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Applied and Environmental Microbiology, November 2006, p. 7253-7259, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00922-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Increased Abundance of IncP-1ß Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1ß Plasmids with a Complex mer Transposon as the Sole Accessory Element{triangledown}

Kornelia Smalla,1* Anthony S. Haines,2 Karen Jones,2 Ellen Krögerrecklenfort,1 Holger Heuer,1 Michael Schloter,3 and Christopher M. Thomas2

Federal Biological Research Centre for Agriculture and Forestry (BBA), Messeweg 11-12, 38104 Braunschweig, Germany,1 School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,2 GSF National Research Center for Environment and Health—Institute for Soil Ecology, Ingolstädter Landstr. 1, 85758 Oberschleissheim, Germany3

Received 19 April 2006/ Accepted 11 September 2006


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ABSTRACT
 
Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1ß replicon-specific sequences detected in total community DNA. The isolation of IncP-1ß plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1ß plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1ß plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRT{Delta}P probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1ß plasmids R751 and pB8. However, this is the first example of an IncP-1ß plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.


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INTRODUCTION
 
Mercury is one of the most toxic heavy metals in the environment. Mercury pollution of areas can be caused by both geological processes and anthropogenic activities. Bacteria play an important role in the global mercury cycle, and they have evolved resistance mechanisms to detoxify several chemical forms of mercury. Molecular analysis of mercury-resistant bacterial isolates has revealed an enormous diversity of mercury resistance genes which has recently been reviewed by Barkay et al. (1). Mercury resistance genes are often located on mobile genetic elements (MGE), which can be located either chromosomally or on plasmids. Mercury resistance genes were reported to occur on a wide range of plasmids belonging to various incompatibility groups. In general, it is proposed that MGE facilitate bacterial adaptability in response to environmental stresses and contribute considerably to their diversity (31). While mercury resistance for many Hg-resistant strains, plasmids, and transposons has been studied at a molecular level in great detail, studies making the link with their ecology and providing data on their abundance and diversity depending on environmental Hg pollution are relatively rare (5). Recently, methods for assessing the prevalence of MGE and of resistance genes in environmental samples independent from cultivation-based techniques have become available (24). PCR amplification of DNA which was directly extracted from environmental samples with primers targeting replicon or resistance gene sequences, when combined with Southern blot hybridization, enables their rapid and specific detection. While PCR-based detection of MGE and resistance genes in community DNA is an ideal method to analyze many samples for the presence and abundance of the sequence targeted (23), no information is obtained on its genomic context. For this, the isolation of the respective MGE is essential. Three methods are presently used which allow the isolation of MGE independent from cultivation: exogenous isolation in either a bi- or triparental mating or transformation of recipient strains with directly extracted plasmid DNA (24, 26).

DNA directly extracted from river sediment samples taken from two mercury-polluted and two nonpolluted areas of the river Nura (region of Termirtau County, central Kazakhstan, 1,200 km north of the capital, Almaty) was analyzed by PCR for merRT{Delta}P and a range of broad-host-range plasmids. PCR and Southern blot analysis revealed that the abundance of IncP-1ß trfA2 and merRT{Delta}P corresponded to the degree of mercury pollution. To find out whether or not mercury resistance genes were indeed located on IncP-1ß plasmids, we attempted to isolate them from the contaminated sites. Isolation of IncP-1 plasmids conferring resistance was attempted from the cultured fraction of Hg-resistant bacteria and by exogenous isolation. In a triparental mating, three IncP-1ß plasmids were isolated based on their gene-mobilizing capacity. These plasmids were characterized in more detail, and the complete sequence for one of the plasmids was determined.


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MATERIALS AND METHODS
 
Strains and plasmids.
The following rifampin-resistant mutants, which were tagged with the gene (gfp) coding for green fluorescent protein (GFP) by Heuer et al. (11) were used as recipients for exogenous isolation and as hosts for testing plasmid transfer and stability in different hosts: Agrobacterium tumefaciens UBAPF2 (Alphaproteobacteria), Cupriavidus necator (formerly Ralstonia eutropha) JMP228 (Betaproteobacteria), Escherichia coli CV601 (Gammaproteobacteria), and Pseudomonas putida KT2442 (Gammaproteobacteria). The IncQ plasmid pIE723 (30) was used as second donor in triparental matings. This plasmid was obtained in E. coli J53 from H. Tschäpe (Robert Koch-Institut, Wernigerode, Germany).

Sampling.
In autumn 1999 and spring 2000, sediment samples were collected from the sediment of the river Nura in Termirtau County, Kazakhstan. For over 150 years, the region has been characterized by high levels of industrial activity, especially coal mines and steel-producing companies. The river Nura is highly contaminated by mercury mainly caused by a chemical plant (catalyst for acetaldehyde formation), a metallurgical plant, and a coal-burning power station. Although most companies have been closed since the breakup of the Soviet Union, the riverbed sediments were still found to contain very high concentrations of mercury, particularly the first 10 km downstream of the pollution source. Composite samples were taken from four sampling points downstream of the pollution area at distances of 5 (A), 10 (B), 15 (C), and 20 (D) km from the main source (degree of longitude, 49° 51' 47" N, latitude 73° 11'37"E). In autumn 1999, 10 separate samples of 5 g each were taken from each site from the upper 5 cm of the sediment in an overall sampling area of 1 m2 and mixed as one composite sample per site. In spring 2000, four separate samples were taken in the same way and stored as four single replicate samples per site. The pHs of sediment samples were 7.8 to 8.0 and 7.6 to 7.7, and the organic carbon contents were 1.6 to 1.9% and 1.7 to 2.0% for sites A/B and C/D, respectively, in both years. The total N content of all sediment samples was 2.1 ± 0.1 mg/kg.

Determination of mercury concentrations.
Total mercury concentrations were analyzed on a specialized mercury atomic absorption spectrophotometer. The mobile (bioavailable) fraction was measured using an ammonium nitrate extraction procedure (DIN 19730). High-performance liquid chromatography coupled with cold vapor-atomic fluorescence spectrometry was used for the analysis of methyl- and ethyl-mercury compounds.

For the measurement of total and bioavailable mercury, HgCl2 (Sigma-Aldrich) served as standard; for the determination of the organomercury species, sodium ethyl mercurithiosalicylate (Sigma-Aldrich) was used for ethyl mercury concentration measurement and methylmercury(II) chloride (Riedel-deHaen) was applied to gauge the methyl-mercury concentration in the sediment samples.

Direct DNA extraction and PCR-based detection of MGE and mercury resistance genes in total community DNA from river sediment.
DNA was extracted directly from 0.5 g of sediment for each composite sample. After a harsh lysis step (bead beater), the DNA was extracted directly using the soil DNA extraction kit from an Ultra clean soil DNA isolation kit (MoBio Laboratories, Solana Beach, CA). The absence of substances inhibiting the PCR was tested by 16S rRNA gene amplification. The same extraction protocol was used to extract the DNA from the bacterial pellets obtained after centrifugation of resuspended bacterial lawn from plate counts. PCR-based detection of IncP1-{alpha}, IncP1-ß, IncQ, IncW, and IncN plasmids was performed according to Götz et al. (10), and that of the merRT{Delta}P was performed according to Osborn et al. (15). PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridizations as described by Götz et al. (10).

Plate counts and Hg resistance quotients.
Plate counts were determined for the sediment samples taken in 2000 by plating serial dilutions onto plate count agar (PCA; Merck, Darmstadt, Germany) at 100 µl of dilutions of 10–1 to 10–5) supplemented with cycloheximide (100 µg ml–1) and onto PCA supplemented with HgCl2 (20 µg ml–1; Merck, Darmstadt, Germany) at 1 ml of a 10–1 dilution and 100 µl of 10–1 to 10–3 dilutions. Plate counts were determined after 24, 48, and 144 h. Colonies grown from the lowest dilution of A and B samples on PCA and PCA supplemented with HgCl2 were resuspended with a Drigalski spatula after addition of sterile saline (0.85% NaCl). Cells were harvested from the suspension by centrifugation. Genomic DNA was isolated from the cell pellet according to the MoBio DNA extraction protocol.

Biparental exogenous isolation of MGE conferring Hg resistance.
The microbial fraction of the contaminated sediment was activated by shaking 10-g samples of sediment (5 g each of A1 and A2, A3 and A4, B1 and B2, and B3 and B4) in 25 ml of 1/10 tryptic soy broth (TSB; Becton Dickinson and Company, Sparks, MD) at room temperature overnight. After settling for 30 min, the supernatant was transferred to a new centrifuge tube. Twenty milliliters of fresh 1/10 TSB and glass beads (15 g) were added to the remaining sediment, and this mixture was vigorously shaken at 155 rpm and room temperature for 10 min. After 15 min of settling, the supernatants were combined and centrifuged at 4,500 rpm. Two hundred microliters of the harvested cell pellet (donor) and 100 µl of overnight broth culture of P. putida KT2442 gfp (recipient) were mixed, and 200 µl was added on a filter placed on PCA as previously described (11). Recipients which captured an MGE conferring mercury resistance were obtained by plating serial dilutions of the resuspended mating mixture from the filter onto PCA plates containing HgCl2 (20 µg ml–1) and rifampin (50 µg ml–1; Sigma-Aldrich Corp., St. Louis, MO). Green fluorescent protein (GFP)-positive colonies were picked. Recipient numbers were determined on PCA supplemented with rifampin (50 µg ml–1).

Triparental exogenous isolation of MGE.
The microbial fraction obtained after overnight incubation of the Hg-contaminated sediment samples in 1/10 TSB as described above was also used as donor in a triparental mating with C. necator JMP228r (Rifr gfp Kmr) as the recipient and E. coli J53(pIE723) as a second donor. After overnight incubation at 28°C, the cell mixture was resuspended from the filter and plated onto PCA supplemented with gentamicin (20 µg ml–1) and rifampin (50 µg ml–1). After 2 days of incubation, all transconjugants were picked with sterile toothpicks and transferred to fresh plates with selective medium.

Antibiotic and Hg resistance patterns of transconjugants carrying IncP-1 plasmids.
Acquired antibiotic resistances were determined by the Robert Koch-Institut in Wernigerode using the microdilution assay according to NCCLS approved standard M100-S8 (14a). Mercury resistance of the transconjugants was assessed by means of paper discs containing 0, 5, 10, and 20 µg HgCl2, which were placed on PCA upon which a cell suspension of the respective transconjugants was spread. Inhibition zones were determined after 5 days of incubation at room temperature.

Plasmid DNA isolation and characterization by Southern blot analysis.
Plasmid DNA was extracted from the transconjugants obtained after the biparental and triparental matings according to the protocol published by Smalla et al. (22). Plasmid DNA was digested with SphI or EcoRI (New England Biolabs, Beverly, MA) and Southern blotted. Hybridization was performed with digoxigenin-labeled probes generated from PCR products obtained for the broad-host-range plasmids IncP, IncN, IncW, and IncQ (10) and merRT{Delta}P according to the instructions of the manufacturer (Roche Applied Science, Mannheim, Germany).

BOX PCR.
To confirm the identity of transconjugants, BOX PCR (17) was performed with genomic DNA as the target and amplicons were analyzed by agarose gel electrophoresis.

Plasmid host range and stability.
To determine the host range and stability in different hosts, plasmids pTP6, pTP7, and pTP8 were transferred from the rifampin- and kanamycin-resistant C. necator strain JMP228 to the rifampin- and Hg-sensitive recipient strain E. coli JM109 in filter matings. Transconjugants were selected by their growth at 37°C. Transconjugants grown on PCA supplemented with 15 µg ml–1 Hg were tested by PCR for the presence of the IncP-1ß plasmids and the absence of pIE723.

Plasmids pTP6 and pTP7 were transferred from E. coli JM109 to the rifampin-resistant and gfp-tagged strains E. coli CV601, P. putida KT2442, A. tumefaciens UBA PF2, and C. necator JMP228 in filter matings as described by Heuer et al. (11). Transconjugants were obtained by plating the resuspended cell mixture onto selective medium supplemented with HgCl2 (15 µg ml–1) and rifampin (50 µg ml–1). Recipient numbers were determined on PCA supplemented with rifampin (50 µg ml–1), and transfer frequencies are given as ratio of numbers of transconjugants and recipients.

To test the stability of plasmids pTP6 and pTP7 in different hosts, a single colony picked from PCA supplemented with HgCl2 (15 µg ml–1) was used to inoculate 100 ml Luria-Bertani (LB) broth (Carl Roth GmbH, Karlsruhe, Germany). After 24 h of shaking at 28°C, serial dilutions were plated on PCA and 100 µl was used to inoculate fresh LB broth. The procedure was repeated twice, and 48 colonies each were picked from PCA plates of appropriate dilutions obtained when grown in LB broth for 24, 48, and 72 h and streaked on PCA supplemented with 15 µg ml–1 HgCl2.

Sequencing of plasmid pTP6.
Large-scale isolation and purification of plasmid DNA were done with the QIAGEN Maxiprep kit (QIAGEN, Hilden, Germany). The purified DNA was sheared, and fragments of around 1.5 to 3 kb were cloned using the TOPO shotgun subcloning kit from Invitrogen (Paisley, United Kingdom). DNA was prepared for sequencing with the QIAGEN MagAttract 96 Miniprep system or, in some cases, Promega Wizard plus SV miniprep kits (Promega, Southampton, United Kingdom). Clones were initially sequenced using universal primers from both insert ends on an ABI3700 sequencer. Finishing reads used custom primers on clones or PCR products. A few PCR products for sequencing were cloned using the Promega pGEM-T Easy vector system. The sequence was produced and assembled using the Phred/Phrap/Consed package (6-9). Analysis and annotation of the sequence used Artemis and ACT (18) and WU-BLAST2 (W. Gish, 1996 to 2004; http://BLAST.wustl.edu).

Nucleotide sequence accession number.
The nucleotide sequence of pTP6 has been submitted to the EMBL database (accession no. AM048832). As a consequence of the analysis of pTP6, two regions of R751 have been resequenced (accession no. U67194) and updated.


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RESULTS
 
Mercury contamination of sediments.
The sediments taken from sites A and B at the river Nura were highly contaminated by mercury in 1999 and 2000 (Table 1). The concentrations drastically decreased the greater the distance from the contamination source. However, for organomercury compounds, the largest amounts were not found at site A but at the more distant site B, and they were still present in large amounts at site C. In the second year (2000) of the investigation, the mercury concentrations were lower than in 1999, especially the mobile bioavailable fraction.


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  		TABLE 1. Mercury concentrations of the sediment samples analyzed

Culturable mercury-resistant bacteria.
To determine how the presence of high mercury concentrations in the sediment affected the proportion of bacteria resistant to mercury, the quotients of the CFU counts on PCA supplemented with and without mercury were determined for sediment samples. The total CFU decreased slightly downstream from site A (log CFU g–1 dry weight, 5.6) to site D (log CFU g–1 dry weight, 4.8). Mercury-resistant bacteria could only be detected for the highly contaminated sites A and B. Resistance quotients were 0.0002 for site A and 0.05 for site B.

PCR-based detection of broad-host-range plasmids in total community DNA and in genomic DNA of cultured bacteria.
To investigate the role of broad-host-range plasmids in the maintenance and spread of selective traits, PCR was performed to amplify broad-host-range plasmid-specific sequences from total community DNA. The finding that the IncP-1ß trfA2 sequence could be only detected in community DNA extracted from sediments taken at sites A and B in 1999 but not from the nonpolluted sites C and D was the reason for undertaking a more comprehensive study with four replicate samples from each site in 2000. IncP-1 plasmids seemed to be highly abundant in all replicates from sites with strong selective pressure by mercury (sites A and B), but could also be detected in two of four samples from site D with low mercury concentrations (Fig. 1). IncQ-specific oriV sequences were detected in DNA from site B only (Table 2), which was also found in 1999. Weak hybridization signals were also obtained with PCR amplicons of IncW oriV-specific PCR for site B (Table 2). IncN or IncP-9 plasmids could not be detected. Interestingly, IncP-1-specific sequences (trfA2) were not detectable by PCR/Southern blot hybridization in genomic DNA extracted from the lowest dilution of plate counts on PCA from mercury-resistant bacteria isolated from sediments of both polluted sites (A and B), and only a weak signal was obtained (and for only one of the replicates) for genomic DNA extracted from the bacterial lawn resuspended from PCA without mercury. This suggested that IncP-1 plasmids were mainly carried in uncultured bacteria of the sediment.


Figure 1
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  		FIG. 1. Detection of broad-host-range IncP-1 plasmids (trfA2) and mercury resistance transposons (merRT{Delta}P) in total DNA of sediment samples by PCR and Southern blot hybridization (sampling from 2000). +, positive control from R751 (IncP-1) or pJP4 (merRT{Delta}P).


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  		TABLE 2. Detection of broad-host-range plasmids,a IncP-9 plasmids, and mercury resistance genes in total DNA of sediment samples from 2000 by PCR and Southern blot hybridization

Abundance of merRT{Delta}P in community DNA.
Since there are a variety of resistance gene sets that can confer resistance against mercury, the prevalence of one of the most common sets, merRT{Delta}P, was determined. PCR-based detection of merRT{Delta}P in community DNA extracted directly from the spring 2000 sediment samples contaminated with different levels of mercury revealed a high abundance of bacteria containing merRT{Delta}P in samples from sites A and B, which showed the highest mercury pollution (Fig. 1). Again, weak signals were obtained in two samples from site D, in which also IncP-1 sequences were detected. In sediment samples from site C, the mercury resistance genes could not be detected. The estimated detection limit is approximately 103 target sequences per gram of sediment. Southern blot hybridization indicated that in both years the abundance of IncP-1 and merRT{Delta}P sequences was higher at sites A and B than at sites C and D.

Isolation of mercury resistance plasmids in biparental matings.
In order to capture IncP-1 plasmids conferring mercury resistance to their host independently from the culturability of their original hosts, direct capture from bacteria of the mercury-contaminated sediment samples (A and B) into P. putida KT2442 was attempted. In biparental matings of bacteria from the mercury-contaminated sediment samples (A and B) with P. putida KT2442 gfp as a recipient, transconjugants were obtained for both sites. Transfer frequencies were higher for the more contaminated site A (A1/A2, 8 x 10–4; A3/4, 5 x 10–3) than for B (B1/B2, 4 x 10–4; B3/4, 8 x 10–5). Plasmid DNA was extracted from 10 GFP-positive transconjugants, and EcoRI digests were hybridized with the merA and the trfA2 probe (data not shown). While one plasmid showed a strong hybridization signal with the merA probe, all other plasmids gave rather weak hybridization signals. Surprisingly, none of these plasmids belonged to the IncP-1ß group, which were detected by PCR amplification from community DNA of sediment samples from sites A and B.

Triparental exogenous isolation of mobilizing plasmids.
Since IncP-1 plasmids are known for their efficient mobilization of IncQ plasmids, a triparental mating was performed to capture IncP-1 plasmids based on their mobilizing activity. Transconjugants were selected on the basis of the antibiotic resistances encoded by pIE723 (gentamicin and streptomycin) and the recipient (rifampin). A total of 17 transconjugant colonies were obtained: 9 from site A and 8 from site B. Plasmid DNA was detected in all transconjugants. SphI-digested plasmid DNA was Southern blotted and consecutively hybridized with the IncP-1ß (trfA2), IncQ (oriV), and merRT{Delta}P probes. While all 17 transconjugants contained the IncQ plasmid for which the transconjugants were selected, only three transconjugants seemed to have also captured the mobilizing plasmid. These plasmids hybridized with both the IncP-1ß trfA2 probe and the merRT{Delta}P probe. The three plasmids originated from site A and were designated pTP6, pTP7, and pTP8 because they had related but still different SphI restriction patterns (data not shown). These findings gave further evidence for the linkage of IncP-1ß plasmids and mercury resistance in the contaminated sediments.

Characterization of pTP6, pTP7, and pTP8.
In order to compare the IncP-1 plasmids isolated in this study with previously described ones, the phenotype and the genotype of the plasmids were characterized in more detail. Furthermore, the full sequence of plasmid pTP6 was determined.

Antibiotic and mercury resistances.
Compared to the recipient strain C. necator, identical resistances and sensitivities against a range of clinically relevant antibiotics were recorded. Thus, none of the three plasmids confers any antibiotic resistance to their host, but all of them confer resistance towards mercury up to a concentration of 30 µg ml–1.

Transfer to a range of hosts.
Only plasmids pTP6 and pTP7 were successfully transferred from the rifampin-resistant C. necator to the rifampin-sensitive E. coli J53. The donor was counterselected by incubation at 37°C, and transconjugant colonies free of the IncQ plasmid pIE723 were selected. Thus, transfer from E. coli to C. necator, A. tumefaciens, P. putida, and E. coli was investigated only for pTP6 and pTP7. Transfer to all recipients was observed (Table 3). Transfer frequencies were in general higher for pTP7 than for pTP6, but for both strains the highest transfer frequencies were observed for P. putida.


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  		TABLE 3. Transfer rates of plasmids pTP6 and pTP7 from E. coli JM109 to diverse recipients in filter matings

Stability of pTP6 and pTP7 in different host backgrounds.
The stability of the plasmids in the different host backgrounds was monitored over approximately 30 generations. All colonies grown on PCA without selection grew after transfer to PCA supplemented with HgCl2, indicating that the plasmid was stably maintained in P. putida, A. tumefaciens, C. necator, and E. coli.

Plasmid-mediated mercury resistance in different host backgrounds.
The levels of mercury resistance conferred by pTP6 and pTP7 in the different host backgrounds were also compared. Plasmids pTP6 and pTP7 conferred resistance to HgCl2 up to 30 µg ml–1 in all hosts tested, except with pTP7 in A. tumefaciens, which seemed to provide a lower level of resistance because growth was observed only up to 15 µg ml–1.

Restriction patterns and Southern blot hybridization.
The restriction patterns of pTP6, pTP7, and R751 are shown in Fig. 2. Southern blot hybridization revealed nearly identical fragment sizes hybridizing with the oriT, korA, and trfA probes generated by PCR with R751. In contrast to this, different fragments of pTP6 and pTP7 hybridized with the merRT{Delta}P probe, indicating that the accessory DNAs of both plasmids are different.


Figure 2
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  		FIG. 2. Comparison of plasmids pTP6, pTP7, and R751 by Southern blot hybridizations of SphI fragments with probes derived from mer genes of pJP4 or backbone genes of R751. L, DIGIII ladder.

Analysis of the pTP6 sequence.
The sequence of pTP6 was determined as described in Materials and Methods. The 42,268-bp backbone of pTP6 (that is, the replication, stable inheritance, and transfer genes with the identifiable MGE removed [see Fig. 3 ], is very similar (99% identity) to the well-studied IncP-1ß plasmid R751 (29), but even more similar to the backbones of pUO1 (25) and pB8 (20): the backbones of pTP6 and pB8 differ by only 40 single-base changes (99.91% identities). Because a number of the discrepancies between R751 and other IncP-1ß plasmids were at highly conserved positions, we resequenced several regions of R751 to confirm whether these were real differences rather than errors in the original sequence. This resequencing led us to identify a single missing base in traJ and several changes, including three separate superfluous bases, in trbM. After these corrections, which have been introduced into the GenBank record, there were few differences between pTP6, pB8, and R751 backbones in the regions checked.


Figure 3
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  		FIG. 3. Genetic map of plasmid pTP6.

By comparison with other IncP-1ß plasmids, pTP6 has suffered a small deletion between two adjacent repeats upstream of upf31.0, which has removed the –35 box of a promoter identified in R751, although this gene may still be expressed from transcription upstream. In R751 a deletion appears to have fused the terminus of upf31.0 to a remnant of parA, a convergently transcribed gene present in pTP6 and other IncP-1 plasmids. Comparing R751 to pB8, there are deletions (21 bp near oriV and 828 bp including parA) adjacent to the insertion sites of two transposable elements and a 46-bp insert in the –10 signal of upf31.0.

A single insertion of a 12.1-kb putative transposable DNA element into the IncP-1 backbone is present between parA and traC. This transposon confers resistance to mercury and appears to carry the genes necessary for broad-spectrum (organomercury) resistance. It is very similar in sequence (99% identity for almost the entire length) to Tn5058; therefore, we have named it Tn50580 (Fig. 4). There are no flanking direct repeats, which corroborates a deletion of 61 bp compared against pJP4, an IncP-1ß plasmid with no insertion in this region (32).


Figure 4
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  		FIG. 4. Alignment of the mercury resistance transposons Tn50580 of pTP6 and Tn5058 of Pseudomonas sp. strain ED23-33 (GenBank accession no. Y17897). IR, inverted repeats similar to the terminal inverted repeat of the transposable element In2 or Tn21.

In conclusion, pTP6 is the first IncP-1 plasmid reported so far which carries only mercury resistance functions, but no antibiotic resistance or biodegradation genes.


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DISCUSSION
 
This study provided further evidence that as a result of mercury pollution the abundance of bacteria carrying merRT{Delta}P target sequences in sediment samples from sites A and B was strongly increased compared to those in nonpolluted or less-polluted sediment samples (sites C and D). It was also observed that the abundance of bacteria with IncP-1ß plasmids was increased in sediment samples taken from polluted sites in 1999 and 2000. However, the PCR amplification of merRT{Delta}P and IncP-1ß trfA2 from DNA extracted directly from sediments provides only an estimate of the abundance of the respective sequences in the sample analyzed, but conclusions on their exact genetic location or their actual hosts cannot be drawn. Considering their broad host range (2, 28) and their transfer frequencies observed in soils and sediments (16), IncP-1 plasmids carrying mer operons could be an efficient means for microbial communities to rapidly adapt to mercury selective pressure. However, surprisingly, we could not detect IncP-1ß plasmids in the genomic DNA extracted from the fraction of cultured bacteria which were resistant to mercury (data not shown). This suggests that either bacteria carrying IncP-1ß plasmids did not belong to the fraction of bacteria accessible by standard cultivation techniques or the IncP-1ß plasmids themselves did not carry or express mercury resistance genes. Biparental exogenous isolation of MGE in P. putida was chosen as a cultivation-independent approach to capture MGE conferring mercury resistance. Although P. putida should be a perfect host for IncP-1ß plasmids, MGE conferring mercury resistance that was captured in the biparental mating did not belong to the IncP-1ß group. These results indicate that different kinds of self-transferable MGE conferring mercury resistance were present in the polluted sediment samples which might contribute to an adaptation of the bacterial populations. It was therefore rather remarkable that when sediment bacteria from the sites A and B were used as donors of plasmids which mobilized the IncQ plasmid pIE723 into C. necator, 3 (all from the most polluted site) of the 17 transconjugants captured an IncP-1ß plasmid. In contrast to the biparental mating, C. necator was used in these experiments because of earlier observations that only in C. necator were IncP-1 plasmids, which were used to mobilize IncQ plasmids into recipients from different phylogenetic groups, stably maintained after growth under nonselective conditions (22). Despite the fact that the mating mixture was plated on a medium without mercury, containing gentamicin to select for transconjugants with pIE723, three IncP-1ß plasmids conferring resistance to mercury were captured, indicating their high abundance in the polluted sediments. It remains unclear why IncP-1ß plasmids were not obtained with the biparental approach. Despite the fact that all three IncP-1ß plasmids conferring mercury resistance were captured from one polluted sample type, plasmids differed in the accessory elements (Fig. 2) as well as in their transfer frequencies to different hosts (Table 3).

The complete sequence of plasmid pTP6 revealed that the backbone of pTP6 was almost identical to that of well-studied IncP-1ß plasmid R751 (29) from a clinical isolate (13) as well as the recently sequenced IncP-1ß plasmid pB8, which encodes multiple antibiotic resistances and originates from sewage (20). Although their backbone sequences seemed to be almost identical, in contrast to pTP6, plasmid pB8 transfers only poorly to E. coli and replicates in E. coli only after significant genetic modifications (20). Most of the sequenced IncP-1 plasmids contain two insertions of accessory DNA (the first between trfA and klcA, and the second downstream of traC), or an additional (third) insertion downstream of trbM (4, 12). Only plasmids pTP6 and pB2/3 (12) have a single insertion between parA and traC, while plasmid pJP4 has a single insertion downstream of trfA (32).

Actually the triparental mating by which pTP6 was captured should have also allowed us to capture the IncP-1 ancestor plasmid without any accessory DNA. Integrated between parA and traC, there is a 12.1-kb putatively transposable element encoding a multitude of genes which should provide the plasmid host with resistance towards inorganic and organic mercury compounds. The fact that no other accessory functions were found on pTP6 suggests that mercury was the main selective force responsible for the increased abundance of IncP-1 plasmids in the polluted sediments. Interestingly, while many IncP-1 plasmids carry mercury resistance genes, pTP6 appears to be the first one that confers only this phenotypic trait: all others confer additional properties—either additional resistance markers or degradative determinants (12, 14, 19, 20, 25, 27, 29, 32, 33). The fact that IncP-1 plasmids have acquired mercury resistance determinants on a number of separate occasions reinforces the view that selection for such genes has been a dominant force in shaping the evolution of the horizontal gene pool.

Considering the kinds and concentrations of mercury compounds contaminating sites A and B, the carriage of such a complex mer operon must have been advantageous. The sequence of the mer genes suggests that it is the result of different recombination and transposition events. The mer operon found in pTP6 showed very high similarity to Tn5058 and was therefore named Tn50580. Interestingly, this transposon Tn5058, was described in a Pseudomonas sp. isolate from permafrost soil in Russia preserved since the upper Pleistocene. This would indeed point to an ancient evolution of mer operons. In addition, the left (until merP) and the right (from merB2) parts of Tn50580 share a high similarity to Tn5718 on mercury resistance plasmid pSB102, recently isolated exogenously from the rhizosphere of alfalfa in Sinorhizobium meliloti (21). The sequence in between resembles a fragment from pMR26 (merAB1D1orf1) and merB2 from pDU1358.

The remarkable conservation of the general features of the IncP-1 backbone may well be due to the extent of evolution this system has already undergone. Not only are the backbone functions physically clustered, but they are incorporated into a relatively small number of transcriptional units, and these are expressed from strong promoters that are tightly regulated by the proteins encoded in the central control region (3). This complex system can only have evolved because it created a sophisticated unit with selective advantages. The integration of the parts of it means that variants that have lost key features will be at a disadvantage and hence will not survive. Therefore, by comparison of the major subtypes of IncP-1 plasmids between which there is a considerable sequence drift, one can identify the key selective features of this group. Since these features clearly do not prevent sequence drift at a local level (for example, in less important and noncoding segments of the replication origin), the highly similar backbone sequences of the IncP-1ß plasmids that recently have been sequenced (12, 14, 19, 20, 25, 27, 29, 32) suggest a very recent common ancestor that has spread across the globe, presumably on particles in the air, and acquired diverse insertions with a variety of phenotypic determinants. Although other MGE carrying mer operons are also present in the sediment samples analyzed, this study provides further evidence of the role of IncP-1ß plasmids as contributors to the maintenance and spread of adaptive traits (mercury resistance) in microbial communities.


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ACKNOWLEDGMENTS
 
We acknowledge the collaborators of the INTAS projects 97-30723 and 2001-2383, in particular the contributions of Sonia Heaven, Kazakh State Academy of Architecture and Construction, and Sevetlava Abdrashitova, Institute of Microbiology and Virology (Almaty, Republic of Kazakhstan) and of several students at the BBA, Braunschweig, Germany.

Sequencing of plasmid pTP6 was supported by The Wellcome Trust grant 063083 and was carried out in the University of Birmingham Functional Genomics Laboratory, funded by BBSRC grant JI6/F13209. Financial support to H.H. was obtained from DFG FOR 566.


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FOOTNOTES
 
* Corresponding author. Mailing address: Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA), Messeweg 11-12, D-38104 Braunschweig, Germany. Phone: 49-531-2993814. Fax: 49-531-2993013. E-mail: K.Smalla{at}bba.de. Back

{triangledown} Published ahead of print on 15 September 2006. Back


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Applied and Environmental Microbiology, November 2006, p. 7253-7259, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00922-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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