BACKGROUND
the phytopathogenic spiroplasmas and phytoplasmas, which cause serious diseases of economically important plant species worldwide , are wall-less prokaryotes phylogenetically related to gram-positive eubacteria with low g+c content  <cit> .
they are transmitted in nature by phloem-feeding insects, predominantly leafhoppers, in a propagative manner  <cit> .
even though there are close to forty recognized spiroplasma species, only three plant pathogenic spiroplasmas have been identified and characterized to date.
s. citri  is the causative agent of stubborn disease of citrus and brittle root disease of horseradish; s. kunkelii  is the etiological agent of corn stunt; and s. phoeniceum  <cit>  causes periwinkle yellows.
unlike phytoplasmas, spiroplasmas can be cultured in vitro.
therefore, the relationships between s. citri and its insect vectors, the beet leafhopper, circulifer tenellus, and the related species, c. haematoceps  <cit> , have been investigated extensively, serving as models for investigating the molecular aspects of mollicute-vector interactions.
spiroplasma binding to insect host and non-host cells, both in tissue-culture and within the intact insect, has been reported .
the loss and restoration of the ability of s. citri to adhere to tissue-cultured c. tenellus cells was associated with degradation and restoration of p <dig> , a spiroplasma membrane protein  <cit> .
due to the possible direct involvement of sarp <dig> in the spiroplasma-leafhopper interaction, it was hypothesized that sarp <dig> is an adhesin.
later, berg et al  <cit>  reported cloning and characterization of arp <dig>  the gene encoding sarp <dig>  from s. citri br3-t. they also reported that mature sarp <dig> protein contains a novel domain at the n-terminus, called "sarpin", made of six repeats of 39– <dig> amino acids each.
s. citri harbors several extrachromosomal dnas with unique restriction patterns .
s. citri lines, derived from a clone, and sister clones of the same lines showed differences in their extrachromosomal dnas  <cit> .
in addition to known plasmids, there are replicative forms  of several viruses and other uncharacterized circular extrachromosomal dnas in s. citri  <cit> .
plasmids have also been noted in strains of s. kunkelii  <cit> .
recently, davis and colleagues  <cit>  reported the complete sequence of the s. kunkelii cr2-3x plasmid psku <dig>  which encodes a homolog of sarp <dig>  sk-arp <dig>  in the present study, we isolated and characterized a related indigenous plasmid, designated pbjs-original , from s. citri br3-3x.
this is a report of the discovery, distribution and characterization of that plasmid.
among other genes, pbjs-o contains arp <dig>  the significance of the discovery of pbjs-o in relation to our current understanding of the s. citri-leafhopper interactions and potential genetic manipulations in mollicutes is discussed.
implications for the evolution of both pbjs-o and psku <dig> are also presented.
RESULTS
detection and analysis of arp2
sarp <dig> has been characterized previously and the gene encoding it, arp <dig>  has been cloned and sequenced  from s. citri br3-t  <cit> .
in the process, an rsai restriction fragment was cloned and sequenced from br3-t genomic dna; the alignment of this fragment with aj <dig> revealed 92% similarity in the 3'  <dig> nucleotides of the former sequence.
however in the 5'  <dig> bases of the total  <dig> bp, upstream from position  <dig> in aj <dig>  the new fragment was not similar to the known sequence.
we designated this gene, which resembles but is not identical to arp <dig>  as arp <dig> and its putative protein product as sarp <dig>  as also noted by bai et al.
<cit> , the s. kunkelii cr2-3x genome contains two sequences similar to those of s. citri br3-t arp genes.
the predicted protein, sk-arp <dig> , encoded by the first sequence, sk-arp <dig>  contains seven rather than six sarpin repeats and has c-terminal domains resembling those of sarp <dig>  <cit> .
the second sequence encodes a putative protein whose c-terminus is homologous to that of sarp <dig>  but has an unrelated n-terminus.
this protein is designated sk-arp <dig>  and the corresponding gene is named sk-arp <dig>  sarp <dig> has sequence similarity with known adhesins.
fleury et al.
<cit>  have shown that the predicted amino acid sequence of p <dig>  a mycoplasma agalactiae cytadhesin, is similar not only to that of sarp <dig> but also to the one of p <dig>  an adhesin of m. hominis.
isolation and distribution of spiroplasma extrachromosomal dna
we isolated extrachromosomal dna from s. citri br3-3x to test the hypothesis that this dna contains an arp-like gene as in s. kunkelii.
restriction of the dna with single enzymes, including bglii and ndei, converted a dna migrating with  <dig> kb into a fragment migrating close to  <dig> kb .
these results were consistent with the presence of a single major plasmid.
we designated the plasmid pbjs-o.
by nucleotide sequencing, we determined that the actual size of the plasmid was  <dig>  bp and deposited the sequence in the embl nucleotide sequence database
to test the conservation of pbjs-o in s. citri strains derived from s. citri br <dig>  plasmid preparations from s. citri br3-3x and from br3-g, br3-t, br3-m and br3-p, lines derived from br3-3x, were probed with a dna fragment derived from arp <dig> .
all hybridized with the probe, producing two or more bands.
to test the conservation of pbjs-o in other s. citri strains, other plant-associated spiroplasmas and the closest relative of s. citri, s. melliferum  <cit> , the plasmids of s. kunkelii cr2-3x, s. melliferum, s. citri strains r8a <dig>  asp- <dig> and beni mellal, s. floricola and s. phoeniceum also were probed with the arp1-derived probe .
only s. kunkelii cr2-3x and s. melliferum reacted in the hybridization.
however, when the same plasmids were probed with the whole pbjs-o plasmid as a probe , all the sample preparations, except those from s. citri beni mellal, s. floricola and s. phoeniceum, hybridized with the probe.
all of the above southern hybridization experiments revealed multiple reactive species in the plasmid preparations and the hybridization patterns of ecori-digested and undigested plasmid samples were very similar to each other.
for comparison, the blots included ecori-digested chromosomal dna of s. citri br3-3x.
a single hybridization signal distinct from those of plasmid preparations was observed .
*s. citri, s. kunkelii, s. phoeniceum and s. melliferum belong to serogroup i, whereas s. floricola belongs to serogroup iii.
the biological features of the spiroplasmas, except for s. phoeniceum, are taken from references  <dig> and  <dig>
**only experimental transmission to the plant host is known for this spiroplasma.
it is unclear whether it can be naturally transmitted by leafhoppers.
arp <dig> and arp <dig> locations in s. citri br3-3x
the southern blot hybridization results suggest that arp-related sequences are present on both a plasmid and the chromosome.
arp <dig> and arp <dig> from br3-t are nearly identical over a considerable portion of their nucleotide sequence.
hence, using a probe containing this conserved region should detect both genes.
nevertheless, arp <dig> and arp <dig> differ at several positions in those regions.
to determine whether the br3-3x plasmid and chromosomal sequences represented arp <dig> or arp <dig> genes, we determined parts of the sequences of br3-3x plasmid and chromosomal dnas by direct sequencing and by sequencing amplified pcr products.
comparison of the br3-3x arp sequences with those of br3-t revealed that the br3-3x arp <dig> sequence had diverged more from the other three sequences than the latter had from each other .
at positions where the two br3-t genes differed from one another, the chromosomal br3-3x sequence had arp <dig> residues in  <dig> positions and arp <dig> residues in only  <dig> positions .
conversely, at arp1- and arp2-specific positions, the br3-3x plasmid dna had no arp <dig> residues and  <dig> arp <dig> residues.
further, at all  <dig> positions at which chromosomal and plasmid sequences differed, the br3-3x plasmid and arp <dig> nucleotides were identical.
hence, we conclude that, in s. citri br3-3x, the arp <dig> gene resides on a plasmid and that the arp <dig> gene most likely resides on the chromosome.
the newly determined arp <dig> sequences from br3-3x and br3-t were deposited in the embl nucleotide sequence database .
complete pbjs-o sequencing and analysis
the  <dig> bp sequence  originally cloned and characterized from s. citri br3-t  <cit>  contains a partial orf soj, followed by orf <dig>  p <dig>  and another partial orf, orf <dig>  aj <dig> was used to design primers and initiate primer walking to determine the complete pbjs-o plasmid sequence and allow its characterization.
during sequencing, a segment  of the assembled sequence proved particularly difficult to sequence.
it contained three of the six oligopurine/oligopyrimidine tracts of  <dig> or more bp in the entire plasmid sequence.
that the sequence of the tracts was consistent with triple-helix formation suggests that this region of the plasmid may readily form triple-helical structures interfering with sequencing.
the total plasmid sequence is  <dig>  bp in length and contains ten predicted orfs , of which orf <dig>  has no homologs, and orf <dig> and orf <dig> appear to have distant relatives .
of the ten, six putative pbjs-o-orfs have homologs in psku <dig>  the recently characterized s. kunkelii cr2-3x plasmid  <cit> : arp <dig> , orf <dig> , trae , orf <dig> , mob  and orf <dig> .
predicted products of trae and mob are similar to proteins involved in conjugative dna transfer in other bacterial genera.
in the regions where the plasmid sequence was available from both br3-3x and br3-t, pbjs-t  sequence had a  <dig>  kb deletion relative to pbjs-o, bringing the orf <dig> gene close to arp <dig> and trae.
in br3-3x, however, arp <dig> and orf <dig> are separated by  <dig> bp.
the nucleotide sequence variations between pbjs-o and pbjs-t were found to be clustered.
two regions of enhanced variation were observed over a  <dig> bp stretch in the orf2-arp <dig> intergenic region .
in a comparable stretch from position  <dig> to  <dig> in the arp1-orf <dig> intergenic region, a single stretch of dissimilarity was found.
* aj <dig>  the sequence originally characterized from s. citri br3-t, is the source of these hits.
algorithm tmhmm v.  <dig>  was used to predict the locations of transmembrane helices and intervening loops in the putative products of trae , mob and orf <dig>  although the trae polypeptide was predicted to contain three transmembrane helices, the third helix was predicted at a lower probability than were the other two.
assuming the presence of three transmembrane helices, the protein was predicted to have the n-terminal region  in the cytosol and almost all of the rest of the protein extracellular.
plasmid psku <dig> from s. kunkelii cr2-3x encodes the s. kunkelii homolog of sarp <dig>  sk-arp <dig>  in addition to sk-arp <dig>  psku <dig> contains  <dig> orfs.
the psku146-orfs having counterparts on pbjs-o were listed above.
however, although both plasmids contain genes encoding the para-like protein, soj, sequences surrounding those genes are more distant from one another than are sequences in any other regions.
further, unlike psku <dig>  pbjs-o lacks the conserved orit region characteristic of the incp group of bacterial plasmids.
also, we were unable to identify a region in pbjs-o resembling a known plasmid origin of transfer.
discussion
in the present study we report isolation, distribution and structural characterization of pbjs-o, an indigenous s. citri br3-3x plasmid.
we also present evidence that pbjs-o harbors arp <dig>  the gene encoding sarp <dig>  and describe the presence on the br3-3x chromosome of arp <dig>  an s. citri homolog of arp <dig>  finally, the sequences of pbjs-o, pbjs-t and the s. kunkelii cr2-3x plasmid, psku <dig>  in relation to plasmid evolution are discussed.
conservation of arp and pbjs-o sequences in spiroplasma
in southern hybridizations, the similarity in the hybridization patterns of ecori digested versus undigested pbjs-o preparations, despite the presence of two gaattc recognition sequences, may be due to an adenine methylation system in s. citri.
restriction site modification in s. citri has been reported elsewhere.
rascoe et al.
<cit>  detected multiple bands of s. citri extrachromosomal dna by southern blotting, which they attributed to incomplete restriction due to variable restriction site modification in the dna, and ye et al.
<cit>  reported protection of an ecori site in the s. citri 16s rdna.
moreover, differential methylation of restriction sites in the rf of the spiroplasma virus, svts <dig>  allowed sha et al.
<cit>  to clone the full-length dna.
s. citri br3-3x showed probe-reactive sequences in both the chromosomal and extrachromosomal dna fractions.
however, that the patterns of hybridization of the two fractions differed significantly from each other demonstrates that the two fractions of br3-3x dna were not appreciably cross-contaminated.
sequence analyses of dna from the two fractions showed that, in br3-3x, arp <dig> resides on pbjs-o and arp <dig> on the chromosome.
hybridization of s. citri asp- <dig> and r8a <dig> plasmid preparations with the pbjs-o probe , but not with the arp <dig> probe , indicates that each of these two strains contained a plasmid related to pbjs-o, which differed from pbjs-o in lacking arp <dig>  although s. citri asp- <dig> and r8a <dig> were originally derived from the same parent strain, both have undergone extensive cultivation in vitro since their first isolation, which may have contributed to the differences between their plasmids and pbjs-o.
moreover, the differences in the maintenance regimes of the various spiroplasmas tested may have contributed to the evolution of their plasmids.
in this paper we could not correlate pbjs-o and pbjs-o like sequences with either transmissibility or phytopathogenicity of the spiroplasmas tested.
however, it is still hypothesized that sarp <dig> is involved in s. citri transmission by the insect vector.
frequent chromosomal rearrangements such as inversions and deletions, leading to genome instability, have been reported in spiroplasmas, such as in the lines derived from s. citri strain br <dig> .
in the present study, we detected a  <dig>  kbp deletion in pbjs-t relative to pbjs-o.
unlike br3-3x, which was stored frozen, s. citri br3-t was maintained for several years in turnip plants via transmission by the natural insect vector c. tenellus, possibly leading to the sequence differences between pbjs-t and pbjs-o.
a recombinational chromosomal rearrangement is indicated by the 5'-sequence differences between arp <dig> and arp <dig> reported above.
recombination likely also played a role in the generation of pbjs-o like plasmids.
the gene organization on pbjs-o is similar to that of the recently characterized incp-like s. kunkelii cr2-3x plasmid, psku <dig>  yet, the two plasmids have substantially different sequences in the region including the soj-like gene in both plasmids and the incp orit sequence in psku <dig>  highly similar sequences in the remainder of the two plasmids suggest that recombination events have occurred during the generation of one or both plasmids.
phage sequences have been implicated in many recombination events in spiroplasmas.
only a short region with similarity to a phage gene was found in pbjs-o.
however, the observation of strong stops to sequencing reactions in the region of nucleotides  <dig> to  <dig> is reminiscent of a strong stop encountered during the sequencing of the svts <dig> phage  <cit> .
this strong stop was attributed to potential secondary structure putatively involved in phage packaging.
it is, thus, possible that pbjs-o has some phage-like properties.
pbjs-o genes
as mentioned above, orf <dig>  encoding sarp <dig>  and adjacent orfs , had been cloned and characterized from s. citri br3-t  <cit> .
orf <dig> was flanked downstream by a partial orf  having no known homologs.
upstream, orf <dig> was flanked by orf <dig>  encoding a hypothetical protein with no similarity to any known protein, and orf <dig>  a partial orf encoding a putative homolog of a para-like protein, soj, which oscillates from pole to pole  <cit>  and is important for chromosome partitioning in bacillus subtilis  <cit> .
in this study, the putative protein product of orf <dig> was predicted to contain eight transmembrane helices.
due to a  <dig>  kb deletion in the derivation of pbjs-t, orf <dig> is possibly a part of the same transcription unit as arp <dig> and trae in this strain.
in br3-3x, arp <dig> and orf <dig> are separated by  <dig> bp, suggesting that they are transcribed separately.
consistent with different translational constraints on this region in br3-t and br3-3x, this region contains a large proportion of the differences between the lines.
the translation start site of trae was predicted to be ten nucleotides upstream of the orf <dig> translation stop site.
consistent with the observation of bai et al.
<cit> , putative products of the other pbjs-o orfs, trae  <cit>  and mob  <cit> , are homologous to proteins that are components of the bacterial type iv secretion system involved in conjugative dna transfer.
members of the trae family of proteins are thought to form pili that, in addition to conjugation, are involved in processes like virus infection and biofilm formation.
bai et al.
<cit>  reported the presence of three conserved transmembrane helices in four trae homologs that they characterized from s. kunkelii m <dig>  ozbek et al.
<cit> , in their transmission electron micrographs, reported the presence of structures resembling fimbriae and pili in s. kunkelii and bai et al.
<cit>  considered whether the structures may be involved in conjugation.
bové  <cit>  reported that rod-shaped spiroplasma viruses, approximately 230– <dig> by 10– <dig> μm in size, can also be surface-associated.
because they can attach perpendicularly to the host membrane at their tips , they might resemble the structures reported as pili/fimbriae.
in the putative trae homolog reported here, unlike its s. kunkelii counterpart, two transmembrane helices were predicted at high probability and a third one at moderate probability.
should the third not actually be a transmembrane helix, the atp binding site would be located intracellularly rather than extracellularly.
pbjs-o gene organization and evolution
unlike psku <dig>  pbjs-o was found to lack the conserved orit region characteristic of the incp group of plasmids.
we were also unable to identify a region in pbjs-o resembling any other known plasmid origins of transfer, suggesting that pbjs-o belongs to a hitherto unidentified group of plasmids.
horizontal transfer of a promiscuous plasmid, such as an incp plasmid, between phylogenetically related and unrelated bacteria would help the hosts quickly adapt to different niches  <cit> .
it is possible that an incp-like plasmid was acquired by the ancestor of s. citri and s. kunkelii.
the plasmid may have co-evolved with the host chromosomes after the divergence of the two species, leading to the emergence of pbjs-o and psku <dig>  respectively, and to the adaptation of the pathogens to phylogenetically distinct leafhopper vectors and plant hosts.
future directions
molecular genetic tools such as cloning and transposon-mediated mutagenesis are available for the study of mollicutes  <cit> .
cloned genes were expressed in s. citri gii- <dig> using artificial plasmids based on the s. citri chromosomal oric .
however, those plasmids tend to integrate into the s. citri chromosome.
when pcj <dig>  a derivative of the oric plasmid pbot <dig>  containing an internal fragment of the gene scm <dig> , was transformed into s. citri gii- <dig> cells it successfully integrated into the host chromosome by homologous recombination and disrupted scm <dig>  resulting in non-motile s. citri gii- <dig> mutants  <cit> .
however, attempts to use pbot <dig> in s. citri br3-3x have been unsuccessful , possibly due to the incompatibility of the plasmid with the host.
the indigenous s. citri br-3x plasmid, pbjs-o, will help us develop a better vector for genetic manipulation not only in s. citri br3-3x but also in other spiroplasmas.
CONCLUSIONS
we have shown that the s. citri br3-3x plasmid, pbjs-o, encodes the putative adhesin sarp <dig>  this is the first report of an s. citri plasmid encoding a putative adhesin.
we have further shown that the arp1-like gene, arp <dig>  resides on the br3-3x chromosome.
the indigenous s. citri br3-3x plasmid, pbjs-o, will be useful for the development of a better vector for genetic manipulation not only in s. citri br3-3x but also in other spiroplasmas.
our data also suggest that pbjs-o is a novel s. citri plasmid that does not belong to any known plasmid incompatibility group.
the differences between pbjs-o and psku <dig> suggest that recombination has contributed to the divergence of the two plasmids.

 the phytopathogenic spiroplasmas and phytoplasmas, which cause serious diseases of economically important plant species worldwide, are wall-less prokaryotes phylogenetically related to gram-positive eubacteria with low g+c content. they are transmitted in nature by phloem-feeding insects, predominantly leafhoppers, in a propagative manner. even though there are close to forty recognized spiroplasma species, only three plant pathogenic spiroplasmas have been identified and characterized to date. s. citri is the causative agent of stubborn disease of citrus and brittle root disease of horseradish; s. kunkelii is the etiological agent of corn stunt; and s. phoeniceum causes periwinkle yellows. unlike phytoplasmas, spiroplasmas can be cultured in vitro. therefore, the relationships between s. citri and its insect vectors, the beet leafhopper, circulifer tenellus, and the related species, c. haematoceps, have been investigated extensively, serving as models for investigating the molecular aspects of mollicute-vector interactions. spiroplasma binding to insect host and non-host cells, both in tissue-culture and within the intact insect, has been reported. the loss and restoration of the ability of s. citri to adhere to tissue-cultured c. tenellus cells was associated with degradation and restoration of p, a spiroplasma membrane protein. due to the possible direct involvement of sarp in the spiroplasma-leafhopper interaction, it was hypothesized that sarp is an adhesin. later, berg et al reported cloning and characterization of arp the gene encoding sarp from s. citri br3-t. they also reported that mature sarp protein contains a novel domain at the n-terminus, called "sarpin", made of six repeats of 39– amino acids each. s. citri harbors several extrachromosomal dnas with unique restriction patterns. s. citri lines, derived from a clone, and sister clones of the same lines showed differences in their extrachromosomal dnas. in addition to known plasmids, there are replicative forms of several viruses and other uncharacterized circular extrachromosomal dnas in s. citri. plasmids have also been noted in strains of s. kunkelii. recently, davis and colleagues reported the complete sequence of the s. kunkelii cr2-3x plasmid psku which encodes a homolog of sarp sk-arp in the present study, we isolated and characterized a related indigenous plasmid, designated pbjs-original, from s. citri br3-3x. this is a report of the discovery, distribution and characterization of that plasmid. among other genes, pbjs-o contains arp the significance of the discovery of pbjs-o in relation to our current understanding of the s. citri-leafhopper interactions and potential genetic manipulations in mollicutes is discussed. implications for the evolution of both pbjs-o and psku are also presented.  detection and analysis of arp2 sarp has been characterized previously and the gene encoding it, arp has been cloned and sequenced from s. citri br3-t. in the process, an rsai restriction fragment was cloned and sequenced from br3-t genomic dna; the alignment of this fragment with aj revealed 92% similarity in the 3' nucleotides of the former sequence. however in the 5' bases of the total bp, upstream from position in aj the new fragment was not similar to the known sequence. we designated this gene, which resembles but is not identical to arp as arp and its putative protein product as sarp as also noted by bai et al., the s. kunkelii cr2-3x genome contains two sequences similar to those of s. citri br3-t arp genes. the predicted protein, sk-arp, encoded by the first sequence, sk-arp contains seven rather than six sarpin repeats and has c-terminal domains resembling those of sarp. the second sequence encodes a putative protein whose c-terminus is homologous to that of sarp but has an unrelated n-terminus. this protein is designated sk-arp and the corresponding gene is named sk-arp sarp has sequence similarity with known adhesins. fleury et al. have shown that the predicted amino acid sequence of p a mycoplasma agalactiae cytadhesin, is similar not only to that of sarp but also to the one of p an adhesin of m. hominis. isolation and distribution of spiroplasma extrachromosomal dna we isolated extrachromosomal dna from s. citri br3-3x to test the hypothesis that this dna contains an arp-like gene as in s. kunkelii. restriction of the dna with single enzymes, including bglii and ndei, converted a dna migrating with kb into a fragment migrating close to kb. these results were consistent with the presence of a single major plasmid. we designated the plasmid pbjs-o. by nucleotide sequencing, we determined that the actual size of the plasmid was bp and deposited the sequence in the embl nucleotide sequence database to test the conservation of pbjs-o in s. citri strains derived from s. citri br plasmid preparations from s. citri br3-3x and from br3-g, br3-t, br3-m and br3-p, lines derived from br3-3x, were probed with a dna fragment derived from arp. all hybridized with the probe, producing two or more bands. to test the conservation of pbjs-o in other s. citri strains, other plant-associated spiroplasmas and the closest relative of s. citri, s. melliferum, the plasmids of s. kunkelii cr2-3x, s. melliferum, s. citri strains r8a asp- and beni mellal, s. floricola and s. phoeniceum also were probed with the arp1-derived probe. only s. kunkelii cr2-3x and s. melliferum reacted in the hybridization. however, when the same plasmids were probed with the whole pbjs-o plasmid as a probe, all the sample preparations, except those from s. citri beni mellal, s. floricola and s. phoeniceum, hybridized with the probe. all of the above southern hybridization experiments revealed multiple reactive species in the plasmid preparations and the hybridization patterns of ecori-digested and undigested plasmid samples were very similar to each other. for comparison, the blots included ecori-digested chromosomal dna of s. citri br3-3x. a single hybridization signal distinct from those of plasmid preparations was observed. *s. citri, s. kunkelii, s. phoeniceum and s. melliferum belong to serogroup i, whereas s. floricola belongs to serogroup iii. the biological features of the spiroplasmas, except for s. phoeniceum, are taken from references and **only experimental transmission to the plant host is known for this spiroplasma. it is unclear whether it can be naturally transmitted by leafhoppers. arp and arp locations in s. citri br3-3x the southern blot hybridization results suggest that arp-related sequences are present on both a plasmid and the chromosome. arp and arp from br3-t are nearly identical over a considerable portion of their nucleotide sequence. hence, using a probe containing this conserved region should detect both genes. nevertheless, arp and arp differ at several positions in those regions. to determine whether the br3-3x plasmid and chromosomal sequences represented arp or arp genes, we determined parts of the sequences of br3-3x plasmid and chromosomal dnas by direct sequencing and by sequencing amplified pcr products. comparison of the br3-3x arp sequences with those of br3-t revealed that the br3-3x arp sequence had diverged more from the other three sequences than the latter had from each other. at positions where the two br3-t genes differed from one another, the chromosomal br3-3x sequence had arp residues in positions and arp residues in only positions. conversely, at arp1- and arp2-specific positions, the br3-3x plasmid dna had no arp residues and arp residues. further, at all positions at which chromosomal and plasmid sequences differed, the br3-3x plasmid and arp nucleotides were identical. hence, we conclude that, in s. citri br3-3x, the arp gene resides on a plasmid and that the arp gene most likely resides on the chromosome. the newly determined arp sequences from br3-3x and br3-t were deposited in the embl nucleotide sequence database. complete pbjs-o sequencing and analysis the bp sequence originally cloned and characterized from s. citri br3-t contains a partial orf soj, followed by orf p and another partial orf, orf aj was used to design primers and initiate primer walking to determine the complete pbjs-o plasmid sequence and allow its characterization. during sequencing, a segment of the assembled sequence proved particularly difficult to sequence. it contained three of the six oligopurine/oligopyrimidine tracts of or more bp in the entire plasmid sequence. that the sequence of the tracts was consistent with triple-helix formation suggests that this region of the plasmid may readily form triple-helical structures interfering with sequencing. the total plasmid sequence is bp in length and contains ten predicted orfs, of which orf has no homologs, and orf and orf appear to have distant relatives. of the ten, six putative pbjs-o-orfs have homologs in psku the recently characterized s. kunkelii cr2-3x plasmid: arp, orf, trae, orf, mob and orf. predicted products of trae and mob are similar to proteins involved in conjugative dna transfer in other bacterial genera. in the regions where the plasmid sequence was available from both br3-3x and br3-t, pbjs-t sequence had a kb deletion relative to pbjs-o, bringing the orf gene close to arp and trae. in br3-3x, however, arp and orf are separated by bp. the nucleotide sequence variations between pbjs-o and pbjs-t were found to be clustered. two regions of enhanced variation were observed over a bp stretch in the orf2-arp intergenic region. in a comparable stretch from position to in the arp1-orf intergenic region, a single stretch of dissimilarity was found. * aj the sequence originally characterized from s. citri br3-t, is the source of these hits. algorithm tmhmm v. was used to predict the locations of transmembrane helices and intervening loops in the putative products of trae, mob and orf although the trae polypeptide was predicted to contain three transmembrane helices, the third helix was predicted at a lower probability than were the other two. assuming the presence of three transmembrane helices, the protein was predicted to have the n-terminal region in the cytosol and almost all of the rest of the protein extracellular. plasmid psku from s. kunkelii cr2-3x encodes the s. kunkelii homolog of sarp sk-arp in addition to sk-arp psku contains orfs. the psku146-orfs having counterparts on pbjs-o were listed above. however, although both plasmids contain genes encoding the para-like protein, soj, sequences surrounding those genes are more distant from one another than are sequences in any other regions. further, unlike psku pbjs-o lacks the conserved orit region characteristic of the incp group of bacterial plasmids. also, we were unable to identify a region in pbjs-o resembling a known plasmid origin of transfer. discussion in the present study we report isolation, distribution and structural characterization of pbjs-o, an indigenous s. citri br3-3x plasmid. we also present evidence that pbjs-o harbors arp the gene encoding sarp and describe the presence on the br3-3x chromosome of arp an s. citri homolog of arp finally, the sequences of pbjs-o, pbjs-t and the s. kunkelii cr2-3x plasmid, psku in relation to plasmid evolution are discussed. conservation of arp and pbjs-o sequences in spiroplasma in southern hybridizations, the similarity in the hybridization patterns of ecori digested versus undigested pbjs-o preparations, despite the presence of two gaattc recognition sequences, may be due to an adenine methylation system in s. citri. restriction site modification in s. citri has been reported elsewhere. rascoe et al. detected multiple bands of s. citri extrachromosomal dna by southern blotting, which they attributed to incomplete restriction due to variable restriction site modification in the dna, and ye et al. reported protection of an ecori site in the s. citri 16s rdna. moreover, differential methylation of restriction sites in the rf of the spiroplasma virus, svts allowed sha et al. to clone the full-length dna. s. citri br3-3x showed probe-reactive sequences in both the chromosomal and extrachromosomal dna fractions. however, that the patterns of hybridization of the two fractions differed significantly from each other demonstrates that the two fractions of br3-3x dna were not appreciably cross-contaminated. sequence analyses of dna from the two fractions showed that, in br3-3x, arp resides on pbjs-o and arp on the chromosome. hybridization of s. citri asp- and r8a plasmid preparations with the pbjs-o probe, but not with the arp probe, indicates that each of these two strains contained a plasmid related to pbjs-o, which differed from pbjs-o in lacking arp although s. citri asp- and r8a were originally derived from the same parent strain, both have undergone extensive cultivation in vitro since their first isolation, which may have contributed to the differences between their plasmids and pbjs-o. moreover, the differences in the maintenance regimes of the various spiroplasmas tested may have contributed to the evolution of their plasmids. in this paper we could not correlate pbjs-o and pbjs-o like sequences with either transmissibility or phytopathogenicity of the spiroplasmas tested. however, it is still hypothesized that sarp is involved in s. citri transmission by the insect vector. frequent chromosomal rearrangements such as inversions and deletions, leading to genome instability, have been reported in spiroplasmas, such as in the lines derived from s. citri strain br. in the present study, we detected a kbp deletion in pbjs-t relative to pbjs-o. unlike br3-3x, which was stored frozen, s. citri br3-t was maintained for several years in turnip plants via transmission by the natural insect vector c. tenellus, possibly leading to the sequence differences between pbjs-t and pbjs-o. a recombinational chromosomal rearrangement is indicated by the 5'-sequence differences between arp and arp reported above. recombination likely also played a role in the generation of pbjs-o like plasmids. the gene organization on pbjs-o is similar to that of the recently characterized incp-like s. kunkelii cr2-3x plasmid, psku yet, the two plasmids have substantially different sequences in the region including the soj-like gene in both plasmids and the incp orit sequence in psku highly similar sequences in the remainder of the two plasmids suggest that recombination events have occurred during the generation of one or both plasmids. phage sequences have been implicated in many recombination events in spiroplasmas. only a short region with similarity to a phage gene was found in pbjs-o. however, the observation of strong stops to sequencing reactions in the region of nucleotides to is reminiscent of a strong stop encountered during the sequencing of the svts phage. this strong stop was attributed to potential secondary structure putatively involved in phage packaging. it is, thus, possible that pbjs-o has some phage-like properties. pbjs-o genes as mentioned above, orf encoding sarp and adjacent orfs, had been cloned and characterized from s. citri br3-t. orf was flanked downstream by a partial orf having no known homologs. upstream, orf was flanked by orf encoding a hypothetical protein with no similarity to any known protein, and orf a partial orf encoding a putative homolog of a para-like protein, soj, which oscillates from pole to pole and is important for chromosome partitioning in bacillus subtilis. in this study, the putative protein product of orf was predicted to contain eight transmembrane helices. due to a kb deletion in the derivation of pbjs-t, orf is possibly a part of the same transcription unit as arp and trae in this strain. in br3-3x, arp and orf are separated by bp, suggesting that they are transcribed separately. consistent with different translational constraints on this region in br3-t and br3-3x, this region contains a large proportion of the differences between the lines. the translation start site of trae was predicted to be ten nucleotides upstream of the orf translation stop site. consistent with the observation of bai et al., putative products of the other pbjs-o orfs, trae and mob, are homologous to proteins that are components of the bacterial type iv secretion system involved in conjugative dna transfer. members of the trae family of proteins are thought to form pili that, in addition to conjugation, are involved in processes like virus infection and biofilm formation. bai et al. reported the presence of three conserved transmembrane helices in four trae homologs that they characterized from s. kunkelii m ozbek et al., in their transmission electron micrographs, reported the presence of structures resembling fimbriae and pili in s. kunkelii and bai et al. considered whether the structures may be involved in conjugation. bové reported that rod-shaped spiroplasma viruses, approximately 230– by 10– μm in size, can also be surface-associated. because they can attach perpendicularly to the host membrane at their tips, they might resemble the structures reported as pili/fimbriae. in the putative trae homolog reported here, unlike its s. kunkelii counterpart, two transmembrane helices were predicted at high probability and a third one at moderate probability. should the third not actually be a transmembrane helix, the atp binding site would be located intracellularly rather than extracellularly. pbjs-o gene organization and evolution unlike psku pbjs-o was found to lack the conserved orit region characteristic of the incp group of plasmids. we were also unable to identify a region in pbjs-o resembling any other known plasmid origins of transfer, suggesting that pbjs-o belongs to a hitherto unidentified group of plasmids. horizontal transfer of a promiscuous plasmid, such as an incp plasmid, between phylogenetically related and unrelated bacteria would help the hosts quickly adapt to different niches. it is possible that an incp-like plasmid was acquired by the ancestor of s. citri and s. kunkelii. the plasmid may have co-evolved with the host chromosomes after the divergence of the two species, leading to the emergence of pbjs-o and psku respectively, and to the adaptation of the pathogens to phylogenetically distinct leafhopper vectors and plant hosts. future directions molecular genetic tools such as cloning and transposon-mediated mutagenesis are available for the study of mollicutes. cloned genes were expressed in s. citri gii- using artificial plasmids based on the s. citri chromosomal oric. however, those plasmids tend to integrate into the s. citri chromosome. when pcj a derivative of the oric plasmid pbot containing an internal fragment of the gene scm, was transformed into s. citri gii- cells it successfully integrated into the host chromosome by homologous recombination and disrupted scm resulting in non-motile s. citri gii- mutants. however, attempts to use pbot in s. citri br3-3x have been unsuccessful, possibly due to the incompatibility of the plasmid with the host. the indigenous s. citri br-3x plasmid, pbjs-o, will help us develop a better vector for genetic manipulation not only in s. citri br3-3x but also in other spiroplasmas.  we have shown that the s. citri br3-3x plasmid, pbjs-o, encodes the putative adhesin sarp this is the first report of an s. citri plasmid encoding a putative adhesin. we have further shown that the arp1-like gene, arp resides on the br3-3x chromosome. the indigenous s. citri br3-3x plasmid, pbjs-o, will be useful for the development of a better vector for genetic manipulation not only in s. citri br3-3x but also in other spiroplasmas. our data also suggest that pbjs-o is a novel s. citri plasmid that does not belong to any known plasmid incompatibility group. the differences between pbjs-o and psku suggest that recombination has contributed to the divergence of the two plasmids. 
the nucleotide sequence of pbjs-o was determined and compared to the sequences of a plasmid from br3-t, which is a multiply passaged leafhopper transmissible derivative of br3-3x, and to known plasmid sequences including that of psku in addition to arp the bp pbjs-o sequence putatively contains nine genes, recognized as open reading frames. finally, we also report the presence on the br3-3x chromosome of arp an s. citri homolog of arp that encodes the predicted protein sarp the c-terminal domain of sarp is homologous to that of sarp but its n-terminal domain is distinct. further, unlike psku pbjs-o lacks the conserved orit region characteristic of the incp group of bacterial plasmids. spiroplasma citri br3-3x and s. kunkelii cr2-3x cause serious diseases worldwide on citrus and maize species, respectively. the s. kunkelii cr2-3x plasmid, psku encodes a homolog of sarp sk-arp due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well. s. citri br3-3x harbors a plasmid, pbjs-original, that encodes the spiroplasma adhesion related protein, a protein implicated in binding of the pathogen to cells of its leafhopper vector, circulifer tenellus. southern blot hybridization of extrachromosomal dna from various s. citri strains and spiroplasma species to an arp-specific probe and a probe made from the entire plasmid dna of br3-3x revealed limited conservation of both sequences in the genus spiroplasma. our data suggest that pbjs is a novel s. citri plasmid that does not belong to any known plasmid incompatibility group. the differences between pbjs-o and psku suggest that one or more events of recombination have contributed to the divergence of the plasmids of the two sister spiroplasma species; the plasmid from s. citri itself has diverged slightly during the derivation of s. citri br3-t from br3-3x. we were unable to identify a region in pbjs-o resembling a known plasmid origin of transfer. in regions where sequence was available for the plasmid from both br3-3x and br3-t, the pbjs-t sequence had a kb deletion relative to its progenitor, pbjs-o. several pbjs-o orfs have homologs on psku however, the sequences flanking soj-like genes on both plasmids were found to be more distant from one another than sequences in any other region.
 spiroplasma citri br3-3x and s. kunkelii cr2-3x cause serious diseases worldwide on citrus and maize species, respectively. s. citri br3-3x harbors a plasmid, pbjs-original, that encodes the spiroplasma adhesion related protein, a protein implicated in binding of the pathogen to cells of its leafhopper vector, circulifer tenellus. the s. kunkelii cr2-3x plasmid, psku encodes a homolog of sarp sk-arp due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well.  the nucleotide sequence of pbjs-o was determined and compared to the sequences of a plasmid from br3-t, which is a multiply passaged leafhopper transmissible derivative of br3-3x, and to known plasmid sequences including that of psku in addition to arp the bp pbjs-o sequence putatively contains nine genes, recognized as open reading frames. several pbjs-o orfs have homologs on psku however, the sequences flanking soj-like genes on both plasmids were found to be more distant from one another than sequences in any other region. further, unlike psku pbjs-o lacks the conserved orit region characteristic of the incp group of bacterial plasmids. we were unable to identify a region in pbjs-o resembling a known plasmid origin of transfer. in regions where sequence was available for the plasmid from both br3-3x and br3-t, the pbjs-t sequence had a kb deletion relative to its progenitor, pbjs-o. southern blot hybridization of extrachromosomal dna from various s. citri strains and spiroplasma species to an arp-specific probe and a probe made from the entire plasmid dna of br3-3x revealed limited conservation of both sequences in the genus spiroplasma. finally, we also report the presence on the br3-3x chromosome of arp an s. citri homolog of arp that encodes the predicted protein sarp the c-terminal domain of sarp is homologous to that of sarp but its n-terminal domain is distinct.  our data suggest that pbjs is a novel s. citri plasmid that does not belong to any known plasmid incompatibility group. the differences between pbjs-o and psku suggest that one or more events of recombination have contributed to the divergence of the plasmids of the two sister spiroplasma species; the plasmid from s. citri itself has diverged slightly during the derivation of s. citri br3-t from br3-3x. our data also show that pbjs-o encodes the putative adhesin sarp the presence of trae and mob on pbjs-o suggests a role for the plasmid in spiroplasmal conjugation. 