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Department of Bacterial Genetics, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
Correspondence
Dariusz Bartosik
bartosik{at}biol.uw.edu.pl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ149577.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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TEs [insertion sequences (ISs) and transposons (Tns)] are the most abundant mobile elements in bacterial genomes. The majority of ISs and non-composite Tns have been identified as a result of various sequencing projects. However, it is much more difficult to identify functional composite Tns through DNA sequence analysis. Theoretically, all DNA is potentially mobile (Campbell, 2002
), because any DNA fragment bordered by two, even different (Ramírez-Romero et al., 2001), TEs, could form a composite Tn. However, whether such DNA segments actually are mobile has to be verified experimentally. Several alternative strategies have been developed for the identification of functional TEs and the detection of their transposition activity. These strategies employ various entrapment vectors, which are convenient tools enabling the direct identification of even phenotypically silent elements (reviewed by Solyga & Bartosik, 2004).
In previous studies we have performed analyses aimed at the identification and characterization of TEs in several strains (DSM 11072, DSM 11073, DSM 65 and LMD 82.5) of a facultative chemolithoautotroph Paracoccus pantotrophus as well as in Paracoccus solventivorans DSM 11592 (Bartosik et al., 2003a, b). These strains show great physiological heterogeneity, which might result from the presence of various metabolic Tns. Through the application of positive-selection entrapment vector pMEC1 (which carries a cI–tetA selective cartridge) we showed that TEs are abundant in Paracoccus spp. We identified eight novel ISs: ISPpa1, ISPso1 (IS256 family), ISPpa2, ISPpa3, ISPpa4, ISPso2, ISPso3 (IS5 family) and ISPpa5 (IS66 family), as well as two closely related Tns of the Tn3 family, cryptic Tn3434 (DSM 11072) and streptomycin-resistant Tn5393 (LMD 82.5) (Bartosik et al., 2003a, b).
The size of the identified TEs [400 tetracycline-resistant (TcR) clones analysed] did not exceed 5·3 kb (Tn5393) (Bartosik et al., 2003a). This raised the question whether the studied strains contained Tns of larger size and whether the tool used was not suitable for their capture. We speculated that the replicator region of pMEC1, derived from a small plasmid pWKS1 (2·7 kb) of P. pantotrophus DSM 11072, might be unable to maintain larger plasmid genomes carrying inserted Tns. To circumvent this potential limiting factor we have constructed a modified version of pMEC1 (pMMB2), carrying a megaplasmid-specific repABC-type replicon.
In the present study, we describe the identification and characterization of a novel large composite TE, TnPpa1 (44·3 kb), isolated from P. pantotrophus DSM 11072 by its transposition to the entrapment vector pMMB2.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All strains were grown in Luria–Bertani (LB) medium at 30 °C (Paracoccus spp.) or 37 °C (Escherichia coli). Where necessary, the medium was supplemented with antibiotics at the following concentrations: kanamycin 50 µg ml–1, rifampicin 50 µg ml–1 and tetracycline 0·5 µg ml–1.
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and when required, purified by CsCl–ethidium bromide gradient centrifugation. Megaplasmid visualization was achieved by in-gel lysis and DNA electrophoresis, as described by Wheatcroft et al. (1990). Total DNA from P. pantotrophus strains was isolated by phenol extraction (Williams et al., 1998). Common DNA manipulation methods were performed as described by Sambrook & Russell (2001). For Southern hybridization, DNA probes were labelled with DIG (Roche). Hybridization and visualization of bound probes were carried out as recommended by the supplier.
Construction of entrapment shuttle vector pMMB2.
The construction of the shuttle entrapment vector was performed in E. coli TG1 in two steps. The E. coli-specific mobilizable vector pABW1 (Bartosik et al., 1997) was digested with PstI and ligated with the 2·9 kb entrapment cartridge from pGBG1 (Schneider et al., 2000). The resulting plasmid (pMMB1) was digested with XbaI and KpnI and ligated with a 5·6 kb linear form of the repABC-containing mini-replicon pTAV320 (Bartosik et al., 1998), recovered from shuttle plasmid pABW22A (Table 1
). The resulting positive selection vector was designated pMMB2 (Fig. 1). The selective cartridge of pMMB2 is composed of a silent TcR gene (tetA) under the control of the pR promoter of bacteriophage 
and the gene encoding the cI repressor. Inactivation of the repressor gene or operator (e.g. through insertion of a TE) results in the constitutive expression of TcR.
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. Triparental mating experiments were conducted as previously described (Bartosik et al., 2003a). Briefly, overnight cultures (pelleted by centrifugation and washed to remove antibiotics) of the donor strain E. coli TG1 carrying the mobilizable vector, the recipient strain P. pantotrophus DSM 11072R, and E. coli DH5
carrying the helper plasmid pRK2013, were mixed at a ratio of 1 : 2 : 1. A 100 µl aliquot of this mixture was spread on a plate of solidified LB medium. After overnight incubation at 30 °C, the bacteria were washed off the plate and suitable dilutions were plated on selective media containing rifampicin (selective marker of the recipient strain) and kanamycin to select transconjugants. Spontaneous resistance of the recipient strains to kanamycin was undetectable under these experimental conditions.
Isolation of insertion mutants.
The entrapment vector pMMB2 was introduced by triparental mating into recipient strain P. pantotrophus DSM 11072R. An overnight culture of a kanamycin-resistant (KmR) transconjugant carrying pMMB2 was spread on plates of solidified LB medium supplemented with tetracycline. Appropriate dilutions of the culture were also spread on tetracycline-free, solidified LB medium in order to determine the frequency of transposition. One hundred and eighty TcR colonies were picked and further analysed for plasmid content and restriction pattern. Spontaneous resistance to tetracycline was undetectable under these experimental conditions.
PCR amplification.
For identification of the target site of TnPpa1 integrated within pMMB2, five nested pairs of cartridge-specific forward and reverse oligonucleotide primers (ALIS/ARIS, BLIS/BRIS, CLIS/CRIS, DLIS/DRIS, ELIS/ERIS) were used as described by Bartosik et al. (2003a). The chromosomal DNA sequences adjacent to TnPpa1 within the P. pantotrophus genome were amplified with the following primers: RTN3434 (5'-TCCTCGCCGCCATCATCAT-3'), LTN3434 (5'-GCAGATTGACACAACGACTG-3') and 147LCD2 (5'-ATCGGCAAGGCAGATTGACC-3') (see Fig. 3 and Results for details). For detection of the inverted form of TnPpa1, the following primers were used: RINV (5'-GAGGCCGTCCATGCTCTTGT-3'), LINV (5'-AGAGAGGTCACGTCACGGTC-3') and LNUC (5'-CGTGTCACCATCAACGAGGC-3') (see Fig. 3 and Results for details). For amplification of the internal fragment of Tn3434 (used as a probe in hybridization) the primers L3434 and R3434, previously described by Bartosik et al. (2003a), were used. Amplification was performed in a Mastercycler (Eppendorf) using the above synthetic oligonucleotides, OptiTaq polymerase (Eurx) (with supplied buffer) and appropriate template DNAs. PCR products were analysed by electrophoresis on 0·8 % agarose gels and, where necessary, purified with a Gel Out kit (A&A Biotechnology) and cloned into the pGEM-T Easy Vector (Promega).
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). Similarity searches were performed using the BLAST programs (Altschul et al., 1997) provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast). The G+C plot was created with the program Artemis (Rutherford et al., 2000) with a window setting of 150 nt. |
RESULTS |
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; see Methods for details). This plasmid was a fusion of: (i) an E. coli-specific vector, pABW1 (Bartosik et al., 1997); (ii) mini-replicon pTAV320, carrying a repABC-type replicon originating from low–copy-number plasmid pTAV1 (107 kb) of Paracoccus versutus UW1 (Bartosik et al., 1998); and (iii) a cI–tetA selective cartridge (Schneider et al., 2000). The functionality of pMMB2 was tested in P. pantotrophus DSM 11072R. This strain was chosen for two reasons: (i) it is rich in TEs, and carries ISPpa1, ISPpa2, ISPpa3, ISPpa5 and Tn3434, elements entrapped by pMEC1 (Bartosik et al., 2003a); and (ii) its natural plasmids are compatible with the repABC replicon of pMMB2 (data not shown).
A pool of TcR mutants of strain DSM 11072 carrying pMMB2 with inserted TEs of various sizes was identified and analysed. The frequency of TcR clones and of transposition was similar to that previously observed with pMEC1 (2·1x10–6). Among 180 tested TcR clones, several classes of plasmids were distinguished carrying: (i) point mutations; (ii) putative co-integrates; (iii) potential ISs and small Tns (inserts of 1–4 kb); and (iv) large Tns (inserts of 
45 kb) (Table 2). Of the tested plasmids, 15 % carried a very large element (45 kb), which was not previously identified in this strain using pMEC1 (Table 2). DNA restriction analysis revealed that all the clones carried the same element, which was designated TnPpa1 (data not shown). One randomly selected TcR clone, carrying a pMMB2-derivative with inserted TnPpa1 (pMMB10), was subjected to further detailed study. PCR analysis with the use of five pairs of nested, cartridge-specific primers confirmed that TnPpa1 had integrated within the cI gene. This enabled precise localization of TnPpa1 within the cassette by sequencing of the flanking regions of the element using appropriate primers.
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37 kb DNA segment, placed between two identical, divergently oriented copies of a cryptic, non-composite Tn3434 (3·7 kb) related to Tn3 (Bartosik et al., 2003a) (Fig. 2). These terminal elements were designated Tn3434L and Tn3434R (Fig. 2a). Tn3434 carried two genes, in divergent orientation, encoding a transposase (tnpA) and a resolvase (tnpR) bordered by terminal inverted repeats of 35 bp. The two ORFs were separated by a putative recombination site (res), which in Tn3-like elements is involved in co-integrate resolution and regulation of the tnpA and tnpR genes (Grindley, 2002) (Fig. 3). Sequencing of the integration site of TnPpa1 revealed that the element had been inserted into an AT-rich sequence within the selective cartridge of pMMB2 (Fig. 2a), which is typical for non-composite members of the Tn3 family (Grindley, 2002). Upon insertion, it generated a 5 bp duplication of the target sequence 5'-GTGTT-3'.
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). Analysis of the TnPpa1 nucleotide sequence indicated the presence of 38 ORFs (Fig. 2b). Sequence database comparisons identified: (i) a 4·2 kb DNA segment of TnPpa1 (encoding ORFs 32–36) similar to part of chromosome cII of Rhodobacter sphaeroides (Table 3); and (ii) two ORFs (18 and 19) homologous to hypothetical genes of Bordetella bronchiseptica (Table 3) as well as to the sequences within the GEI of E. coli O157 : H7 EDL933 (z0891 and z0890; accession numbers AAG55060 and AAG55059, respectively; data not shown). The presence of these elements exemplifies the mosaic structure of TnPpa1.
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Identification of the TnPpa1 integration site
The target site of TnPpa1 within the DSM 11072 genome was determined by inverse PCR (IPCR). For this purpose, two Tn3434-specific, divergently priming, oligonucleotide primers LTN3434 and RTN3434, were designed (Fig. 3). The template DNA was prepared by NruI digestion of total DNA of strain DSM 11072 followed by ligation of the mixture of DNA fragments. The NruI endonuclease does not cleave Tn3434, therefore ligation should result in the circularization of DNA fragments containing entire copies of the Tn, together with adjacent sequences. This template was used in IPCR reactions with primers LTN3434 and RTN3434. Two IPCR products were expected (one for each copy of Tn3434), but only one of size 1 kb was obtained. DNA sequencing of the amplified fragment (with the primers used for IPCR) established the presence of the terminal Tn3434 sequences as well as: (i) an internal sequence of TnPpa1 adjacent to the right copy of Tn3434; and (ii) a short genomic sequence, separated by the NruI site created by ligation (Fig. 3).
Possibly the NruI site in genomic DNA flanking Tn3434L is located far away from the Tn, which would preclude obtaining a PCR product. Therefore, an additional IPCR experiment was performed, employing a second set of divergently priming primers, RTN3434 and 147LCD2 (Fig. 3), and template DNA of strain DSM 11072 prepared by SphI digestion and ligation. As shown in Fig. 3, a SphI site was present within Tn3434. Therefore, the desired restriction fragment contained terminal parts of Tn3434 together with the adjacent target sequence. This revised strategy allowed amplification and determination of the genomic sequence (277 bp) flanking the left copy of Tn3434.
TBLASTX comparison of the amplified genomic DNA sequence with the GenBank protein sequence databases revealed the presence of a truncated ORF (disrupted by insertion of TnPpa1) with similarity to a number of bacterial nucleases. The highest similarity (60 %) was observed with a 104 aa product encoded by gene yci present on an octopine-type Ti plasmid of Agrobacterium tumefaciens (accession no. NP_059691) (data not shown).
Inversion of TnPpa1 in the DSM 11072 genome
When two res sites are located on the same DNA molecule in an inverted orientation (as observed in TnPpa1), recombination between them (mediated by resolvase) should result in the inversion of the flanked DNA segment (Mahillon, 1998). This mechanism may result in the rotation of a huge portion of TnPpa1. To confirm this possibility we performed PCR with total DNA of strain DSM 11072 as a template, using two sets of primers, LNUC and LINV, and LNUC and RINV, each of which should amplify DNA fragments specific for a different orientation of TnPpa1. As shown in Fig. 3(a) the former primer pair should amplify a DNA fragment of the sequenced version of TnPpa1, while with the latter pair a PCR product would be produced only following inversion of the element. With both sets of primers we observed amplicons of the expected size (Fig. 3b). DNA sequence analysis demonstrated that the internal DNA region of TnPpa1 adjacent to the Tn3434R, may also lie in the opposite orientation, close to Tn3434L (data not shown), thus confirming recombinational ‘flip-flop’ of TnPpa1 in the host genome.
Genomic localization of TnPpa1 in DSM 11072 and its distribution in other strains of P. pantotrophus
In order to identify the location of TnPpa1 in the strain DSM 11072 genome, a PCR-amplified DNA fragment of Tn3434 (amplified with primers L3434 and R3434) was used to probe a Southern blot of the mega-sized DNA replicons of DSM 11072, visualized by in-gel cell lysis and gel electrophoresis. It has previously been shown that strain DSM 11072, besides pWKS1 (2·7 kb), also carries the megaplasmid pWKS3 (Baj et al., 2000). We found, however, that this strain contained an additional high–molecular-mass replicon (designated pWKS5) of >640 kb, which was readily visible when the strain was cured of pWKS3 (strain DBB1; Fig. 4). On the Southern blot, the Tn3434-specific probe produced a hybridization signal with DNA of pWKS5, which revealed the location of TnPpa1 (Fig. 4).
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). To investigate the distribution of sequences in the core region of TnPpa1, a DIG-labelled mixture of restriction fragments of TnPpa1 was used to probe a Southern blot of EcoRI-digested total DNA from the above P. pantotrophus strains. A positive hybridization signal was observed exclusively with the DNA of the host strain DSM 11072, which proves that the TnPpa1 sequences were strain specific (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This demonstrates that repABC modules (in which the partitioning and replication genes are clustered in a single operon; Ramírez-Romero et al., 2001) can maintain large replicon genomes and should, therefore, be suitable for the construction of large TE entrapment vectors.
Using pMMB2, we captured TnPpa1, which is the first known composite Tn of Paracoccus spp. and, to our knowledge, is the largest TE that has ever been identified by transposition to an entrapment vector. The results summarized in Table 2 show that it was not possible to capture TnPpa1 using the previously constructed entrapment vector pMEC1 carrying the ori region of a small plasmid pWKS1 that naturally resides in strain DSM 11072 (Bartosik et al., 2002). This observation indicates that the nature of the vector replicator region acts as a natural selection for the size of the integrated elements.
We have shown that TnPpa1 resides in strain DSM 11072 within a very large replicon significantly exceeding 640 kb in size (Fig. 4). Replicons of this size have also been detected in other strains of P. pantotrophus (data not shown). Since many representatives of the Alphaproteobacteria contain more than one chromosome (Jumas-Bilak et al., 1998), the question of whether pWKS5 is a chromosome or a mega-sized plasmid remains open.
TnPpa1 has an atypical structure and possesses at each terminus a divergently oriented copy of a non-composite transposon, Tn3434 (Tn3 family). Tn3434 (like ISs) carries only genetic information sufficient for its own transposition. In contrast, the closest homologue of Tn3434, Tn5393, identified in P. pantotrophus LMD 82.5, contains two additional streptomycin-resistance genes (strA and strB) located downstream of the tnpR gene (Bartosik et al., 2003a).
TnPpa1 encodes three clusters of putative transporters. (i) ORFs 14 and 15 encode putative products similar to ChrA transporters, which have been shown, in some cases, to be responsible for chromate resistance (Aguilera et al., 2004). (ii) ORFs 25–27 show sequence similarities to uptake systems of the tripartite, ATP-independent, periplasmic transporter (TRAP-T) family (Kelly & Thomas, 2001). The TRAP-T systems were initially identified in R. sphaeroides (Jacobs et al., 1996) and Rhodococcus capsulatus (Forward et al., 1997), where they catalyse the uptake of glutamate and C4 dicarboxylates, respectively. (iii) ORFs 8–10 encode putative ABC-type transporters, with significant similarity to proteins involved in Fe3+ transport. Iron, which is a limiting factor in the environment, is an essential nutrient for all living organisms, being a component of key enzymes such as cytochromes, ribonucleotide reductase and other metabolically linked molecules (Köster, 2001). The presence of iron transporters within the Tn and their dissemination by lateral transfer may be highly advantageous to many bacterial hosts. The specific function of the putative transporters encoded by TnPpa1 awaits experimental confirmation.
There are two possible scenarios by which TnPpa1 might have arisen in strain DSM 11072. One scenario assumes the acquisition of Tn3434 by lateral transfer and then two independent transposition events of this cryptic Tn into the host genome. In this case TnPpa1 would be composed of a segment of DSM 11072 chromosome flanked by two copies of Tn3434. The other scenario assumes acquisition of the whole TnPpa1 (e.g. on a plasmid) followed by transposition of TnPpa1 into the host genome, or integration of the plasmid molecule into the genome. In this case, TnPpa1-encoded genes might not be conserved in other strains of P. pantotrophus. According to the first scenario, each of the integrated copies of Tn3434 should be bordered by different repeated target sequences (DRs), while in the case of the latter, DRs should only be observed flanking both termini of TnPpa1. As seen in Fig. 3, the entire TnPpa1 (but not the individual copies of Tn3434) is flanked by 5 bp DRs in the strain DSM 11072 genome, which favours the second scenario. Moreover, hybridization analysis revealed that, of the three tested strains of P. pantotrophus (DSM 65, DSM 11073 and LMD 82.5), none carried sequences homologous to TnPpa1, which unequivocally proves that TnPpa1 was acquired by strain DSM 11072 through lateral transfer. Since this element is present only in one strain, it would appear to be a relatively recent acquisition that occurred after branching of the tested P. pantotrophus strains.
Taking into account the similar G+C content of TnPpa1 and the genomic DNA of P. pantotrophus, and the presence of a DNA segment homologous to chromosome cII of R. sphaeroides, it seems that TnPpa1 has been gained from a phylogenetically closely related bacterial species. The most external ORFs of the core region of TnPpa1 (ORFs 3 and 36), located adjacent to both copies of Tn3434, are truncated, and the missing parts of the ORFs could not be identified outside the Tn. These disruptions are probably remnants of the ancient Tn3434 transposition events which led to the formation of TnPpa1 in its native host.
In strain DSM 11072, the two copies of Tn3434 reside within the same DNA molecule despite the phenomenon of Tn immunity (where a replicon containing one copy of a Tn is highly resistant to further insertions of the same element) which is thought to apply to members of the Tn3 family (Grindley, 2002). The divergent orientation of the Tn3434 copies appears to ensure the stable maintenance of TnPpa1 within the host genome. Tn3434, like other Tn3-like elements, encodes a resolvase module, which promotes efficient site-specific recombination between res sequences carried by two Tns residing within the same DNA molecule. Resolvase-mediated recombination should result in the loss of the core region of TnPpa1 (when Tn3434L and Tn3434R are present in the same orientation) or its inversion (when they are arranged divergently). We confirmed that inversion does occur, resulting in rotation of the Tn within the host genome. This rearrangement does not change the local genetic environment, although more detailed studies are required to confirm that the effect of such a recombinational flip-flop of the Tn is neutral.
The identification of TnPpa1 provides evidence for the transposition and lateral transfer of a large segment of DNA of chromosomal origin. Transposition of housekeeping genes into co-residing plasmids or megaplasmids may potentially have a significant impact on the structure and evolution of bacterial genomes (e.g. it may stimulate the formation of multi-chromosome genomes). Additionally, duplication of sets of genes (e.g. by replicative transposition) is considered an important mechanistic antecedent of gene innovation and, consequently, of genetic novelty (Coenye et al., 2005). The duplicated genetic information may then be spread by lateral transfer among bacterial populations, producing a variety of phenotypic effects.
Although TnPpa1 appears not to posses the complete genetic information for any particular metabolic pathway, it carries a number of genes (including housekeeping genes and membrane transporters) whose presence may improve the ecological fitness of the host cells. It is noteworthy that some bacterial catabolic pathways seem to have evolved by patchwork assembly, i.e. by acquisition of various genes following separate lateral transfer events. In such cases, the acquisition of even a single gene may initiate a novel metabolic activity (Copley, 2000; Springael & Top, 2004).
Taking into account all our findings, we suggest that TnPpa1 may be considered a transposable GEI. GEIs are defined as strain-specific, large chromosomal regions that can be excised from the genome and transferred to other recipients. They contain mobility genes encoding integrases or transposases (required for integration and excision), as well as one or more genes that can increase the adaptability and versatility of the bacterium (Dobrindt et al., 2004). The majority of the identified GEIs encode an integrase, involved in integration of GEIs close to tRNA genes. However, in the case of TnPpa1, transposition is responsible for the movement of the element. The same mechanism of translocation is also suggested for a 44·8 kb long pathogenicity island of Bacillus anthracis which is flanked by inverted copies of IS1627 (Okinaka et al., 1999).
Physiologically, Paracoccus species are highly versatile. For example, different strains are able to use a variety of organic compounds as a source of carbon. Although there is no evidence linking the presence of TEs with vital phenotypic characteristics of P. pantotrophus, the possibility that their physiological heterogeneity might result from the presence of various TEs cannot be ruled out. To investigate the role of Tns in genome evolution, we plan to employ the entrapment vector pMMB2 to search for such elements in other strains of Paracoccus and in other members of the Alphaproteobacteria.
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ACKNOWLEDGEMENTS |
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Received 18 October 2005;
revised 19 December 2005;
accepted 19 December 2005.
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