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1 INRA, UMR 1090 Génomique Diversité et Pouvoir Pathogène, F-33883 Villenave d'Ornon, France
2 Université de Bordeaux 2, UMR 1090 Génomique Diversité et Pouvoir Pathogène, F-33883 Villenave d'Ornon, France
Correspondence
Joël Renaudin
renaudin{at}bordeaux.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Five supplementary figures and a supplementary table are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), Spiroplasma kunkelii (Whitcomb et al., 1986) and Spiroplasma phoeniceum (Saillard et al., 1987), have been associated with plant diseases. S. citri is the aetiological agent of the citrus stubborn and horseradish brittle root diseases and was the first plant mollicute to be cultured in vitro (Fletcher et al., 1981; Saglio et al., 1971). Since then, S. citri has been studied extensively (Bové et al., 1989, 2003). The availability of both an experimental transmission assay via the leafhopper vector (Foissac et al., 1996) and gene transfer systems (Foissac et al., 1997; Renaudin, 2002; Renaudin & Lartigue, 2005) has made it a unique model for studying plant mollicute–host interactions (André et al., 2005; Berho et al., 2006a; Bové et al., 2003; Killiny et al., 2005).
In spiroplasmas, plasmids were first detected as extrachromosomal, covalently-closed-circular DNAs (Ranhand et al., 1980) and some of them were mapped and/or cloned in Escherichia coli (Archer et al., 1981; Mouchès et al., 1983a). Extrachromosomal DNAs, including plasmids and viral DNAs, were most frequently detected in group I spiroplasmas (Gasparich et al., 1993), especially in S. citri strains. However, these plasmids have for a long time been designated cryptic since no phenotypic character had been associated with their presence. In addition, due to the lack of sequence and functional data, the engineering of natural plasmids to produce cloning vectors has had very limited success (Salvado et al., 1989). More recently sequencing of plasmids pBJS-O from S. citri strain BR3-3X (Joshi et al., 2005), and pSciA and pSci1–6 from S. citri GII3 (Saillard et al., 2008), revealed that they encode proteins, including adhesin-like molecules, which have been tentatively associated with insect transmission (Killiny et al., 2006; Yu et al., 2000). Furthermore, it was shown that plasmids pSci1–6 from the insect-transmissible strain GII3 were absent in the non-insect-transmissible strains of S. citri (Berho et al., 2006b). However, the first clear-cut evidence of a phenotypic trait associated with spiroplasmal plasmids was the finding that pSci6 from S. citri GII3 conferred insect-transmissibility to the plasmid-free, non-insect-transmissible strain 44 (Berho et al., 2006a). Besides assessing their role in insect transmission, the successful transfer of pSci plasmids between strains of S. citri raised their potential as genetic tools. In addition to adhesin-like proteins, pSci1–6 encode proteins with similarities to components of bacterial type IV secretion systems, suggesting the possibility of conjugative transfer of these plasmids. In turn, none of the plasmid-encoded proteins shared similarity with known replication proteins (Saillard et al., 2008).
Previously, we showed that sequences essential for replication and maintenance of pSci2 in S. citri were located within an 8 kbp restriction fragment (Berho et al., 2006a). In the present study, we describe the functional characterization of the replication and stability regions of S. citri pSci-derived plasmids, with the aim of further investigating their role in spiroplasmal biology. We also demonstrate the usefulness of pSci-derived plasmids as vectors for gene transfer and expression in plant-pathogenic spiroplasmas other than S. citri.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). S. citri 44 was isolated from a stubborn-diseased sweet orange tree in Iran (Hosseini Pour, 2000). Other spiroplasma strains used in this study are listed in Supplementary Table S1, available with the online version of this paper. Spiroplasmas were grown at 32 °C in SP4 medium (Whitcomb, 1983) from which fresh yeast extract was omitted. Spiroplasma cells were transformed by electroporation (Stamburski et al., 1991) using 2–10 µg purified plasmid or various ligation mixtures. Spiroplasmal transformants were selected in the presence of 2 µg tetracycline ml–1, and further propagated in broth medium containing 5 µg tetracycline ml–1. The Spiroplasma species of tetracycline-resistant transformants were confirmed by PCR-RFLP analysis of the spiralin genes, using amplification with primer pair SR28/SR29 (Table 1) and digestion with MboI and/or RsaI.
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). Plasmid pSRT1 differs from pSRT2 in that the tetM cassette is in the opposite orientation. Plasmids pSci1NT and pSci6PT were obtained by inserting the tetM cassette (as a 2.3 kbp PstI fragment) into the NsiI and PstI sites of S. citri pSci1 and pSci6, respectively, whereas pSci21NT resulted from the combination of the tetM cassette with the 8034 bp NsiI fragment of pSci2 (Berho et al., 2006a). In pSci1NT
S, the scarp3d gene was disrupted by inserting the 20 bp oligonucleotide duplex SBamF1/SBamR1 (Table 1
) into the NheI site of pSci1NT. Similarly, in pSci21NTsoj, the soj gene was disrupted by insertion of the 19 bp SojBamF1/SojBamR1 duplex into the SmlI site of pSci21NT. As a result, the Soj coding sequence (CDS) encodes a truncated polypeptide of 67 amino acids as compared with 260 in the wild-type. Plasmid pFL3 was obtained by self-ligation of the 6675 bp EcoRI–BglII fragment of pSci21NT. Plasmid pFL4 was constructed by deleting the 891 bp HincII–HaeIII fragment within the mob gene of pFL3, and pFL5 by deletion of the 504 bp AclI–EcoRV fragment of pFL4. Plasmid pBS1 was derived from pBS+ by inserting the EPBg1/EPBg2 duplex at the EcoRI site to introduce a BglII site. Plasmid pBST1 was then obtained by combining the BglII-linearized pBS1 with the tetM cassette (as a 2311 bp BamHI–BglII fragment) of pSRT2. Plasmid pFL6, containing the pE gene and the flanking, intergenic regions, resulted from the ligation of the 2265 bp PacI fragment from pFL4 to the 2329 bp PacI fragment of pBST1 containing the tetM cassette. Plasmid pFL7 (4259 bp) was obtained by deleting the 341 bp AclI–BglII fragment of pFL6. The shuttle (S. citri/E. coli) plasmid pBSFL6 resulted from the combination of PstI-linearized pBS+ and pFL6. To construct pSDE1, the amplification product of pSci2 with primer pair PEF1/IEFR2 was inserted into pSRT1 after they were both digested with BamHI+EcoRI. Similarly, the Rep1-F1_BglII/Rep1-R1_BglII amplification product, comprising the pE gene and the flanking intergenic regions of pSci1, was digested with BglII, and the 1963 bp fragment was inserted into BglII-linearized pSRT1 to yield pMBRE. While pBSFL6 comprised the pE region of pSci2, pMBRE contained the comparable pE region of pSci1 having a MluI restriction site within the pE gene. To construct pMBRE1, pMBRE was linearized with MluI, and recircularized by ligation after the 4 bp cohesive ends were made blunt-ended. In this plasmid, disruption of pE led to a truncated polypeptide of 97 amino acids instead of 217 in the wild-type. Similarly, in pMBRE2 inserting the 19 bp oligonucleotide duplex EBamF1/EBamR1 into the MluI site led to a truncated PE of 95 amino acids. Plasmid pNAB32H resulted from insertion of the p32-HA tagged gene inserted into pBSFL6. Each plasmid construct was confirmed by restriction mapping and sequencing the appropriate regions.
DNA isolation, Southern blot hybridization and Western immunoblotting.
Spiroplasma genomic DNA was prepared from 10 ml cultures using the Wizard genomic DNA purification kit (Promega), whereas plasmid DNA was purified from 25 ml cultures with the Wizard SV minipreps DNA purification kit (Promega). Southern blot hybridization of spiroplasmal DNA with appropriate digoxigenin-dUTP-labelled probes has been described elsewhere (André et al., 2003). Hybridization signals were detected with anti-digoxigenin–alkaline phosphatase conjugate and 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP) as the substrate, following the supplier's instructions (Roche Diagnostics). Fluorescent signals were detected using a Fluor-S Multimager Phosphoimager (Bio-Rad). Probes specific to ScARPs, P32 and PE were generated by PCR amplification with primer pairs S235F/S235R, P32F/P32R and PEF1/PER1, respectively (Table 1).
Spiroplasmal proteins were separated by SDS-PAGE and further analysed by immunoblotting as described previously (Duret et al., 2003) except that the proteins reacting with the primary antibodies were visualized by using a goat anti-rabbit immunoglobulin G-peroxidase conjugate and the SuperSignal West Pico chemiluminescent substrate (Pierce).
In silico analyses.
The BLAST program (Altschul et al., 1997) was used to search for homologies in general databases (http://www.ncbi.nlm.nih.gov/blast/), the mollicute dedicated database MolliGen (Barré et al., 2004), and the S. kunkelii partially sequenced genome (http://www.genome.ou.edu/blast/spiro_blastall.html). Conserved domains were detected by CD-Search against the NCBI's conserved domain database (Marchler-Bauer & Bryant, 2004). Global sequence alignments were accomplished using a Needleman–Wunsch algorithm implemented in the Needle program (Needleman & Wunsch, 1970). Multiple alignments were done with T-Coffee (Notredame et al., 2000) and/or CLUSTAL W (Thompson et al., 1994). For subsequent phylogenetic analyses, poorly aligned positions and divergent regions were eliminated using the alignment curation program Gblocks (Castresana, 2000). Neighbour-joining trees based on these alignments were constructed using the MEGA 3.1 program (Kumar et al., 2004) and maximum-likelihood trees were computed with the WAG substitution model using the PhyML program (Guindon & Gascuel, 2003). All trees were bootstrapped 100 times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), which share a large number of highly conserved regions and display a mosaic gene organization (Fig. 1). Most of the CDS are present on several plasmids and, reciprocally, very few of them are specific to one given plasmid. Such an exception is p32, encoding a hydrophilic protein specific to pSci6 (Killiny et al., 2006). Surprisingly, most of the plasmid CDS have no orthologues in other bacterial genera, with the exceptions of soj, trsE, traG and mob, the naming of which indicates that they encode proteins closely related to proteins involved in partitioning (Soj/ParA family COG1192) and transfer (TrsE, TraG and Mob) of DNA molecules. The scarp genes of pSci1–5 encode proteins sharing strong similarities with the adhesin-like protein SARP1 from S. citri BR3 (Berg et al., 2001). Plasmid pSci2 (14.4 kbp) seems to represent the ‘core plasmid’ as it displays a gene organization (pE, pF, pA, soj, pB, scarp, pC, pD, trsE, p4, mob) which is conserved in pSci1 (13 kbp) and larger plasmids pSci3 (19.3 kbp) and pSci4 (20.2 kbp) (Fig. 1). Plasmid pSci3 possesses an additional CDS (pG), whereas pSci4 carries two scarp genes, two copies of soj, and the additional CDS pH, which is absent in the other three plasmids pSci1–3. In pSci1, pSci5 and pSci6, the mob-like gene is disrupted. The large plasmids pSci5 and pSci6 probably result from recombination between plasmids and/or from gene duplication. Whereas pSci1–3 contain a single copy of pE and soj, pSci4–6 have two copies of soj, and multiple copies of pE are present in pSci5 (2 copies) and pSci6 (4 copies). Interestingly, pE and soj are the only two CDS that are conserved, uninterrupted, in all seven plasmids, pSciA and pSci1–6, suggesting that these genes may be essential for replication and maintenance of the plasmids.
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). This fragment encodes seven putative polypeptides, including a Mob-like ATPase and a Soj-like protein (Fig. 2). Polypeptides PA and PE, as well as the truncated PB and PF, are hypothetical proteins that have counterparts in other pSci plasmids, whereas the hypothetical polypeptide HP shares no homology with other proteins. None of these polypeptides share homology with known replication proteins. To identify the replication region of pSci2, various pSci2 derivatives were constructed by successive deletions of pSci21NT, and tested for their ability to replicate through transformation of S. citri 44, a strain which contains no plasmid. Plasmid constructs are described in Methods. The finding that pFL3, pFL4, pFL5, pFL6 and pFL7 all replicated in S. citri showed that the replication region was located within the 1876 bp HaeIII–AclI fragment of pFL3 (Fig. 2). Interestingly, this DNA fragment comprised a single CDS, suggesting that the pE-encoded polypeptide might be the replication protein and that the flanking intergenic regions contained the cis-acting binding sites. In silico analyses revealed the region upstream of pE to contain specific features including the imperfect repeats ATTTAATCCCCC and ATTTAACCCCC (at positions 10963 and 11084 of pSci2) and four sequences (GTTTCCATA, ATTTCCACA, TCTACCACA and TTTTCCAAA, at positions 10833, 10899, 10977 and 11268, respectively) resembling the DnaA boxes (TTTTCCACA) of the S. citri chromosomal replication origin (oriC) (Ye et al., 1994). However, in contrast to the situation at oriC, these sequences did not cluster within a short region. Immediately downstream of pE, two tandem repeated sequences of 11 nucleotides (TAACTCCCCTA), as well as an A-T rich region, were detected (Fig. 2). However, none of the six inverted repeat sequences identified in the intergenic region downstream of pE (Saillard et al., 2008) was located within the 1876 bp minimal replicon, indicating that these sequences, which putatively lead to hairpin structures, are not essential for plasmid replication. To further delineate the replication region, DNA fragments were retrieved from pFL6 by digestion or PCR amplification and inserted into ColE1 replicons by cloning in E. coli. Whereas the recombinant plasmid pBSFL6, which contained the pE gene and the flanking intergenic regions, readily replicated in S. citri, plasmid pSDE1 did not, indicating that replication probably requires sequences upstream of primer PEF1.
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E1 and pMBRE2 were used to transform S. citri 44. As expected, transformation by pMBRE yielded tetracycline-resistant transformants at relatively high frequency (
10–4 transformants c.f.u.–1µg–1), indicating that the plasmid replicated in these cells. The presence of the relevant plasmid in the spiroplasmal transformants was confirmed by PCR and Southern blot hybridization (data not shown). In contrast, no transformants were obtained through transformation with pMBRE1 and pMBRE2, in which the pE gene was disrupted (see Methods and Fig. 2). As a control, disruption of the scarp gene in pSci1NTS had no effect on the ability of the plasmid to replicate.
Considering PE as the replication protein, one would expect pMBRE1 and pMBRE2 to replicate in S. citri GII3, a strain with plasmids, from which wild-type PE was known to be expressed, as indicated by proteome analyses (unpublished data). However, transformation of S. citri GII3 by these plasmids failed repeatedly, indicating that replication of pMBRE1 and pMBRE2 cannot be driven by the PE protein when provided in trans from endogenous plasmids.
Alignments of the PE proteins from pSci1–6 showed that they share strong similarities to each other, with the conserved regions being distributed all along the protein sequence. Identities ranged from 76.1 % between pSci1-PE and pSci6-PE4 to 97.7 % between pSci3-PE and pSci5-PE1 (Fig. 1 and Supplementary Fig. S1). The pSciA-PE was much less conserved with identities ranging from 25.2 % with pSci1-PE to 28.4 % with pSci6-PE1. Unexpectedly, despite the PE protein being essential for plasmid replication, no putative DNA-binding motif such as helix-turn-helix, coiled coil, zinc finger, or leucine zipper was predicted in the PE sequence. It is noteworthy that most of the PE polypeptides end with an identical C-terminus (ELDNLD), except that from pSciA and three of the four PE from pSci6, which have distinct C-termini (Supplementary Fig. S1). The autonomous replication of the 16195 bp circularized BstBI fragment of pSci6PT (unpublished data) indicated that at least one PE of pSci6 (pSci6 E2 in Fig. 1) functioned as the replication protein. Whether the other three are functional or not has not yet been investigated. Studying the genetic relatedness of the S. citri GII3 proteins PEs showed that they clustered within three distinct groups (Fig. 3). The largest group consisted of PEs from pSci1–5 and the PE counterparts of pBJS-O from S. citri BR3 (Joshi et al., 2005) and pSKU146 from S. kunkelii CR2-3X (Davis et al., 2005). Unexpectedly, whereas one PE (PE2) from pSci6 also fell in this group, the other three (PE1, 3 and 4) formed a distinct, heterogeneous group. Such variability is consistent with the hypothesis that, in contrast to PE2, PE1, 3 and 4 might not be functional. Whether the occurrence of multiple copies of the pE gene in pSci6 resulted from gene duplication or recombination between plasmids is unclear. Intriguingly, the pSciA PE did not cluster with any other PE proteins, suggesting that pSciA had a distinct origin. This is in agreement with the fact that, except for PE and Soj, the pSciA CDS have no counterparts in any other S. citri plasmids.
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). To further confirm the presence of the six plasmids in every cell, the spiroplasma strain was further subcloned twice, and the plasmid contents of 10 isolated colonies were characterized by Southern blot hybridization of total DNA. The hybridization patterns of all 10 subclones tested were identical to each other and to that of the original culture (Supplementary Fig. S2), indicating that each spiroplasma cell contained the whole set of plasmids, pSci1–6. Identical hybridization patterns were obtained with spiroplasma cultures isolated from the leafhopper vector and from the host plant after experimental transmission of GII3-3X, by injection, to its leafhopper vector (data not shown).
As indicated above, each S. citri plasmid contains at least one soj-like gene, encoding a polypeptide with significant similarities to proteins of the Soj/ParA, ATPase family involved in chromosome partitioning. Similarities to the chromosomal ParA of S. citri and the Soj protein of Bacillus subtilis ranged from 41.3 to 46.3 % and from 41.6 to 50.8 %, respectively. In the plasmid-encoded Soj, COG1192 (Soj) and COG0455 (ATPases involved in chromosome partitioning) were predicted with significant probabilities (e-values ranging from 3x10–10 to 1x10–28). Like the chromosomal ParA, the pSci-encoded Soj all possessed the deviant Walker-type ATPase consensus motif with the typical A, A', and B boxes (Koonin, 1993) (Supplementary Fig. S3).
To investigate the role of Soj in S. citri plasmid partitioning, we assessed the stability of various pSci2 derivatives in S. citri 44 transformants. In the experiment shown in Fig. 4, S. citri 44 cells transformed with pSci21NT (containing soj) or pFL3 (lacking soj) were propagated in the absence of selection pressure. After 5, 15 and 30 passages (30 passages correspond to 100 generations), the plasmid contents of 10 individual clones were analysed by Southern blot hybridization. The results showed that pSci21NT was detected in each of the 10 clones tested, regardless of the passage number (5, 15 or 30). In contrast, pFL3 was lost in three clones out of ten, as early as the fifth passage. After 15 passages, pFL3 was detected in only two clones, and after 30 passages, none of the ten clones carried pFL3. The finding that, in the absence of selection pressure, pSci21NT was stably maintained for many generations whereas pFL3 was rapidly lost strongly suggested that the soj-encoded protein was indeed involved in partitioning of pSci2. In accordance with these results, plasmid loss was also noticed in spiroplasmas transformed with pSci2 derivatives pFL4, pFL5 and pFL6, all of which lack soj. However, when compared with pSci21NT these plasmids lack not only soj but also three additional CDS, the two conserved hypothetical proteins PA and PB as well as the orphan CDS HP (Fig. 2). The role of Soj was further demonstrated by evaluating the stability of pSci21NTsoj, a plasmid in which the soj gene was disrupted, in S. citri 44 transformants. After propagation for up to 30 passages in the absence of selection pressure, the presence of the plasmid was checked by monitoring the percentage of spiroplasma cells growing on selective (tetracycline) agar medium. As expected from the above experiment, nearly 100 % (285 /294) of cells from pSci21NT transformants were still tetracycline-resistant after 30 passages. In contrast, in pSci21NTsoj transformants, the ratio of tetracycline-resistant to total cells progressively decreased to 81 % (515/638) at the fifth passage, 27 % (132/496) at the 15th passage, and less than 0.2 % (less than 1/417) after 30 passages. These results clearly indicated that the soj gene product is involved in the stability of pSci plasmids.
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) indicates that plasmid-encoded Soj were distinct from the chromosomal Soj/ParA and clustered in two separate groups: Soj from pSci1–5 (S. citri GII3) and pBJS-O (S. citri BR3) in one group, and Soj from pSciA and pSci6 (S. citri GII3), and pSKU146 (S. kunkelii E275) in the other, suggesting that these groups had evolved separately and disjointedly from the chromosomal ParA. In an effort to understand the origin of these plasmids, phylogenetic analyses of spiroplasma Soj proteins were extended to a set of representative ParA homologues (Gerdes et al., 2000) including the 23 annotated ParA homologues from mollicute genomes (http://cbi.labri.fr/outils/molligen/). The phylogenetic tree showed that, as in many bacteria (McLeod et al., 2006), the chromosomally encoded ParA proteins of spiroplasmas had phylogenies congruent with those of their hosts. In contrast, the plasmid-encoded Soj/ParA proteins were more diverse and their clustering did not follow the organismal phylogeny (Supplementary Fig. S4). When restricted to the mollicutes and several other firmicutes, these analyses identified four major clusters supported by strong bootstrap values (Supplementary Fig. S5). One coherent cluster was made from the S. citri chromosomal ParA and other chromosomal ParA proteins of firmicutes (except those of mycoplasmas). A second cluster consisted of the mycoplasmal chromosome-encoded ParA/Soj proteins, whose genes are located in the vicinity of the chromosome replication origin. These proteins are clearly distinct from ParA/Soj proteins of other firmicutes and the spiroplasmas in particular. The third cluster comprised plasmid-encoded Soj proteins from S. citri pSci1–5 and pBJS-O; the fourth cluster consisted of Soj proteins from S. citri pSciA and pSci6, S. kunkelii pSKU146, and chromosomal Soj proteins that are encoded by putative or demonstrated mobile elements of mycoplasmas (Marenda et al., 2006; Sirand-Pugnet et al., 2007).
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). To investigate the occurrence of PE replicating plasmids in spiroplasmas further, genomic DNAs from various strains of S. citri and other spiroplasma species were hybridized with probes scarp and pE (Fig. 6, Table 2). Both scarp and pE hybridization signals were detected in strains GII3, Corse, Palmyre, Hinckley, IP85 and Israel. In contrast, no hybridization signal was detected in ASP-1, R8A2 and 44. Detection of pE but not scarp sequences in strain Alcanar 254 is consistent with the fact that this strain carries a single plasmid nearly identical to pSci6 (unpublished data), which contains no scarp genes. Interestingly, pE-hybridizing signals were also detected in the plant-pathogenic spiroplasmas S. kunkelii E275 (subgroup I-3) and S. phoeniceum P40 (subgroup I-8), indicating that PE replicons were not restricted to the species S. citri, i.e. subgroup I-1 of the spiroplasmal classification (Williamson et al., 1998). In addition, the pE probe was found to hybridize with genomic DNAs from the bee pathogen Spiroplasma melliferum (subgroup I-2) strains BC3, G1 and AS576, the rabbit tick spiroplasma 277F (I-4), the plant bug spiroplasma sp. LB-12 (I-5), and the plant surface Spiroplasma sp. N525 (I-7) but not with DNAs from strains B13 and B31 of the bee pathogen Spiroplasma apis (group IV) and strain 23.6 of the plant surface Spiroplasma floricola (group IV). From these data, the PE replicons appear to be restricted to species of group I spiroplasmas. Unlike pSci1–5 from S. citri GII3, these replicons do not necessarily contain conserved scarp genes. Similarly to S. citri Alcanar 254, PE replicons from S. melliferum did not hybridize with the scarp probe.
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) was repeatedly transformed by pSci21NT and pFL6 at frequencies close to 10–5 transformants c.f.u.–1 µg–1. Interestingly, transformation of S. kunkelii E275 and S. phoeniceum P40 by pSci21NT, pFL6, and its shuttle derivative pBSFL6 also yielded tetracycline-resistant transformants at frequencies of 10–5–10–4 transformants c.f.u.–1 µg–1. The possibility that these tetracycline-resistant cells could be spontaneous mutants or contaminants was excluded by showing that they did contain the relevant plasmid, by PCR and Southern blot hybridization, and by confirming the spiroplasma species through PCR-RFLP analysis of the spiralin gene (Methods).
pSci-derived plasmids as gene vectors
To demonstrate the usefulness of pSci-derived plasmids as gene vectors, the plasmid construct pNAB32H, in which the p32 gene fused to a HA tag was inserted into the shuttle plasmid pBSFL6, was introduced into S. kunkelii E275 by electrotransformation. The presence of pNAB32H in the spiroplasmal transformants was confirmed by PCR and Southern blot hybridizations (data not shown). In the experiment shown in Fig. 7, total proteins were probed by Western immunoblotting with P32- and HA-specific, polyclonal antibodies (Fig. 7a and b, respectively). Cells from S. citri GII3 and S. kunkelii E275 transformed by pSci1NT were used as controls. As expected, P32 was detected in S. citri GII3 (Fig. 7a, lane 1) but not in untransformed S. kunkelii (lane 2) or in S. kunkelii transformed with pSci1NT (lane 6). In contrast, P32-related proteins were detected in S. kunkelii cells transformed by pNAB32H, carrying the p32–HA fusion gene (lanes 3 and 4), or pSci6PT containing the wild-type p32 gene (lane 5). As expected, the HA-tagged protein was clearly detected in pNAB32H-transformed cells, indicating that the fusion protein was expressed from the recombinant plasmid in S. kunkelii E275 (Fig. 7b, lanes 3 and 4). In addition, the relative intensities of the P32 detection signals were consistent with an expression level of the P32–HA fusion protein from pNAB32H similar to that of the wild-type P32 encoded by pSci6 in S. citri GII3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tran-Nguyen & Gibb, 2006). S. citri plasmids pSciA and pSci1–6 all possess genes soj and pE, which are also conserved in plasmids pBJS-O from S. citri BR3 (Joshi et al., 2005) and pSKU146 from S. kunkelii CR2-3X (Davis et al., 2005). In this study, we showed that pE is essential and the only plasmid-encoded protein required for plasmid replication. The finding that inactivation of pE by a single 4 bp insertion prevented replication clearly indicates that the polypeptide, rather than the nucleotide sequence, is required. The fact that the pE-disrupted plasmids failed to replicate in S. citri GII3 (a strain that produces PE from endogenous plasmids) suggests that PE, unlike other replication initiator proteins (Emond et al., 2001) might be a cis-acting protein, unable to support replication when provided in trans. In this experiment, however, the hypotheses of a retro-control system specific for S. citri GII3, or incompatibility between plasmids having identical replication regions, cannot be excluded (Bouet et al., 2007). Therefore, complementation of the mutation carried by the pE-disrupted plasmids in S. citri 44 will be required to definitely establish whether or not PE is a cis-acting protein.
Although the PE protein was found to be essential for replication, it lacks the leucine zipper and helix-turn-helix motifs found in many Rep proteins (Del Solar et al., 1998). The lack of sequence and/or structural similarity between PE and known replication proteins suggests that replication of spiroplasma plasmids pSci involves a distinctive mechanism. Circular bacterial plasmids use two modes of DNA replication known as rolling circle (RCR) and theta. RCR plasmids are generally recognized by their small size (less than 12 kbp), their high copy number, the generation of single-stranded intermediates, and the presence of specific functional features comprising a rep gene, encoding the replication initiator protein, and the replication origin sequences (Khan, 2005). None of these typical features of RCR plasmids apply to pSci plasmids from S. citri GII3 (Saillard et al., 2008), suggesting that pSci plasmids replicate through a theta mechanism. The presence of four putative DnaA boxes and an AT-rich region upstream of pE is consistent with this hypothesis. Also, the 11 bp tandem repeated sequences separated by 9 bp and located immediately downstream of pE might represent iterons, i.e. binding sites for the replication initiator protein. Interaction of the replication protein with its cognate iteron repeat is an important determinant of plasmid incompatibility (Bouet et al., 2007). Obviously more work is needed to determine whether or not these sequences that are strictly conserved in plasmids pSci1–6 and pBJS-O of S. citri and pSKU146 of S. kunkelii are the binding site of the replication protein PE.
Partitioning protein Soj
In bacteria, active segregation of low-copy-number plasmids into daughter cells relies on plasmid-encoded active partitioning (par) systems. In S. citri, the fact that pSci1–6 were stably maintained both in vitro and in vivo indicates that, in spite of highly conserved sequences, they exhibit no incompatibility with each other and hence suggests that each plasmid has its own partitioning system. The finding that, provided they carry a functional soj gene, the pSci2-derived plasmids are stably maintained in the absence of selection pressure indicates that S. citri plasmids possess an active partition system involving the Soj protein. In general, the par locus encodes two trans-acting proteins expressed from an operon and one or more cis-acting centromere-like sites (Bouet et al., 2007; Ebersbach & Gerdes, 2005). The par loci are divided into two main groups depending on the type of ParA ATPase encoded by the first gene of the parA/B operon. Type I loci encode Walker box ATPases and type II loci encode actin-like ATPases. The type I systems can be further divided into type Ia and type Ib based on the similarities and sizes of the Par proteins, the localization of the partition site, and the mechanism of the transcriptional regulation (Gerdes et al., 2000). S. citri pSci plasmids have soj genes predicted to encode ParA superfamily ATPases. These ParA homologues possess the deviant Walker-type ATPase motif of the type I plasmid partitioning system (Koonin, 1993) and the genetic organization of soj loci is more reminiscent of type Ib. The pSci-encoded Soj proteins have reduced sizes (255–260 residues) and lack the N-terminal extension containing a DNA-binding domain, usually present in type Ia. Considering that mollicutes are phylogenetically related to Gram-positive bacteria (Weisburg et al., 1989), this is in agreement with the fact that type Ia loci have only been reported in plasmids from Gram-negative bacteria, whereas loci of type Ib have been described in both Gram-negative and Gram-positive bacteria (Gerdes et al., 2000). The hypothesis that CDS such as pB located immediately downstream of soj in pSci2 could encode a ParB-like component cannot be excluded as these components frequently exhibit no conserved sequences (Ebersbach & Gerdes, 2005). Phylogenetic analyses revealed that Soj proteins from spiroplasma plasmids clustered within two distinct groups that are clearly unrelated to spiroplasma chromosomal ParA/Soj (see Supplementary Fig. S4). Clustering of Soj from S. citri pSciA and pSci6 and S. kunkelii pSKU146 with those of the mycoplasmal mobile elements is consistent with previous observations that the arrangement of three CDS of pSKU146 paralleled that of their counterparts in the integrated conjugative element (ICE) of Mycoplasma fermentans (Calcutt et al., 2002; Davis et al., 2005), and suggests that spiroplasma plasmids and mycoplasma ICEs might have a common ancestor. It is noteworthy that, in contrast to spiroplasmas, most mycoplasmas do not possess parA/soj genes (Livny et al., 2007), and that mycoplasmas having parA/soj-like sequences all belong to the ‘pneumoniae’ phylogenetic group. However, the absence of parB and parS equivalents in these mycoplasmas raises the question of whether the mycoplasmal ParA function as chromosome partitioning proteins.
Distribution of PE replicons
The PE replicons were found to be widely distributed in group I spiroplasmas, including the three plant-pathogenic spiroplasmas S. citri, S. kunkelii and S. phoeniceum. In S. citri, however, strains R8A2 and 44 do not contain plasmids pSciA and pSci1–6 (Berho et al., 2006b). In contrast, ASP-1 is known to carry plasmid pIJ2000 (Archer et al., 1981), highly similar if not identical to pSciA. In this case, the lack of hybridization signal probably resulted from the fact that pE sequences are poorly conserved in pSciA (Supplementary Fig. S1). Therefore, in our experiments, the failure to hybridize with the pE probe does not exclude the presence of pSciA-related plasmids. In S. phoeniceum, both scarp and pE probes were found to hybridize with total DNA from S. phoeniceum P40, indicating the presence of PE replicons in this strain. For unknown reasons, these data do not fit with those of Joshi et al. (2005) showing that undigested plasmid preparations from S. phoeniceum did not hybridize with the whole pBJS-O probe.
Expanding the genetic toolbox of spiroplasmas
Limited cloning tools are available for the genetic modification of S. citri. Artificial oriC plasmids have been used as vectors to complement mutants (Gaurivaud et al., 2000; Jacob et al., 1997). However, due to incompatibility with the chromosomal oriC, the spiroplasmal transformants have a reduced growth rate and the plasmids are unstable, as they tend to integrate into the host chromosome. In addition, transformation frequencies of S. citri by oriC plasmids are relatively low, and some S. citri strains cannot be transformed consistently (Renaudin, 2002; Renaudin & Lartigue, 2005). Characterizing the replication and stability regions of pSci2 proved the S. citri plasmids and their shuttle derivatives to hold considerable advantages for the development of new vectors: (i) they transform the various S. citri strains tested at relatively high frequencies (10–4–10–3 transformants c.f.u.–1 µg–1), (ii) the doubling times of the transformants are not significantly affected as compared with the wild-type, (iii) according to the demand they are either stably maintained or not, depending on the presence of soj, and never integrate into the chromosome, and (iv) their host range is not restricted to S. citri. Indeed, they were shown to replicate in S. phoeniceum P40 and S. kunkelii E275, in which expression of the foreign protein P32-HA was demonstrated. To our knowledge this is the first report of transformation and expression of foreign genes in plant-pathogenic mollicutes other than S. citri.
In pathogenic bacteria, plasmids are usually considered as being non-essential genetic elements that confer a selective advantage under specific conditions (Stewart et al., 2005; Vivian et al., 2001). Whether plasmids are required for S. citri survival in nature is unknown. Nevertheless, plasmid-encoded determinants have been tentatively associated with insect transmission (Berg et al., 2001; Berho et al., 2006a; Killiny et al., 2006). In S. citri GII3, 10 copies of each plasmid co-exist within the same cell. Transformation of S. citri 44 by extrachromosomal DNA from S. citri GII3 also revealed that many transformants contained more than one plasmid (Berho et al., 2006a). Therefore, the compatibility of the S. citri plasmids would permit the design of experiments in which two distinct plasmids must be stably maintained in the same cell. Conversely, from the preliminary observation that transformation of S. citri GII3 by selectable pSci2 derivatives led to rapid loss of the wild-type pSci2, incompatibility of identical replication regions would provide a means to displace natural plasmids by their mutated/deleted derivatives and hence to investigate the biological function of plasmid-encoded determinants.
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ACKNOWLEDGEMENTS |
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Edited by: I. K. Toth
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REFERENCES |
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Received 11 April 2008;
revised 12 June 2008;
accepted 16 June 2008.
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), repeated sequences (
), iteron-like sequences (small white arrows), and an AT-rich region (|||||) are indicated in the minimum replicon pFL7 only. A, AclI; B, BglII; E, EcoRI; EV, EcoRV; H, HincII; Ha, HaeIII; M, MluI; N, NsiI; P, PacI; Ps, PstI; S, SmlI; nd, not done. + and – indicate the ability or inability to replicate or to be stably maintained in the absence of selection pressure. * indicates truncated CDS of pSci2.
