Sequence comparisons of plasmids pBJS-O of Spiroplasma citri and pSKU146 of S. kunkelii: implications for plasmid evolution

Background: 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 (pBJS-O), that encodes the spiroplasma adhesion related protein 1 (SARP1), a protein implicated in binding of the pathogen to cells of its leafhopper vector, Circulifer tenellus. The S. kunkelii CR2-3X plasmid, pSKU146, encodes a homolog of SARP1, SkARP1. Due to the close phylogenetic relationship of the two pathogens, we hypothesized that the two plasmids are closely related as well. Results: The nucleotide sequence of pBJS-O was determined and compared to the sequences of a plasmid from BR3-T (pBJS-T), which is a multiply passaged leafhopper transmissible derivative of BR3-3X, and to known plasmid sequences including that of pSKU146. In addition to arp1, the 13,374 bp pBJS-O sequence putatively contains nine genes, recognized as open reading frames (ORFs). Several pBJS-O ORFs have homologs on pSKU146. 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 pSKU146, 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 0.4 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 arp2, an S. citri homolog of arp1 that encodes the predicted protein SARP2. The C-terminal domain of SARP2 is homologous to that of SARP1, but its N-terminal domain is distinct. Conclusion: 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 pSKU146 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 SARP1. The presence of traE and mob on pBJS-O suggests a role for the plasmid in spiroplasmal conjugation.