatic species that can also be isolated from soils and animals, and is phylogenetically related to Gammaproteobacteria (Table 2). Both strains are opportunistic pathogens carrying multidrugresistant genes, insertion sequences, transposons and an ACCase (NCBI accession numbers NC_011586.1 and NC_010410.1). Similarities to both chloroplastic sequences were detected in 30 cyanobacterial strains (Table 2). Most of these Cyanobacteria (70%) are found in an aquatic environment, but some genera such as Cyanothece and Nostoc are also common in terrestrial environments in close contact with plants with which they have developed nitrogen-fixing symbiotic relationships (NCBI, Genome Project, Cyanothece sp. and Nostoc punctiforme). The distance-based phylogenetic analysis of complete chloroplastic and bacterial genomes showed that the chloroplast genomes were most closely related to Cyanobacteria [START_REF] Chu | Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes[END_REF] and chloroplast promoters were shown to initiate gene expression in Cyanobacteria [START_REF] Dzelzkalns | Chloroplast promoter driven expression of the chloramphenicol acetyl transferase gene in a cyanobacterium[END_REF][START_REF] Christopher | Expression of a higher-plant chloroplast psbD promoter in a cyanobacterium (Synechococcus sp. strain PCC7942) reveals a conserved cis-element, designated PGT, that differentially interacts with sequence-specific binding factors during leaf development[END_REF]. Consequently, homologous recombination could theoretically occur in these strains. However, considering the fact that the smallest physical distance between both sequences was of 506 361 bp for the terrestrial strain Cyanothece sp. PCC 7425 and 105 564 bp for the aquatic strain Synechocystis sp. PCC 6803 (Table 1), the likelihood of homologous recombination is considerably reduced. The mechanism to integrate transplastomic DNA could be by homology-facilitated illegitimate recombination (HFIR) or by illegitimate recombination (IR) favored by microhomologies. The frequency of IR is very low (2 Â 10 À10 ), but HFIR provide intermediate levels of integration frequencies (3 Â 10 À3 ) compared with the high frequency of homologous integration [START_REF] Brigulla | Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues[END_REF]. Considering all these data, the probability of gene transfer from plants to bacteria would occur (if any) by HFIR. As a consequence, gene introduction in plants should target chromosomal or plastidal sequences with very low or no similarities to bacterial genomes.

Occurrence of the aadA gene in soil isolates

A previous study [START_REF] Monier | Detection of potential transgenic plant DNA recipients among soil bacteria[END_REF] demonstrated that bacterial isolates able to colonize decaying tobacco plant tissue possessed DNA sequence similarities to chloroplastic genes accD and/or rbcL flanking the transplastomic transgene. We further studied these isolates concerning their natural resistance to spectinomycin and streptomycin, and the presence of the aadA gene in their genome.

To test the natural resistance to streptomycin and spectinomycin of soil isolates, overnight cultures were spread on LBm plates supplemented with both antibiotics. Only five isolates (CA11, CO03, MC02, PA07, PO01) were sensitive to streptomycin and spectinomycin, three isolates (AA12, AO13, MS01) presented an intermediate resistance, meaning that one to 100 colonies were able to develop on LBm supplemented with streptomycin and spectinomycin. The eight other isolates were resistant to both antibiotics (Table 3).

The second step included a PCR screening test in order to detect the presence of the aadA gene. None of the 16 isolates yielded a positive PCR signal (Table 3), indicating that the 11 soil isolates that resisted spectinomycin and streptomycin used mechanisms that did not involve products of the aadA gene. This is in accordance with the fact that aadA is not a gene commonly found in soil, unless it was introduced via manure or wastewater treatment plant spreading [START_REF] Tennstedt | Sequence of the 68,869 bp IncP-1alpha plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements[END_REF][START_REF] Srinivasan | Distribution of tetracycline and streptomycin resistance genes and class 1 integrons in Enterobacteriaceae isolated from dairy and nondairy farm soils[END_REF][START_REF] Binh | Diverse aadA gene cassettes on class 1 integrons introduced into soil via spread manure[END_REF]. Consequently, this gene should not represent a genomic site of insertion for gene transfer between transplastomic plants and soil bacteria.

In vitro detection of HGT between transplastomic transgene and selected bacteria

The probability of gene transfer from transplastomic plant to soil isolates able to colonize decaying tobacco plant tissue and possessing DNA sequence similarities to chloroplastic genes accD and/or rbcL flanking the transplastomic transgene aadA was then monitored experimentally. Two mechanisms could allow the transfer of genomic DNA into bacteria in the environment: natural transformation or electrotransformation. Natural transformation is restricted to a narrow range of bacteria fitted with adequate genes [START_REF] Johnsborg | Natural genetic transformation: prevalence, mechanisms and function[END_REF]. Moreover, different bacteria require different conditions such as a specific growth phase to develop their competence state [START_REF] Lorenz | Bacterial gene transfer by natural genetic transformation in the environment[END_REF]. To cope with these requirements, transformation experiments were carried out on plates so that bacteria were in contact with DNA during all growth phases. The second mechanism, electrotransformation, is based on the (Demane `che et al., 2001a;[START_REF] Cérémonie | Isolation of lightning-competent soil bacteria[END_REF].

The broad host-range plasmid pBHCrec [START_REF] Cérémonie | Isolation of lightning-competent soil bacteria[END_REF], a pBBR1MCS-3 derivative containing the transplastomic transgene aadA with flanking sequences accD and rbcL and coding resistance to tetracycline, was used for transformation and electroporation assays. Plasmid pBBR1MCS-3 was evidenced to replicate in at least 16 bacterial strains [START_REF] Kovach | Four new derivatives of the broadhost-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes[END_REF]. Two isolates, Ochrobactrum sp. AA12 and Acinetobacter sp. CA11, were able to acquire the plasmid by natural transformation with a respective frequency of 7.9 Â 10 À9 and 9.8 Â 10 À9 and six isolates (Ochrobactrum sp. AA12, three Acinetobacter sp. CA11, MC02 and PO01, Bosea sp. GA05 and Pseudomonas sp. SO15) were able to acquire this plasmid by electroporation with a frequency of c. 1.0 Â 10 À8 (Table 3). Hybridization experiments on transformant genomic DNA demonstrated that the transgene cloned in pBBR1MCS-3 was never integrated into the chromosome, but was propagated as a plasmid (data not shown). This was in accordance with the absence of transformants when the PCR product and transplastomic plant DNA were used as donor DNA (data not shown). The absence of a sufficient similarity and/ or too wide a length between homologous regions in the bacterial genomes for recombination to occur may explain these results.

Conclusion

Transgene transfer from plants to bacteria has been detected under greenhouse conditions with specifically selected donor and recipient organisms, but without anchor sequences, transfer could not be detected [START_REF] Gebhard | Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA[END_REF]Kay et al., 2002b;[START_REF] De Vries | Transfer of plastid DNA from tobacco to the soil bacterium Acinetobacter sp. by natural transformation[END_REF][START_REF] Pontiroli | Long-term persistence and bacterial transformation potential of transplastomic plant DNA in soil[END_REF]. In spite of these experiments that simulated an environment under optimized conditions for HGT to occur (transgene construction or genetically modified recipient strains, for example), plant bacteria HGT events remain undetected under field conditions [START_REF] Badosa | Lack of detection of ampicillin resistance gene transfer from Bt176 transgenic corn to culturable bacteria under field conditions[END_REF][START_REF] Demane `che S, Sanguin | Antibiotic-resistant soil bacteria in transgenic plant fields[END_REF]. These experimental results are in accordance with theoretical monitoring of the likelihood of gene transfer by examining the similarities between transplastomic plants and associated bacteria and contribute to establish that the probability of gene transfer from plants to bacteria, integration of transferred DNA into bacterial genomes and expression of the carried genes remain very unlikely to occur in nature. Finally, our results provide no indication that the high number of transgene copies in transplastomic plants could cause HGT at rates above those found in other studies with nuclear transgenes.

Table 1 .

 1 Primers used in this study

		Primer		Amplicon
	Target	name	Primer sequence (5 0 -3 0 )	size (bp)	T h ( 1C)	T e	References
	16S rRNA gene	pA	AGAGTTTGATCCTGGCTCAG	1534	55	1 min 30 s	Edwards et al. (1989)
		pH	AAGGAGGTGATCCAGCCGCA
	aadA	p415	ATTCCGTGGCGTTATC	382	60	30 s	Ceccherini et al. (2003)
		p416	TGACGGGCTGATACT
	accD-aadA-rbcL	accDF	ACCCACAAATGCCTGTATTTTTG	4113	56	4 min 30 s	This study
		rbcLR	TAGCTGCCGAATCTTCTACTGGTA
	Transformants	rbcL455r	GCTTTGTTGATTTACTGCGTG	1487	50	1 min 30 s	Monier et al. (2007)
		p416	TGACGGGCTGATACT
	T h , hybridization temperature; T e , elongation time.

FEMS Microbiol Ecol 78 (2011) 129-136 FEMS Microbiology Ecology c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. No claim to original French government works

Table 2 .

 2 Strains possessing more than 60% identity to accD and aadA or rbcL genes

	Strain	Accession number	accD	aadA	rbcL	Lineage	Length (bp) Ã
	Acaryochloris marina MBIC11017	NC_009925.1	1		1	Cyanobacteria	2 158 668
	Acinetobacter baumannii AB0057	NC_011586.1	1	1		Gammaproteobacteria	3 163 708
	Acinetobacter baumannii AYE	NC_010410.1	1	1		Gammaproteobacteria	2 997 610
	Anabaena variabilis ATCC 29413	NC_007413.1	1		1	Cyanobacteria	4 616 865
	Cyanothece sp. ATCC 51142	NC_010546.1	1		1	Cyanobacteria	1 160 075
	Cyanothece sp. PCC 7424	NC_011729.1	1		1	Cyanobacteria	3 659 837
	Cyanothece sp. PCC 7425	NC_011884.1	1		1	Cyanobacteria	506 361
	Cyanothece sp. PCC 8801	NC_011726.1	1		1	Cyanobacteria	1 479 116
	Cyanothece sp. PCC 8802	NC_013161.1	1		1	Cyanobacteria	1 471 856
	Microcystis aeruginosa NIES-843	NC_010296.1	1		1	Cyanobacteria	905 608
	Nostoc punctiforme PCC 73102	NC_010628.1	1		1	Cyanobacteria	1 795 936
	Nostoc sp. PCC 7120	NC_003272.1	1		1	Cyanobacteria	1 063 322
	Prochlorococcus marinus strain AS9601	NC_008816.1	1		1	Cyanobacteria	205 629
	Prochlorococcus marinus strain MIT 9211	NC_009976.1	1		1	Cyanobacteria	352 325
	Prochlorococcus marinus strain MIT 9215	NC_009840.1	1		1	Cyanobacteria	211 361
	Prochlorococcus marinus strain MIT 9301	NC_009091.1	1		1	Cyanobacteria	229 637
	Prochlorococcus marinus strain MIT 9303	NC_008820.1	1		1	Cyanobacteria	776 528
	Prochlorococcus marinus strain MIT 9312	NC_007577.1	1		1	Cyanobacteria	224 419
	Prochlorococcus marinus strain MIT 9313	NC_005071.1	1		1	Cyanobacteria	711 762
	Prochlorococcus marinus strain MIT 9515	NC_008817.1	1		1	Cyanobacteria	170 376
	Prochlorococcus marinus strain NATL1A	NC_008819.1	1		1	Cyanobacteria	208 055
	Prochlorococcus marinus strain NATL2A	NC_007335.2	1		1	Cyanobacteria	208 046
	Prochlorococcus marinus ssp. marinus	NC_005042.1	1		1	Cyanobacteria	259 007
	Prochlorococcus marinus ssp. pastoris	NC_005072.1	1		1	Cyanobacteria	226 966
	Synechococcus sp. CC9902	NC_007513.1	1		1	Cyanobacteria	762 712
	Synechococcus sp. JA-3-3Ab	NC_007775.1	1		1	Cyanobacteria	914 137
	Synechococcus sp. PCC 7002	NC_010475.1	1		1	Cyanobacteria	1 819 094
	Synechococcus sp. RCC307	NC_009482.1	1		1	Cyanobacteria	835 843
	Synechococcus sp. WH 8102	NC_005070.1	1		1	Cyanobacteria	885 127
	Synechocystis sp. PCC 6803	NC_000911.1	1		1	Cyanobacteria	105 564
	Thermosynechococcus elongatus BP-1	NC_004113.1	1		1	Cyanobacteria	143 798
	Trichodesmium erythraeum IMS101	NC_008312.1	1		1	Cyanobacteria	2 639 725

à The length corresponds to the number of nucleotides in the chromosome of strains between the two genes presenting more than 60% identity with accD and aadA or rbcL. Bold, terrestrial strains; underscoring, strains possessing similarities with aadA and accD genes. 1, presence of the gene. FEMS Microbiol Ecol 78 (2011) 129-136 FEMS Microbiology Ecology c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. No claim to original French government works

Table 3 .

 3 Identification, antibiotic resistance and transformation frequencies of the isolates studied Electroporation and transformation frequencies were calculated as the mean number of transformant mL À1 divided by the mean total number of cells mL À1 ; all experiments were conducted in triplicate. 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. No claim to original French government works electroporation principle. It was previously shown that gene transfer could occur during lightning discharge and may target the broadest range of recipient bacteria due to the absence of genetic requirements

		Strain	Accession		Antibiotic		Electroporation	Transformation
	Isolate	identification Ã	numbers	Lineage Ã	resistance w	PCR aadA	with pBHCrec z	with pBHCrec z
	AA12	Ochrobactrum sp.	HQ670701	Alphaproteobacteria	I	-	6.5E À 08	7.9E À 09
	A013	Acinetobacter sp.	HQ670702	Gammaproteobacteria	I	-	o 10E À 08	o 10E À 09
	CA01	Ochrobactrum sp.	HQ670703	Alphaproteobacteria	R	-	o 10E À 08	o 10E À 09
	CA11	Acinetobacter sp.	HQ670704	Gammaproteobacteria	S	-	1.9E À 08	9.8E À 09
	CO03	Pseudomonas sp.	HQ670705	Gammaproteobacteria	S	-	o 10E À 08	o 10E À 09
	GA05	Bosea sp.	HQ670706	Alphaproteobacteria	R	-	5.0E À 08	o 10E À 09
	MA06	Stenotrophomonas sp.	HQ670707	Gammaproteobacteria	R	-	o 10E À 08	o 10E À 09
	MC02	Acinetobacter sp.	HQ670708	Gammaproteobacteria	S	-	1.7E À 08	o 10E À 09
	MG02	Alcaligenes sp.	HQ670709	Betaproteobacteria	R	-	o 10E À 08	o 10E À 09
	MG07	Alcaligenes sp.	HQ670710	Betaproteobacteria	R	-	o 10E À 08	o 10E À 09
	MS01	Stenotrophomonas sp.	HQ670711	Gammaproteobacteria	I	-	o 10E À 08	o 10E À 09
	MP07	Stenotrophomonas sp.	HQ670712	Gammaproteobacteria	R	-	o 10E À 08	o 10E À 09
	PA03	Stenotrophomonas sp.	HQ670713	Gammaproteobacteria	R	-	o 10E À 08	o 10E À 09
	PA07	Bacillus sp.	HQ670714	Firmicutes	S	-	o 10E À 08	o 10E À 09
	PO01	Acinetobacter sp.	HQ670715	Gammaproteobacteria	S	-	6.4E À 08	o 10E À 09
	SO15	Pseudomonas sp.	HQ670716	Gammaproteobacteria	R	-	7.9E À 08	o 10E À 09

à Strain identification and lineage were based on the sequence of their 16S rRNA gene BLASTed (BLASTN) against the nr NCBI database. w Strains were tested for their resistance to spectinomycin and streptomycin; I, intermediate resistant strain; R, resistant strain; S, sensitive strain. z FEMS Microbiol Ecol 78 (2011) 129-136 FEMS Microbiology Ecology c

Acknowledgements

This work was supported in part by the Rho ˆne-Alpes Region, the Haut Conseil des Biotechnologies and the Programme National de Recherches sur les Organismes Génétiquement Modifiés from the Agence Nationale de la Recherche for the projects Ploben Grant ANR-05-POGM-004-01 and Septante Grant ANR-07-POGM-002. We are grateful to Prof. T.M. Vogel for his critical reading of the manuscript.