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Applied and Environmental Microbiology, October 2006, p. 6687-6692, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00013-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cultivation-Independent Examination of Horizontal Transfer and Host Range of an IncP-1 Plasmid among Gram-Positive and Gram-Negative Bacteria Indigenous to the Barley Rhizosphere

Sanin Musovic,1 Gunnar Oregaard,1 Niels Kroer,2 and Søren J. Sørensen1*

University of Copenhagen, Institute of Biology, Department of Microbiology, Sølvgade 83H, 1307K Copenhagen K, Denmark,1 National Environmental Research Institute, Department of Environmental Chemistry and Microbiology, Frederiksborgvej 399, 4000 Roskilde, Denmark2

Received 3 January 2006/ Accepted 24 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10–2 ± 3.84 x 10–2. This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Horizontal gene transfer between bacteria has gained considerable interest during the past 2 decades due to impacts on microbial adaptation to natural and anthropogenic stress and concerns related to the spreading of recombinant DNA. Bacteria are known to horizontally exchange genetic material by transformation, transduction, and conjugation (7). The mechanism of bacterial conjugation relies on exchange of mobile genetic elements (e.g., plasmids and conjugative transposons) and is believed to be one of the main mechanisms responsible for short-term microbial adaptation (34), transferring genetic material even among phylogenetically remote organisms (7, 21).

Horizontal plasmid transfer has traditionally been studied by culture-dependent approaches, which are limited to the culturable fraction of environmental bacteria. However, since most bacteria are difficult to culture, with estimates that 1 to 5% of all bacteria are readily culturable (1, 25, 36), not only the transfer frequency, but also the host range of the plasmid may be grossly underestimated (34). The reluctant culturability of many soil bacteria requires that conjugative plasmid transfer be examined by cultivation-independent methods. Cultivation-independent approaches using fluorescent reporter genes (e.g., the green fluorescent protein gene, gfp) have previously been used to study the locations of transconjugants on the phylloplane (22, 23) and in biofilms (4, 11).

The aim of the present study was to investigate in a cultivation-independent manner the host range and transfer frequency of a conjugative plasmid, pKJK10, among indigenous barley rhizosphere bacteria. To do this, we applied single-cell flow cytometry (FCM) analysis and cell sorting for cultivation-independent detection and quantification of plasmid transfer (32).

The rhizosphere supports dense bacterial communities (20, 24, 33), and rhizosphere environments have been classified as hot spots for horizontal gene transfer (HGT) (7, 17, 20). Furthermore, the high cell density in the rhizosphere makes this environment well suited for flow cytometric analysis of gene transfer and cell sorting.

A new approach for the detection of plasmid transfer by single-cell flow cytometry analysis has recently been developed by our group (32). In brief, a natural barley rhizosphere plasmid, pKJK10 (IncP-1), was tagged with gfp downstream from a synthetic lac promoter (30). The expression of the gfp gene is repressed in the plasmid donor strain by a chromosomal lacIq1 insert but is not repressed if the conjugative plasmid is transferred to a recipient cell (i.e., a transconjugant). Large bacterial populations can quickly be analyzed by flow cytometry according to cell size, surface structure, and fluorescence. Using the fluorescence-labeled reporter plasmids described above, horizontal gene transfer was detected and quantified by flow cytometry (32). In the present study, we have expanded this approach by combining it with automated cell sorting of green fluorescent transconjugant cells. 16S rRNA genes from sorted transconjugant cells were subsequently PCR amplified and sequenced to determine the identities of the transconjugants without cultivation. To our knowledge, this is the first study of plasmid transfer from an introduced donor strain to indigenous bacterial rhizosphere communities using a flow cytometry-based cultivation-independent approach and the first study demonstrating that gram-positive Actinobacteria are recipients of a natural conjugative rhizosphere plasmid.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Site description and soil water-holding capacity.
Soil samples were collected in mid-June 2003 from a field with spring barley (Hordeum vulgare L.) at the Højbakkegaard (Taastrup, Denmark) experimental station. Barley plants with undisturbed soil around the roots were collected separately. The top 3 to 5 cm of surface soil was discarded. The soil samples were processed in the laboratory by careful removal of plant parts and stored at +5°C. The water content of the stored soil samples was determined as weight loss after 24 h of incubation of sieved soil at 80°C and was found to be 12.2%. The water-holding capacity was measured using dried soil saturated with distilled water on top of sand beds and was found to be 28.2%. The field's pH had previously been determined to be 6.4 ± 0.2 (15).

Bacterial strain, conjugal plasmid, and growth media.
Pseudomonas putida KT2440::lacIq1, harboring the conjugative plasmid pKJK10, was used as the donor strain. Plasmid pKJK10 is a derivative of the 54-kb natural rhizosphere plasmid pKJK5 carrying natural tetracycline resistance (30). pKJK10 has the gfp(mut3b) gene inserted downstream of the lac-repressible promoter PA1-04/03 (22, 30). The plasmid donor strain was grown in Luria-Bertani (LB) broth (10 g tryptone, 5 g yeast extract, 10 g NaCl in 1 liter distilled water, pH 7.2) supplemented with tetracycline (20 µg/ml). The broth cultures were grown with vigorous shaking (250 rpm) at 30°C for 20 h. The cells were washed twice and resuspended in sterile saline (9{per thousand} NaCl). The plasmid donor cells were verified for repression of the gfp gene by flow cytometry prior to inoculation into microcosms.

Microcosm setup and inoculation.
Microcosms consisted of 20-ml plastic cups containing 14 g of 2-mm-pore-size sieved soil inoculated with 3 x 106 donor bacteria/g. The water content was adjusted to 68% of the maximum water-holding capacity and mixed thoroughly. Two sterile barley seeds were planted in each plastic cup. Barley seed sterilization was performed by sequential treatment with 50% H2SO4 (1 h) and 1% AgNO3 (7.5 min), as described by Schwaner and Kroer (29). The seed bracts were manually removed after 50% H2SO4 treatment. For each of the three independent HGT experiments, five replicate microcosms and three control microcosms, to which no donor bacteria were added, were prepared and packed in transparent plastic bags with a plastic cup containing water for maintenance of humidity. The microcosms were incubated at room temperature. On day 4, 0.5 ml sterile Milli-Q water was added to each microcosm.

Extraction of rhizosphere bacteria for flow cytometry analysis and sorting.
For each HGT experiment, three selected replicate barley microcosms and three control microcosms were sampled 7 days after inoculation. Roots with tightly associated rhizosphere soil were gently separated from the bulk soil, weighed, immersed in 3 ml 0.05% Tween 80-50 mM tetrasodium pyrophosphate (38), and homogenized in a mortar.

The resulting rhizosphere extract was vortexed for 2 min at 2,500 rpm and sonicated for 10 min (3) in a 5-ml Becton Dickinson Falcon tube to increase the release of bacterial cells from clay and soil particles. The bacterial fraction was purified by Nycodenz extraction (16). Briefly, the rhizosphere extract was carefully transferred on top of the autoclaved Nycodenz solution (Nycomed Pharma, Norway; final density, 1.3 g/ml) and centrifuged (10,000 x g for 10 min). The upper and middle phases containing a bacterial layer on top of the Nycodenz layer were carefully pipetted off and resuspended in 5 ml sterile 0.05% Tween 80-50 mM tetrasodium pyrophosphate in a 9-ml plastic centrifuge tube (Sarsteds, Numbrecht, Germany). After centrifugation at 7,000 x g for 10 min, the pellet was resuspended in 1 ml sterile-filtered (0.2 µm) saline, followed by filtration through sterile 100-µm-pore-size filters, to remove large soil particles that could be detrimental to the later flow cytometry analysis, generating purified rhizosphere extract. The saline was 0.2-µm filtered to reduce the number of false events during flow cytometric analysis. The total cell loss during this four-step Nycodenz process was determined to be 76% ± 1.7% (n = 2) of the initial bacterial numbers in rhizosphere extracts.

Enumeration and sorting of bacteria by FCM.
A FACSCalibur flow cytometer (Becton Dickinson, ICS, San Jose, CA) equipped with an argon laser (20 mW) emitting blue light (488 nm) and with a modified mechanical sorting device for collecting cells from a continuous stream (38) was used for enumeration of bacterial cells in the purified rhizosphere extract. The extract was diluted in sterile saline to ensure that the number of events by FCM analysis was below 2,000 per second.

The FCM analysis was carried out by thresholding on side scatter (SSC). The instrumental settings were as follows: FSC = E01; SSC = 350; FL1 = 510. Flow cytometry analysis was conducted, and two regions (forward scatter versus side scatter and side scatter versus green fluorescence) were used to define the bacteria, enabling discrimination between bacterial cells and soil debris. Prior to use, the flow cytometer was rinsed with FACSrinse and FACSclean solutions (Becton Dickinson). The flow cytometry analysis allowed enumeration of fluorescent bacteria. The total number of cells was determined after the cells were stained with SYBR Green II (Molecular Probes, Inc., Eugene, OR). The numbers of donor cells (P. putida KT2440) in rhizosphere extracts were determined after derepression of the lac promoter. This was done by addition of 1/10-strength LB broth, nalidixic acid (80 µg/ml), Savinase (5 µl/ml; 16 U/g; product P331; Sigma Aldrich), and IPTG (isopropyl-ß-D-thiogalactopyranoside) (10 mM) and incubation for 5 h with shaking (250 rpm) at 30°C prior to flow cytometric analysis. Appropriate concentrations of IPTG and nalidixic acid, necessary for the derepression of the gfp gene and inhibition of bacterial proliferation, respectively, were determined in an initial experiment (data not shown). Enumerations of transconjugants were done using diluted rhizosphere extracts. Analyses of rhizosphere extracts from microcosms to which no plasmid donor bacteria were added were performed as a control for false-positive transconjugant-like particles. The number of metabolically active cells was determined by staining them with nonfluorescent 6-carboxyfluorescein diacetate, a nonspecific substrate for intracellular esterases that, after cleavage, intracellularly releases a green fluorescent carboxyfluorescein molecule (6, 13, 39). The staining process involved fivefold dilution of the sample into sterile-filtered Tris-EDTA buffer (1 mM, pH 7.5) and carboxyfluorescein diacetate (10 µM final concentration) for 20 min in the dark at room temperature (6). The numbers were corrected with a negative control, an esterase-inactivated cell suspension, as recommended by Tanaka et al. (35). All bacterial samples were analyzed in triplicate. Analysis of sterile water was performed in every run as a control for background noise in the flow cytometer.

For sorting purposes, a region was defined in a dot plot with SSC versus green fluorescence (FL1), discriminating green fluorescent cells from nonfluorescent cells and debris. The threshold was set on SSC at a value of 52, and all data were analyzed using CellQuest software (BD Bioscience). The flow rate was determined prior to analysis and at 2-hour intervals during analysis of extracted rhizosphere bacteria. Prior to sorting of green fluorescent transconjugant cells, the capillary tubes inside the flow cytometer were sterilized and cleaned by injection of 50 ml 50% chlorine (sodium hypochlorite) and rinsed twice with the same volume of sterile-filtered Milli-Q water. The sheath fluid tank was also washed and filled with sterile-filtered saline. The purified rhizosphere extract samples were diluted in order to obtain an event rate of 300 to 400 events per second, resulting in approximately two sorted cells per minute (single-cell sorting mode). The sorted cells were collected by vacuum filtration (7 to 10 mm Hg) onto a 0.22-µm-pore-size cellulose acetate membrane filter (Advantec MFS Inc., United Kingdom). The filters were prewashed with 70% ethanol and twice in sterile Milli-Q water. For verification of correct sorting, a few selected filters were examined by epifluorescence microscopy using a Zeiss Axioskop 2 equipped with a 100-W mercury lamp and a no. 10 filter set (Carl Zeiss). These filters, carrying detectable green fluorescent transconjugant cells, were not used for molecular analysis (see below). As a control for false sorting of nontransconjugant bacteria to the filters, sorting was also performed on the rhizosphere extracts from microcosms with no donor bacteria added and on sterile water in every sorting experiment.

After sorting of approximately 400 cells, the filter was removed and the cells were dislodged from the filter by vortexing them (2,500 rpm; 2 min) in 1 ml autoclaved sterile-filtered Milli-Q water, previously shown not to contain amplifiable DNA (DNA-free water). The cells were pelleted (10,000 x g; 10 min) and resuspended in 10 µl DNA-free water. A double boiling-freezing protocol was used for cell lysis (100°C for 10 min, followed by –80°C for 10 min). The lysates were stored at –20°C.

Amplification of 16S rRNA genes from sorted transconjugant bacteria.
Bacterial 16S rRNA gene fragments were amplified with broad-specificity primers for the bacterial domain: 27F, 5'-AGAGTTTGATCCTGGCTCAG-3', and 1492R, 5'-GGA/TTACCTTGTTACGACTT-3', or 907R, 5'-CCGTCAATTCATTTA/GAGTTT-3' (19, 27). PCR mixtures contained 10 µl bacterial lysate, 20 pmol of each primer, 50 mM KCl, 30 mM Tris-HCl, 1.5 mM MgCl2, 12.5 mM of each nucleotide (dATP, dCTP, dGTP, and dTTP), and 1 U Taq polymerase. The final sample volume of 25 µl was adjusted using DNA-free water. Amplification involved initial denaturation at 92°C for 2 min, followed by 35 cycles of 30 s at 92°C, 30 s at 51°C, and 2 min at 72°C. Finally, a primer extension reaction was performed for 6 min at 72°C. The PCR negative control consisted of DNA-free water. The PCR products were analyzed on 1.5% (wt/vol) Nusieve Agarose gels (Medinova, Hellerup, Denmark) in 1x Tris-borate-EDTA buffer.

DGGE.
The heterogeneity of the amplified 16S rRNA gene fragments from the sorted transconjugant bacteria was determined by denaturation gradient gel electrophoresis (DGGE) analysis, using internal primers with a GC clamp, 338F (5'-GCTGCCTCCCGTAGGAGT-3') and 518R (5'-ATTACCGCGGCTGCTGG-3') (27).

PCR mixtures (25 µl) contained 1 µl amplified bacterial DNA, 20 pmol of each primer, 12.5 mM of each deoxynucleoside triphosphate, 1.5 mM MgCl2, and 1 U Taq polymerase. After PCR (2 min at 94°C; 30 cycles of 30 s at 92°C, 30 s at 53°C, and 2 min at 72°C; and finally, 10 min at 72°C, followed by cooling at 6°C), the product was loaded on a DGGE gel. The denaturation gradient of the DGGE gel ranged from 30% to 60%, and electrophoresis was run at 60°C and 70 V for 17 h. The gel was stained with SYBRGold (Molecular Probes) for 45 min in the dark.

Cloning, sequencing, and phylogenetic analysis.
Amplified 16S rRNA genes from the transconjugant bacteria, showing several bands on the DGGE gel, were selected for cloning. To increase the amount of product, 1 µl amplified 16S rRNA genes was reamplified as described above and purified using the High Pure PCR product purification kit (Roche). Purified 16S rRNA gene fragments were inserted into the pCR4-TOPO vector using the TOPO-TA Cloning Kit for Sequencing (Invitrogen Ltd., United Kingdom). pCR4-TOPO vectors with inserted 16S rRNA genes were transformed into One Shot Chemical Competent Escherichia coli cells. Positive selection for transformant E. coli/pCRR4::16S rRNA gene cells was achieved by plating the cells on LB plates containing 50 µg/ml kanamycin and incubating them at 37°C for 24 h. Plasmid DNA was prepared from transformant cells using the High Pure plasmid isolation kit (Roche). The concentration of plasmid template DNA was adjusted to 60 ng/µl in a solution containing 5 pmol of a primer and the recommended amount of sequencing mix (DYEnamic ET dye terminator cycle sequencing kit; Nycomed Amershham, United Kingdom) prior to sequencing. PCRs were performed on a MEGA BACE 1000 sequencer (Molecular Dynamics, Sunnyvale, CA). The sequencing reactions were carried out in parallel with the 16S eubacterial primers 27F and 907R (19).

The quality of the partial sequences was evaluated with Sequencer 4.1 software (Genes Codes Corp., Ann Arbor, Mich.), and ambiguous sequence regions were deleted. The partial (over 500-bp) 16S rRNA gene sequences were examined by BLASTN against the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/BLAST) and identified by their best match to a known 16S rRNA gene sequence.

Nucleotide sequence accession numbers.
The newly determined 16S rRNA gene sequences were deposited in GenBank under accession numbers DQ342226 to DQ342234.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell densities and transfer frequency.
Conjugal transfer of plasmid pKJK10 to indigenous bacteria of the barley rhizosphere was examined in rhizosphere microcosms. Flow cytometry analysis allowed parallel enumeration of green fluorescent transconjugants, donors after derepression of the gfp gene, total cells by SYBR green II staining, and metabolically active cells from the rhizosphere. The plasmid transfer frequency, determined as transconjugant/donor ratio (T/D), corresponded to 7.03 x 10–2 ± 3.84 x 10–2 (n = 9), and the number of metabolically active cells exceeded 60%, supporting the role of the barley rhizosphere as a hot spot stimulating high bacterial activity (Table 1). As expected, no transconjugant-like events were encountered in the negative controls with no donors added.


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  		TABLE 1. Conjugal transfer of pKJK10 plasmid from inoculated P. putida KT2440::lacIq to bacteria indigenous to the barley rhizospherea

 
Host range of pKJK10.
The PCR amplification efficiency on sorted cells (approximately 400), using universal eubacterial 16S primers, was low. Agarose gel electrophoresis revealed that only about one-fourth of the PCRs were successful. Subsequent DGGE analysis showed that around 60% of the successful PCRs had more than one PCR amplicon, indicating that the PCR mixture contained a mixture of transconjugants. No PCR products were detected in the control sortings, indicating that only the green fluorescent transconjugant bacteria were collected in the cell sorting from microcosms with transconjugants. The 16S rRNA gene amplicons were cloned and transformed for sequencing purposes. A total of 39 transformants (i.e., clones) were obtained from three independent HGT cell-sorting experiments.

DGGE analysis of PCR-amplified 16S rRNA gene fragments prior to and after cloning showed that the cloned 16S fragments corresponded to the initial 16S DGGE bands. Some of the 16S rRNA gene fragments analyzed on DGGE gels are shown in Fig. 1.


Figure 1
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  		FIG. 1. DGGE gel showing PCR-amplified transconjugal 16S rRNA gene fragments after cloning, obtained during HGT cell-sorting experiment S3.

 
Phylogenetic identities of barley rhizosphere transconjugants by partitial 16S sequencing.
The transconjugant 16S rRNA gene sequences showed high similarity (>97% identical) to sequences previously deposited in GenBank. The identities of the cloned sequences are shown in Table 2. The sequenced clones were affiliated with seven different genera, belonging to the Alphaproteobacteria (Bradyrhizobium sp. and Mesorhizobium sp.), Betaproteobacteria (Acidovorax sp., Burkholderia cepacia, Burkholderia pyrrochinia, and Diaptorobacter sp.), Gammaproteobacteria (Acinetobacter calcoaceticus), an uncharacterized rhizosphere-associated bacterium, and, most surprisingly, Actinobacteria (Arthrobacter sp.). The Arthrobacter, Mesorhizobium, Acidovorax, and Burkholderia clones were found in two out of the three independent HGT experiments. The Arthrobacter 16S fragments were, as shown in Table 2, the most frequent among the clones (13 out of a total of 39).


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  		TABLE 2. Taxonomic identification of transconjugants from the barley rhizosphere

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first plasmid transfer study in which indigenous rhizosphere transconjugants were identified and enumerated by a cultivation-independent approach. Combining flow cytometry analysis and fluorescence-activated cell sorting allowed us to culture-independently assess the transconjugant diversity by cloning and sequencing 16S rRNA genes of sorted cells. Sequencing of the 16S rRNA genes of the sorted transconjugants revealed a broad host range for plasmid pKJK10, including Alpha-, Beta-, and Gammaproteobacteria and also, surprisingly, gram-positive Actinobacteria (Table 2). The efficiencies at some steps in transconjugant analysis, including DNA extraction, 16S rRNA gene amplification, and cloning, vary for different bacteria. Therefore, the reported transconjugant distribution is a conservative estimate of the total transconjugant diversity. No 16S rRNA gene sequences were obtained from the negative controls from rhizosphere microcosms without donor bacteria added, indicating that in the absence of transconjugants, no bacteria were sorted to the filter. Furthermore, it has been shown that 99 to 100% purity with regard to cell sorting is achieved with a low event rate (300 events per second) (8). We used a slightly higher event rate of 300 to 400 per second, but most of the detected events in the present study were not from bacteria but from soil particles, in contrast to the study by Ferrari et al. (8), where pure cell suspensions were used. Hence, the sorting procedure used here is most likely very pure. These controls underline the fact that the obtained 16S rRNA genes must originate from transconjugants.

IncP-1 plasmids have previously been shown to have a broad host range, transferring between many gram-negative species (7, 12, 31). Previous studies examining the conjugative properties of the pKJK10 plasmid revealed transfer between different Pseudomonas strains (22, 30, 32) and E. coli strains (32) and from Pseudomonas to Erwinia species (22). Geisenberger et al. (9) demonstrated that pMA119 (RP4 tagged with gfp) transferred to Acidovorax sp. and Acinetobacter sp. in an activated-sludge environment. Transconjugants belonging to these genera were also found in the present study.

The clone library of transconjugants in this study revealed that several of the transconjugants belonged to the gram-positive Actinobacteria (Arthrobacter sp.). Two out of three independent HGT cell-sorting experiments revealed Arthrobacter sp. as the recipient. The venerable paradigm that conjugative plasmid exchange occurs exclusively between members of the gram-negative or gram-positive bacteria has been based partly on differences in their conjugal mechanisms (e.g., initiation of bacterial aggregation) (10). This paradigm has recently been challenged. For instance, Schäfer et al. (28) reported the transfer of a mobilizable shuttle plasmid, pECM1, from E. coli to Corynebacterium glutamicum (gram-positive bacteria). The pECM1 plasmid had an oriT region on an integrated RP4 fragment, and the donor (E. coli) had the RP4-derived tra genes inserted into the chromosome. pECM1 carried, in addition, the origins of vegetative replication in both E. coli (oriVE) and C. glutamicum (oriVC) bacteria. Trieu-Cuot et al. (37) demonstrated conjugative transfer of a mobilizable shuttle plasmid, pAT187, from E. coli to various gram-positive bacteria belonging to the genera Enterococcus, Streptococcus, Bacillus, Listeria, and Staphylococcus. Plasmid pAT187, which was successfully mobilized by the helper plasmid pRK212.1 (IncP), contained the origin of transfer (oriT) of the RP2 plasmid (i.e., RP4) (26), an origin of replication for E coli (i.e., pBR322) (14, 18), and, additionally, a broad-host-range origin of replication for gram-positive streptococcal bacteria (i.e., pAMß1) (18). Furthermore, Bertram et al. (2) reported conjugative transfer and integration of the conjugative transposon Tn916 from diverse gram-positive to gram-negative bacteria, and vice versa. Interestingly, both Schäfer et al. (28) and Trieu-Cuot et al. (37) used shuttle plasmids containing the origin of transfer (oriT) and transfer functions of IncP plasmids. However, these studies were performed with recombinant shuttle vectors, while the present study is the first to show transfer from gram-negative to gram-positive bacteria by a native IncP plasmid replicon.

Thus, the limitation of IncP-1 plasmid transfer to gram-positive bacteria is not due to the conjugative transfer as such but most likely to the inability of IncP-1 plasmids to replicate in these hosts. To be stably maintained in a new host, it is important that the plasmid not be degraded by the restriction enzymes of the host and that it can be replicated in the new host (5). That was the reason why Bertram et al. (2) used the chromosomal integration of transferred Tn916, while Trieu-Cuot et al. (37) and Schäfer et al. (28) used shuttle plasmids containing additional origins of replication known to replicate in gram-positive bacteria. The single-cell detection of transconjugants used in this study does not require stable replication and vertical spread of plasmids in transconjugants. It is therefore possible that pKJK10 does not replicate in the Arthrobacter transconjugant cells but that gfp is expressed. Chromosomal integration of plasmid pKJK10 could also explain the stable expression of pKJK10 selectable traits, like the gfp gene. Investigation of the stability of pKJK10 in transconjugant cells is needed to address this issue further.

Flow cytometry detection of donor and transconjugant cells also provided an easy and precise estimation of plasmid transfer efficiency in the rhizosphere without the bias of cultivation. Indigenous transconjugant bacteria that are not able to express the gfp gene carried by pKJK10 will not be detected by this approach, and therefore, the plasmid transfer quantified by flow cytometry analysis might slightly underestimate the occurrence of gene transfer. The transfer frequency of plasmid pKJK10 in the barley rhizosphere microcosm was determined as T/D on the day of extraction: 7.03 x 10–2 ± 3.84 x 10–2 (n = 3). This relatively high transfer frequency in the rhizosphere can be attributed to nutrient richness and to the root surface, ideal for bacterial aggregation. The aggregation of bacterial cells has previously been shown to increase plasmid transfer (17, 23), especially that of IncP, IncW, and IncN plasmids, due to their rigid pili (7). Sengeløv et al. (30) also observed a high transfer frequency of pKJK10 between P. putida strains in the barley rhizosphere. The decreasing number of plasmid donor cells extracted from microcosms on day 7 is also a contributing factor to the high transfer frequency.

This report demonstrates the potential of a newly developed technique for culture-independent examination of horizontal gene transfer between bacteria in natural environments, overcoming the disadvantages of traditional cultivation-based HGT determination. Furthermore, we have shown that conjugative transfer of the pKJK10 (IncP-1) plasmid involves both gram-positive and gram-negative recipients. The potential of mobile genetic elements, like conjugative plasmids, crossing large phylogenetic distances, i.e., gene swapping between Actinobacteria and Proteobacteria, could be an important issue when bacterial evolution and the acquisition of new traits in nature are considered.


   ACKNOWLEDGMENTS
 
This research was partly supported by funding to Søren J. Sørensen and Niels Kroer by the Natural and Accelerated Bioremediation Research (NABIR) Program, Biological and Environmental Research (BER), U.S. Department of Energy.


   FOOTNOTES
 
* Corresponding author. Mailing address: University of Copenhagen, Institute of Biology, Dept. of Microbiology, Sølvgade 83H, 1307K Copenhagen K, Denmark. Phone: 45 35 32 20 53. Fax: 45 35 32 20 40. E-mail: sjs{at}bi.ku.dk. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, October 2006, p. 6687-6692, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00013-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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