Department of Bacterial Genetics, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland1
Author for correspondence: Dariusz Bartosik. Tel: +48 22 554 13 44. Fax: +48 22 554 14 02. e-mail: bartosik{at}biol.uw.edu.pl
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ABSTRACT |
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Keywords: plasmid replication, mobilization
Abbreviations: Km, kanamycin; oriT, origin of conjugative transfer; oriV, origin of replication; Rif, rifampicin
The GenBank accession number for the sequence reported in this paper is AF482428.
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INTRODUCTION |
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The increasing interest in bacteria belonging to the genus Paracoccus is paralleled by an increasing demand for appropriate tools (e.g. suitable advanced cloning vectors) that would facilitate the genetic analysis of these bacteria. With the construction of an optimal vector for Paracoccus spp. in mind, an analysis of the plasmid content of paracoccal strains has been conducted by Baj et al. (2000 ). A study of strains representing 11 paracoccal species revealed the presence of over 30 plasmids, including megaplasmids (Baj et al., 2000
). So far, only three Paracoccus-carried plasmids have been studied in great detail pTAV1 (107 kb; Bartosik et al., 1998
, 2001a
) and pTAV3 (approx. 400 kb; Bartosik et al., 2002
) of Paracoccus versutus, and pALC1 of Paracoccus alcaliphilus (70 kb; Bartosik et al., 2001b
). However, the mini-replicons of these three plasmids are not suitable for vector construction.
Our attention was drawn to the small multicopy plasmid pWKS1 harboured by Paracoccus pantotrophus DSM 11072 that is capable of utilizing carbon disulphide (Jordan et al., 1997 ). In this study, we present an analysis of the genetic structure of pWKS1. Two functional modules of the plasmid were distinguished that are responsible for its replication and mobilization. These modules show significant similarity to analogous cassettes found in different replicon combinations in plasmids residing in Gram-positive and Gram-negative bacteria.
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METHODS |
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DNA manipulations.
Plasmid DNA was isolated according to Birnboim & Doly (1979) , and if required purified by CsCl/ethidium bromide gradient centrifugation. Other molecular biological procedures were done as described by Sambrook et al. (1989)
. All enzymes were purchased from either Promega or Roche. DNA restriction fragments were recovered from agarose gels by using the DNA Gel-Out Kit (DNA Gdansk). For Southern hybridization (Sambrook et al., 1989
), DNA probes were labelled with digoxigenin (Roche). Hybridization and visualization of the hybridization products was done as recommended by the supplier.
Electroporation and transformation.
Electroporation was carried out at 2500 V, 25 µF and 200 (for E. coli) or 400
(for P. pantotrophus KL100) in a gene pulser apparatus (Bio-Rad), according to the modified Bio-Rad procedure (Wlodarczyk et al., 1994
). Electrotransformants were selected on solidified LB medium supplemented with the appropriate antibiotic. Competent cells of E. coli TG1 were prepared and transformed as described by Kushner (1978)
.
Mating.
The overnight cultures were spun down and washed twice to remove antibiotics. For triparental mating, the donor strain E. coli TG1 (carrying a mobilizable vector), a suitable recipient strain and E. coli DH5(pRK2013) were mixed at a ratio of 1:2:1. An aliquot (100 µl) of this mixture was spread onto TY or LB agar, depending on the recipient strain. After overnight incubation of the plates at 30 °C, the bacteria were washed off of the plates and suitable dilutions of the cultures were plated onto selective media containing Rif or streptomycin. Km had also been added to the media. Rif and streptomycin were selective markers for the recipient strains; Km was added to the media to select for transconjugants. Diparental matings were made with E. coli S17-1 carrying a mobilizable plasmid (as a donor) and a suitable recipient strain of E. coli or Paracoccus sp. The strains were mixed at a ratio of 1:2 and plated onto LB agar. Transconjugants were selected as described above. The plasmid pattern of the transconjugants was verified by screening several colonies by using a rapid alkaline extraction procedure and agarose-gel electrophoresis. Spontaneous resistance of the recipient strains to the selective markers was undetectable under these experimental conditions.
Plasmid stability.
The stability of plasmids during growth under non-selective conditions was tested as previously described (Bartosik et al., 1998 ). Briefly, stationary-phase cultures were diluted in fresh medium without antibiotic selection and cultivated for approximately 10, 20 and 30 generations. Samples taken at these times were diluted and plated onto solid medium in the absence of selective drugs. Two-hundred colonies were tested with the use of a Km-resistance marker by replica plating. The retention of plasmids after approximately 30 generations was defined as the percentage of Km-resistant colonies.
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RESULTS AND DISCUSSION |
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We did not identify regions matching the consensus promoter sequence of E. coli (Greener et al., 1992 ) upstream of ORF1 and ORF2 of pWKS1. Also, we did not find (downstream of the two ORFs) any sequences resembling
-independent transcriptional terminators. However, we did localize two 17 bp long inverted-repeat sequences (mapped at positions 25482591, and separated by 10 bp) 85 bp downstream of ORF2 that were able to form a potential stemloop structure (data not shown).
In summary, the data obtained from sequence analysis suggest the presence of two modules responsible for (i) replication (REP) and (ii) mobilization for conjugative transfer (MOB) in pWKS1. Such modules are commonly found, in different combinations, within many small multicopy plasmids residing in Gram-positive bacteria, such as those in Lactobacillus spp. (Bates & Gilbert, 1989 ), Streptococcus spp. (Priebe & Lacks, 1989
), Staphylococcus spp. (Projan & Novick, 1988
) and Bacillus spp. (Meijer et al., 1998
). These modules also occur in some plasmids from Gram-negative hosts, e.g. those from Bordetella bronchiseptica (Antoine & Locht, 1992
) and Ruminobacter amylophilus (Ogata et al., 1999
). To confirm the presence of such modules in pWKS1, a detailed functional analysis of the plasmid was carried out in a later part of this study.
Replication of pWKS1
To investigate the role of ORF1 and ORF2 in the replication of pWKS1, mutational analysis was performed. First, ORF1 was mutated by the insertion of a Km-resistance cassette into the unique PstI (located within ORF1) and EcoRI (located 5 bp upstream of ORF1) sites of pWKS1, respectively. The EcoRI site is situated between a putative RBS and the start codon of ORF1, therefore a mutation within this site was supposed to inhibit the expression of the ORF1-encoded product. As expected for both cases, the replication and stability of the resulting plasmids pWKS18 (PstI) and pWKS19 (EcoRI) (Table 1), tested in P. pantotrophus KL100, was not affected. Conversely, we were unable to obtain a replicative form of pWKS1 that contained a Km-resistance cassette integrated into the unique SmaI site located within ORF2, which might suggest that ORF2 encodes a protein which is crucial for pWKS1 replication. To confirm this hypothesis, we cloned a SmaI-digested linear form of pWKS1 (plasmid pWKS21) and a 1·8 kb EcoRIPstI restriction fragment of pWKS1 (Fig. 1
) with complete ORF2 (plasmid pWKS25) into the multiple-cloning site of an E. coli-specific (unable to replicate in P. pantotrophus) vector, pBGS18. After the electroporation of pWKS21 and pWKS25 (constructed in E. coli) into P. pantotrophus KL100, we obtained Km-resistant transformants containing only pWKS25. Based on the results of our mutational analysis and sequence similarity data, we conclude that ORF2 encodes the replication initiator protein (Rep) of pWKS1.
Analysis of the structural features of the pWKS1 sequence revealed the presence of an A+T-rich region located within the intergenic region upstream of rep. As shown in Fig. 1(b), this region is highly saturated with directly repeated sequences. The longest repeats (DR1DR3; Fig. 1b
), each of which contains a 21 bp sequence (5'-AAGTGGGGAATCCAGCCGCAA-3'), are tandemly repeated (without any spacer sequences) three times. In many
-replicating plasmids, direct repeats (iterons), located within oriV, are required for the binding of the Rep protein and for the initiation of replication (Chattoraj, 2000
). It is conceivable that the DR1DR3 repeats might play a similar role in pWKS1 replication. However, no sequence corresponding to DnaA or IHF binding sites (also present within the oriV of many plasmids) were identified within this region. To check whether the intergenic sequence mentioned above contains oriV, we cloned this region (on a 380 bp Sau3AI fragment of pWKS1) into the BamHI site of an E. coli-specific (non-replicating in P. pantotrophus), mobilizable vector, pABW1. The resulting plasmid (pWKS24) was introduced via triparental mating (in the presence of the helper plasmid pRK2013) into two host strains P. pantotrophus DSM 11072R(pWKS1) and UWP1 (pWKS1-less). Transconjugants were obtained only with strain DSM 11072R, which indicates that the rep gene of pWKS1, when supplied in trans, can support the replication of pWKS24 that lacks a functional copy of its own rep gene. To verify that the Km-resistant transconjugants were not the result of recombination between pWKS1 and pWKS24, plasmid DNA from several transconjugants was isolated and used to transform E. coli. Ten of the transformants were analysed for their plasmid content and plasmid-restriction pattern. All of these transformants contained intact pWKS24. This experiment provides evidence for the localization of oriV within the pWKS1 genome. The stable maintenance of co-residing autonomous forms of pWKS24 and pWKS1 in P. pantotrophus cells (under selective conditions for pWKS24) indicates that the cloned fragment carrying oriV (together with the potential promoter region of rep) does not carry incompatibility determinants that are frequently involved in the regulation of initiation of plasmid replication (del Solar et al., 1998
).
Mobilization of pWKS1
The observed similarity of the ORF1 product to Mob proteins suggests the potential role of this protein in the conjugative transfer of pWKS1. The compact structure of pWKS1 (and its lack of convenient restriction sites) precludes the cloning of a selective marker into this plasmid without disruption of the replication or putative mobilization regions. This prevents studies on the functionality of the ORF1 protein in the mobilization of pWKS1 between different strains of Paracoccus spp. Consequently, this analysis was performed with strains of E. coli and plasmids pWKS10 and pWKS12 (both carrying an inactivated mob gene), and pWKS21 (with a disrupted rep gene). These plasmids were introduced, respectively, into E. coli S17-1 by transformation (E. coli S17-1 carried transfer genes of the IncP-plasmid RP4 which had been integrated into its chromosome). The Km-resistant transformants obtained were used as donors in diparental matings with the recipient strain E. coli DH5R. We observed that only pWKS21 could be mobilized for transfer. The frequency of transfer was, however, relatively low and averaged 2x10-5 transconjugants (donor cell)-1. The presence of pWKS21 in the transconjugants was confirmed as described in Methods. The lack of transconjugants carrying pWKS10 and pWKS12 points to the role of ORF1 (mob) in plasmid mobilization. We can state that the MOB module of pWKS1 enables the transfer of a hybrid plasmid (by donation) between various E. coli strains (in the presence, in trans, of transfer genes of the IncP-plasmid RP4) although the replication system of pWKS1 is not functional in this host (as demonstrated in a later part of this study).
Mobilizable plasmids possess a specific sequence (origin of conjugative transfer, oriT) from which the initiation of conjugative transfer occurs. A key role in this process is played by Mob proteins (relaxases) which mediate cleavage of the phosphodiester bond within the oriT sequence. Analysis of the nucleotide sequence of pWKS1 revealed the presence of a sequence, located upstream of mob, with significant similarity to oriT of pBBR1 and to the recombination site A sequence involved in the mobilization and recombination processes (mediated by Mob/Pre proteins) of several plasmids from Gram-positive organisms, e.g. pMV158, pE194, pT181 and pUB110 (van der Lelie et al., 1989 ; Gennaro et al., 1987
; McKenzie et al., 1986
). Sequences analogous to the oriT sequence of pWKS1 were also found in conjugative transposons (Crellin & Rood, 1998
). The putative oriT sequence of pWKS1, like other sequences of this type, comprises inverted repeats (Fig. 3
). As has already been shown for pBBR1 (Szpirer et al., 2000
) and pMV158 (Farías et al., 1999
), these inverted repeats are placed in close proximity to the -10 boxes of mob promoters. The nick site determined for the oriT sequence of pBBR1 is also strongly conserved in the potential oriT sequence of pWKS1 (Fig. 3
). This analogous localization of nick sites has also been demonstrated in the oriT sequence of pMV158 from Streptococcus agalactiae (Farías & Espinosa, 2000
) and in oriT sequence of the mobilizable transposon Tn4555 of Bacteroides sp. (Smith & Parker, 1998
).
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The results obtained indicate that the RP4 transfer system in co-operation with the Mob protein of pBBR1 provides functions needed for conjugative mobilization of the plasmid containing oriT of pWKS1. The visible similarity of the oriT sequences observed between a number of mobilizable plasmids (and transposable elements) and the analogous localization of the strand-specific nick site (Fig. 3) may suggest that trans-mobilization by heterologous Mob proteins, derived from co-residing plasmids, might be common in natural genetic systems. It is worth mentioning that some plasmids do not carry a complete MOB module and only code a sequence similar to oriT/ recombination site A, e.g. pA1 from Lactobacillus plantarum A112 (Vujcic & Topisirovic, 1993
) or pCI411 from Leuconostoc lactis 533 (Coffey et al., 1994
). Thus, the transfer of these plasmids depends fully on the presence in trans of a plasmid(s) that provides both mobilization and conjugation functions.
Similarity of pWKS1 to other paracoccal plasmids and its host range
To determine whether pWKS1 carries sequences related to other plasmids from Paracoccus spp., pWKS1 was digoxigenin-labelled and probed against the plasmid profiles of 15 strains of Paracoccus that represented 11 paracoccal species (Table 1). These strains had been shown previously to carry plasmids ranging from 5·6 to over 100 kb in size (Baj et al., 2000
). Under the condition of high stringency, hybridization was observed only for pSOV1 (5·6 kb) of Paracoccus solventivorans (data not shown), which suggests that pWKS1 and pSOV1 carry related MOB and/or REP module(s). Also, pWKS1 did not hybridize with the total DNA from the strains tested, suggesting the absence of related sequences within the megaplasmids or chromosomes of these strains.
We did not succeed in introducing the Km-resistant derivatives of pWKS1 (pWKS18 and pWKS19) into E. coli TG1 by electroporation. To allow more detailed studies on the host range of pKWS1, and to overcome the restriction barrier of this plasmid, we constructed a convenient mobilizable hybrid plasmid that was composed of an E. coli-specific, mobilizable pABW1 vector and the replicator region of pWKS1. The resulting plasmid, pWKS20, was introduced by triparental mating into Rif-resistant derivatives of 10 different species belonging to the genus Paracoccus (P. alcaliphilus, Paracoccus alkenifer, Paracoccus aminophilus, Paracoccus aminovorans, Paracoccus denitrificans, Paracoccus methylutens, P. pantotrophus, P. solventivorans, Paracoccus thiocyanatus and P. versutus) as well as into Rhizobium leguminosarum, Agrobacterium tumefaciens and Rhodobacter sphaeroides (all of which belong to the -Proteobacteria). The transfer of pWKS1 into the paracoccal hosts did not result in the loss of their natural plasmids, which suggests that the incoming plasmid was compatible with them.
The small size of pWKS1 and its compatibility with all of the previously studied plasmids of Paracoccus spp. indicates that this plasmid can serve as a base for the construction of vectors specific for this group of bacteria.
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ACKNOWLEDGEMENTS |
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Received 26 February 2002;
revised 14 May 2002;
accepted 31 May 2002.