Molecular characterization of functional modules of plasmid pWKS1 of Paracoccus pantotrophus DSM 11072

Dariusz Bartosik1, Jadwiga Baj1, Marta Sochacka1, Ewa Piechucka1 and Miroslawa Wlodarczyk1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined. The G+C content of the sequence of this plasmid was 62 mol%. Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44·1 and 37·8 kDa, respectively. Mutational analysis showed that ORF2 was crucial for plasmid replication. The translational product of ORF2 shared local homologies with replication proteins of several {theta}-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts. An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV). ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria. A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli. MOB modules of pWKS1 and pBBR1 were functionally complementary to each other. Hybridization analysis revealed that only plasmid pSOV1 (6·5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1. Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E. coli.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The genus Paracoccus, currently embracing 14 described species (Baj, 2000 ; Kelly et al., 2000 ), belongs to the {alpha}-Proteobacteria. Paracocci are very interesting organisms because of their versatile metabolism. They are chemo-organotrophs or facultative chemolithoautotrophs, which oxidize reduced sulphur compounds as well as molecular hydrogen. They can also grow methylotrophically. Moreover, most species of Paracoccus can use nitrate as an alternative electron acceptor. Some species belonging to this genus have been isolated from polluted soil, water or sewage purification units. All of these features indicate the potential application of paracocci to bioremediation. So far, very little is known about the genomic localization (chromosomes, megaplasmids or plasmids) and genetic structure of the loci responsible for the majority of the versatile metabolic traits of paracocci.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
All of the bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were grown in Luria–Bertani (LB) medium (Sambrook et al., 1989 ) at 30 °C (all paracoccal strains, Agrobacterium tumefaciens and Rhodobacter sphaeroides) or 37 °C (Escherichia coli) and in TY medium (Beringer, 1974 ) at 30 °C (Rhizobium leguminosarum). Antibiotics included in media were used at the following final concentrations (µg ml-1): kanamycin (Km), 50; rifampicin (Rif), 50; streptomycin, 400 for Rhizobium leguminosarum; tetracycline, 3 for P. pantotrophus and P. versutus, and 20 for E. coli. All Rif-resistant derivatives of the strains used in this work were obtained as spontaneous mutants selected on LB agar supplemented with Rif.


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Table 1. Bacterial strains and plasmids used in this study

 
DNA sequencing and sequence analysis.
To determine the complete nucleotide sequence of pWKS1, the plasmid was digested with PstI or EcoRI and then cloned into the pBGS18 vector; the derived plasmids were designated pWKS10 and pWKS12, respectively. The derived plasmids were then sequenced on both strands using an automated DNA sequencer (model 377; Perkin Elmer). Sequence analysis was done by using programs included within the GCG Package (Wisconsin Genetics Computer Group Sequence Analysis Software Package, version 8.1). Comparison searches through the databases were performed by using the BLAST program (Altschul et al., 1990 ) provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST).

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 {Omega} (for E. coli) or 400 {Omega} (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{alpha}(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.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Nucleotide sequence of pWKS1
The complete nucleotide sequence of pWKS1 was determined (accession no. AF482428). The G+C content of this plasmid was found to be 62 mol%, which is slightly lower than the G+C content of the total DNA of P. pantotrophus (64–68 mol%) (Rainey et al., 1999 ). No significant DNA sequence similarity was found between pWKS1 and any of the nucleotide sequences held within the databases. Computer analysis of the sequence of pWKS1 (2697 nt) revealed the presence of two major ORFs (ORF1 and ORF2), which were in the same transcriptional orientation (Fig. 1a) and together covered 82% of the plasmid genome.



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Fig. 1. Genetic organization of pWKS1. (a) Circular map of pWKS1. The unique EcoRI site was arbitrarily assigned as co-ordinate 1. Only the unique restriction sites used for the construction of plasmid derivatives are given on the pWKS1 restriction map. The arrows indicate two ORFs, designated ORF1 (mob) and ORF2 (rep), and their transcriptional orientation. The positions of oriV and oriT are also indicated. (b) Nucleotide sequence of the A+T-rich intergenic region containing oriV of pWKS1. Several short repeated sequences identified within this region are indicated by different lines. Three long iteron-like repeated sequences are marked with thick lines and are designated DR1–DR3. Numbers on the right-hand side of the sequence refer to the nucleotide sequence of pWKS1 deposited within GenBank.

 
ORF1 (mapped at positions 7–1194; Fig. 1a) encodes a 395 aa polypeptide with a predicted molecular mass of 44·1 kDa and a pI value of 7·98. The transcriptional start codon of ORF1 (ATG) is preceded (7 bp) by a potential ribosome-binding site (RBS) AGG (Shine & Dalgarno, 1975 ). A BLAST comparison revealed that the ORF1 translational product showed the highest sequence similarity to a Mob protein involved in mobilization of the broad-host-range plasmid pBBR1 (46% similarity and 37% identity) from Bordetella bronchiseptica (Antoine & Locht, 1992 ). Similarities were also observed between the ORF1 product and the Mob proteins of several plasmids from Gram-negative bacteria, e.g. pTS1 from Treponema denticola (accession no. NP_073756), pCC7120{delta} from Anabaena sp. PCC 7120 (Kaneko et al., 2001 ) and pFL1 from Flavobacterium sp. KP1 (Ashiuchi et al., 1999 ), and between the ORF1 product and the Mob/Pre protein family (recombinases involved in plasmid mobilization/recombination processes) encoded by many plasmids found in Gram-positive hosts (Fig. 2). The highest similarities between the translational product of ORF1 and other proteins were observed for the Mob proteins of plasmids p1414 (37 % similarity and 25% identity) (Thorsted et al., 1999 ), pTA1015 and pTA1040 from Bacillus subtilis (Meijer et al., 1998 ), the Mob protein of pMV158 from Streptococcus agalactiae (Priebe & Lacks, 1989 ) and the Pre protein of pT181 from Staphylococcus aureus (Gennaro et al., 1987 ). The ORF1 product was also similar to the mobilization protein BmpH of the small mobilizable transposon Tn5520 from Bacteroides fragilis (Vedantam et al., 1999 ). A sequence comparison of various Mob proteins of plasmids residing in Gram-positive bacteria showed that the highest similarities in these proteins were observed within two regions (termed A and B) located in their N-terminal parts (Guzman & Espinosa, 1997 ). These conserved sequences correspond to the catalytic domains previously described by Koonin & Ilyina (1993) and Pansegrau et al. (1994) . As shown in Fig. 2, regions analogous to those described by Guzman & Espinosa (1997) can also be distinguished in the N-terminal part of the Mob protein of pBBR1 and the ORF1 product of pWKS1. In their studies on the mobilization mechanism of pBBR1, Szpirer et al. (2001) identified two amino acids (aspartate and glutamate) in the Mob protein, located in the B region, which were necessary for the activity of this protein. These residues are also conserved in the ORF1 product of pWKS1 and in sequences derived from other plasmids (Fig. 2).



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Fig. 2. Alignment of the deduced amino-acid sequences of the Mob proteins from several plasmids. pWKS1 is shown in bold. The other plasmids included in this alignment were: pBBR1 from Bordetella bronchiseptica (accession no. S25246); pTS1 from T. denticola (accession no. NP_073756); pCC7120{delta} from Anabaena sp. PCC 7120 (accession no. NP_489420); pMV158 from Streptococcus agalactiae (accession no. P13925); pFL1 from Flavobacterium sp. KP1 (accession no. NP_052877). Dots indicate gaps introduced to maximize the alignment. Amino acids identical to those in the Mob sequence of pWKS1 are shown against a dark background. An asterisk at the end of a sequence indicates a stop codon. Amino-acid numbering is shown at the right-hand side of the sequence lines. Two conserved regions (termed A and B), as described by Guzman & Espinosa (1997) , are shown boxed. The conserved residues (aspartate and glutamate) shown to be crucial for the mobilization of pBBR1 (Szpirer et al., 2001 ) are indicated by vertical arrows. The sequences of pCC7120{delta} and pMV158 have been truncated at their C termini.

 
The second ORF (ORF2) of pWKS1, located between nucleotides 1444 and 2463 (250 bp downstream of ORF1), encodes a 339 aa polypeptide with a predicted molecular mass of 37·8 kDa and a pI value of 9·51. The start codon of ORF2 (ATG) is not preceded by a typical sequence resembling a consensus RBS. Searches done using the BLASTP program demonstrated that the putative product of ORF2 showed local similarity with replication proteins of many {theta}-replicating plasmids from Lactococcus lactis, e.g. pFV1201, pJW565 and pJW566 (accession nos CAA65650, CAA65652 and CAA65651, respectively; Gravesen et al., 1997 ) or pUCL22 (accession no. S40058; Frere et al., 1993 ), with the Rep proteins of plasmid pOM1 from Francisella tularensis (accession no. NP_052243; Pomerantsev et al., 2001 ) and with plasmid pFA3 of Neisseria gonorrhoeae (accession no. A35257; Gilbride & Brunton, 1990 ). The similarities between the ORF2 product and these proteins were located mainly in the central part of the proteins (data not shown). Analogous similarities between different Rep proteins of various plasmids from Gram-positive and Gram-negative hosts have been observed by Gravesen et al. (1997) , which suggests that the replication systems of these plasmids [in spite of certain differences in the organization of their origins of replication (oriV)] may be distantly related. However, in the sequence of the ORF2-encoded protein, we could find neither an obvious DNA-binding domain (helix–turn–helix motif) nor a leucin-zipper motif, which are typical for many Rep proteins of {theta}-replicating plasmids (del Solar et al., 1998 ).

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 {rho}-independent transcriptional terminators. However, we did localize two 17 bp long inverted-repeat sequences (mapped at positions 2548–2591, and separated by 10 bp) 85 bp downstream of ORF2 that were able to form a potential stem–loop 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 EcoRI–PstI 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 (DR1–DR3; Fig. 1b), each of which contains a 21 bp sequence (5'-AAGTGGGGAATCCAGCCGCAA-3'), are tandemly repeated (without any spacer sequences) three times. In many {theta}-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 DR1–DR3 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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Fig. 3. DNA sequence alignment of the putative oriT sequence of pWKS1 with the oriT sequences of pBBR1 from Bordetella bronchiseptica (accession no. X66730), pMV158 from Streptococcus agalactiae (accession no. X15669) and mobilizable transposon Tn4555 (accession no. U75371). Inverted-repeat sequences are indicated by horizontal arrows. The nick sites are shown by vertical arrows. Conserved nucleotides are shown against a dark background. The -10 boxes determined for the mob promoters of pBBR1 and pMV158 are boxed.

 
The visible similarity between the nucleotide sequences of the oriT genes of pWKS1 and pBBR1, as well as the similarity of the amino-acid sequence motifs of Mob, suggests that the transfer systems of both of these plasmids are very closely related phylogenetically. The conjugative transfer of pBBR1 has been studied intensively (Szpirer et al., 2000 , 2001 ); it has been demonstrated that pBBR1 is mobilized most efficiently by IncP plasmids (e.g. RP4). We decided to check whether the conjugative-transfer systems of pBBR1 and pWKS1 were complementary – i.e. whether Mob of the broad-host-range plasmid pBBR1 was able to interact with oriT of pWKS1. To this end we used pWKS25, a mob-deficient plasmid carrying oriT and the complete replicator region. pWKS25 was introduced into E. coli S17-1(pBBR1MCS), whose pBBR1MCS plasmid carried a compatible chloramphenicol-resistant Mob-positive derivative of pBBR1. As expected, pBBR1MCS was transferred efficiently between E. coli strains, as well as between E. coli and P. pantotrophus KL100 or E. coli and P. versutus UW1R. Interestingly, we also obtained Km-resistant transconjugants that contained pWKS25; this indicates that the plasmid was mobilizable in the constructed system. To exclude the possibility that the transfer of pWKS25 was due to conduction (recombinational fusion and transfer of plasmid co-integrates), 100 Km-resistant transconjugants were plated onto medium containing chloramphenicol. As many as 87% of the examined transconjugant colonies contained only pWKS25 (with this being confirmed by electrophoretic analysis of the plasmid DNA of the transconjugants). The possibility of the mobilization of vector pBGS18 by pBBR1MCS was excluded in a control experiment, as was the transfer of pWKS25 alone (without co-residing pBBR1MCS) between strains of E. coli.

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 {alpha}-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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the State Committee for Scientific Research, Poland (grant no. 6 P04A 048 21).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-410.[Medline]

Antoine, R. & Locht, C. (1992). Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol Microbiol 6, 1785-1799.[Medline]

Ashiuchi, M., Zakaria, M. M., Sakaguchi, Y. & Yagi, T. (1999). Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium. FEMS Microbiol Lett 170, 243-249.[Medline]

Baj, J. (2000). Taxonomy of the genus Paracoccus. Acta Microbiol Pol 49, 185-200.[Medline]

Baj, J., Piechucka, E., Bartosik, D. & Wlodarczyk, M. (2000). Plasmid occurrence and diversity in the genus Paracoccus. Acta Microbiol Pol 49, 265-270.[Medline]

Bartosik, D., Bialkowska, A., Baj, J. & Wlodarczyk, M. (1997). Construction of mobilizable cloning vectors derived from pBGS18 and their application for analysis of replicator region of a pTAV202 mini-derivative of Paracoccus versutus pTAV1 plasmid. Acta Microbiol Pol 46, 379-383.

Bartosik, D., Baj, J. & Wlodarczyk, M. (1998). Molecular and functional analysis of pTAV320, a repABC type replicon of the Paracoccus versutus composite plasmid pTAV1. Microbiology 144, 3149-3157.[Abstract]

Bartosik, D., Szymanik, M. & Wysocka, E. (2001a). Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1. J Bacteriol 183, 6234-6243.[Abstract/Free Full Text]

Bartosik, D., Witkowska, M., Baj, J. & Wlodarczyk, M. (2001b). Characterization and sequence analysis of the replicator region of the novel plasmid pALC1 from Paracoccus alcaliphilus. Plasmid 45, 222-226.[Medline]

Bartosik, D., Baj, J., Bartosik, A. A. & Wlodarczyk, M. (2002). Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for the plasmid-encoded traits. Microbiology 148, 871-881.[Abstract/Free Full Text]

Bates, E. E. & Gilbert, H. J. (1989). Characterization of a cryptic plasmid from Lactobacillus plantarum. Gene 85, 253-258.[Medline]

Beringer, J. E. (1974). R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84, 188-198.[Medline]

Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1519.[Abstract]

Chattoraj, D. K. (2000). Control of plasmid DNA replication by iterons: no longer paradoxical. Mol Microbiol 37, 467-476.[Medline]

Coffey, A., Harrington, A., Kearney, K., Daly, C. & Fitzgerald, G. (1994). Nucleotide sequence and structural organization of the small, broad-host-range plasmid pCI411 from Leuconostoc lactis 533. Microbiology 140, 2263-2269.[Abstract]

Crellin, P. K. & Rood, J. I. (1998). Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ. Mol Microbiol 27, 631-642.[Medline]

del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M. & Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62, 434-464.[Abstract/Free Full Text]

Ditta, G., Stanfield, S., Corbin, D. & Helinski, D. R. (1980). Broad host range DNA cloning system for gram-negative bacteria: construction of a bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77, 7347-7351.[Abstract]

Farías, M. E. & Espinosa, M. (2000). Conjugal transfer of plasmid pMV158: uncoupling of the pMV158 origin of transfer from the mobilization gene mobM, and modulation of pMV158 transfer in Escherichia coli mediated by IncP plasmids. Microbiology 146, 2259-2265.[Abstract/Free Full Text]

Farías, M. E., Grohmann, E. & Espinosa, M. (1999). Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp. lactis. FEMS Microbiol Lett 176, 403-410.[Medline]

Frere, J., Novel, M. & Novel, G. (1993). Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22. Mol Microbiol 10, 1113-1124.[Medline]

Gennaro, M. L., Kornblum, J. & Novick, R. P. (1987). A site-specific recombination function in Staphylococcus aureus plasmids. J Bacteriol 169, 2601-2610.[Medline]

Gilbride, K. A. & Brunton, J. L. (1990). Identification and characterization of a new replication region in the Neisseria gonorrhoeae ß-lactamase plasmid pFA3. J Bacteriol 172, 2439-2446.[Medline]

Gravesen, A., von Wright, A., Josephsen, J. & Vogensen, F. K. (1997). Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids. Plasmid 38, 115-127.[Medline]

Greener, A., Lehman, S. M. & Helinski, D. R. (1992). Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria. Genetics 130, 27-36.[Abstract/Free Full Text]

Guzman, L. M. & Espinosa, M. (1997). The mobilization protein, MobM, of the streptococcal plasmid pMV158 specifically cleaves supercoiled DNA at the plasmid oriT. J Mol Biol 266, 688-702.[Medline]

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]

Hirsch, P. R., van Montagu, M., Johnston, A. W. B., Brewin, N. J. & Schell, J. (1980). Physical identification of bacteriocinogenic, nodulation and other plasmids in strains of Rhizobium leguminosarum. J Gen Microbiol 120, 403-412.

Jordan, S. L., McDonald, I. R., Kraczkiewicz-Dowjat, A. J., Kelly, D. P., Rainey, F. A., Murrell, J. C. & Wood, A. P. (1997). Autotrophic growth on carbon disulfide is a property of novel strains of Paracoccus denitrificans. Arch Microbiol 168, 225-236.[Medline]

Kaneko, T., Nakamura, Y., Wolk, C. P. & 19 other authors (2001). Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8, 205–213, 227–253.[Medline]

Kelly, D. P, Rainey, F. A. & Wood, A. P. (2000). The genus Paracoccus. In The Prokaryotes, 3rd edn. Edited by B. Balows, H. G. Trüper, M. Dworkin, W. Harder and K.-H. Schleifer. Springer Verlag Electronic Publication (http://www.springer-ny.com).

Koekman, B. P., Hooykaas, P. J. & Schilperoort, R. A. (1982). A functional map of the replicator region of the octopine Ti plasmid. Plasmid 7, 119-132.[Medline]

Koonin, E. V. & Ilyina, T. V. (1993). Computer-assisted dissection of rolling circle DNA replication. Biosystems 30, 241-268.[Medline]

Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M.2nd & Peterson, K. M. (1994). pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16, 800-802.[Medline]

Kushner, S. R. (1978). An improved method for transformation of E. coli with ColE1-derived plasmids. In Genetic Engineering , pp. 17-23. Edited by H. B. Boyer & S. Nicosia. Amsterdam:Elsevier.

McKenzie, T., Hoshino, T., Tanaka, T. & Sueoka, N. (1986). The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15, 93-103.[Medline]

Meijer, W. J., Wisman, G. B., Terpstra, P., Thorsted, P. B., Thomas, C. M., Holsappel, S., Venema, G. & Bron, S. (1998). Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria. FEMS Microbiol Rev 21, 337-368.[Medline]

Ogata, K., Sekizaki, T., Aminov, R. I., Tajima, K., Nakamura, M., Nagamine, T., Matsui, H. & Benno, Y. (1999). A small cryptic plasmid from Ruminobacter amylophilus NIAH-3 possesses functional mobilization properties. FEMS Microbiol Lett 181, 41-48.[Medline]

Ohara, M., Katayama, Y., Tsuzaki, M., Nakamoto, S. & Kuraishi, H. (1990). Paracoccus kocurii sp. nov., a tetramethylammonium-assimilating bacterium. Int J Syst Bacteriol 40, 292-296.[Abstract]

Pansegrau, W., Schroder, W. & Lanka, E. (1994). Concerted action of three distinct domains in the DNA cleaving–joining reaction catalyzed by relaxase (TraI) of conjugative plasmid RP4. J Biol Chem 269, 2782-2789.[Abstract/Free Full Text]

Pomerantsev, A. P., Obuchi, M. & Ohara, Y. (2001). Nucleotide sequence, structural organization, and functional characterization of the small recombinant plasmid pOM1 that is specific for Francisella tularensis. Plasmid 46, 86-94.[Medline]

Priebe, S. D. & Lacks, S. A. (1989). Region of the streptococcal plasmid pMV158 required for conjugative mobilization. J Bacteriol 171, 4778-4784.[Medline]

Projan, S. J. & Novick, R. (1988). Comparative analysis of five related staphylococcal plasmids. Plasmid 19, 203-221.[Medline]

Rainey, F. A., Kelly, D. P., Stackebrandt, E., Burghardt, J., Hiraishi, A., Katayama, Y. & Wood, A. P. (1999). A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb. nov. Int J Syst Bacteriol 49, 645-651.[Abstract]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Shine, J. & Dalgarno, L. (1975). Determination of cistron specificity in bacterial ribosomes. Nature 254, 34-38.[Medline]

Simon, R., Priefer, V. & Puhler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1, 784-791.

Smith, C. J. & Parker, A. C. (1998). The transfer origin for Bacteroides mobilizable transposon Tn4555 is related to a plasmid family from gram-positive bacteria. J Bacteriol 180, 435-439.[Abstract/Free Full Text]

Spratt, B. G., Hedge, P. J., te Heesen, S., Edelman, A. & Broome-Smith, J. K. (1986). Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 41, 337-342.[Medline]

Szpirer, C. Y., Faelen, M. & Couturier, M. (2000). Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob. Mol Microbiol 37, 1283-1292.[Medline]

Szpirer, C. Y., Faelen, M. & Couturier, M. (2001). Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1. J Bacteriol 183, 2101-2110.[Abstract/Free Full Text]

Thorsted, P. B., Thomas, C. M., Poluektova, E. U. & Prozorov, A. A. (1999). Complete sequence of Bacillus subtilis plasmid p1414 and comparison with seven other plasmid types found in Russian soil isolates of Bacillus subtilis. Plasmid 41, 274-281.[Medline]

van der Lelie, D., Bron, S., Venema, G. & Oskam, L. (1989). Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158. Nucleic Acids Res 17, 7283-7294.[Abstract]

Vedantam, G., Novicki, T. J. & Hecht, D. W. (1999). Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli. J Bacteriol 181, 2564-2571.[Abstract/Free Full Text]

Vieira, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268.[Medline]

Vujcic, M. & Topisirovic, L. (1993). Molecular analysis of the rolling-circle replicating plasmid pA1 of Lactobacillus plantarum A112. Appl Environ Microbiol 59, 274-280.[Abstract]

Wlodarczyk, M., Jagusztyn-Krynicka, E. K., Bartosik, D. & Kalinowska, I. (1994). Electroporation of Thiobacillus versutus with plasmid DNA. Acta Microbiol Pol 43, 223-227.

Received 26 February 2002; revised 14 May 2002; accepted 31 May 2002.