The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome

Université Victor Ségalen Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, BP 81, F-33883 Villenave d'Ornon, France.
BMC Genomics (Impact Factor: 3.99). 02/2008; 9(1):195. DOI: 10.1186/1471-2164-9-195
Source: PubMed


Spiroplama citri, the causal agent of citrus stubborn disease, is a bacterium of the class Mollicutes and is transmitted by phloem-feeding leafhopper vectors. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of S. citri strain GII3-3X is currently being deciphered.
Assembling 20,000 sequencing reads generated seven circular contigs, none of which fit the 1.8 Mb chromosome map or carried chromosomal markers. These contigs correspond to seven plasmids: pSci1 to pSci6, with sizes ranging from 12.9 to 35.3 kbp and pSciA of 7.8 kbp. Plasmids pSci were detected as multiple copies in strain GII3-3X. Plasmid copy numbers of pSci1-6, as deduced from sequencing coverage, were estimated at 10 to 14 copies per spiroplasma cell, representing 1.6 Mb of extrachromosomal DNA. Genes encoding proteins of the TrsE-TraE, Mob, TraD-TraG, and Soj-ParA protein families were predicted in most of the pSci sequences, in addition to members of 14 protein families of unknown function. Plasmid pSci6 encodes protein P32, a marker of insect transmissibility. Plasmids pSci1-5 code for eight different S. citri adhesion-related proteins (ScARPs) that are homologous to the previously described protein P89 and the S. kunkelii SkARP1. Conserved signal peptides and C-terminal transmembrane alpha helices were predicted in all ScARPs. The predicted surface-exposed N-terminal region possesses the following elements: (i) 6 to 8 repeats of 39 to 42 amino acids each (sarpin repeats), (ii) a central conserved region of 330 amino acids followed by (iii) a more variable domain of about 110 amino acids. The C-terminus, predicted to be cytoplasmic, consists of a 27 amino acid stretch enriched in arginine and lysine (KR) and an optional 23 amino acid stretch enriched in lysine, aspartate and glutamate (KDE). Plasmids pSci mainly present a linear increase of cumulative GC skew except in regions presenting conserved hairpin structures.
The genome of S. citri GII3-3X is characterized by abundant extrachromosomal elements. The pSci plasmids could not only be vertically inherited but also horizontally transmitted, as they encode proteins usually involved in DNA element partitioning and cell to cell DNA transfer. Because plasmids pSci1-5 encode surface proteins of the ScARP family and pSci6 was recently shown to confer insect transmissibility, diversity and abundance of S. citri plasmids may essentially aid the rapid adaptation of S. citri to more efficient transmission by different insect vectors and to various plant hosts.

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    • "Nonetheless, our conservative approach in assembly and annotation mainly removed viral proteins and short hypothetical proteins, thus was not expected to introduce biases in the downstream metabolic analysis. Intriguingly, in contrast to the abundance of plasmids in S. melliferum KC3 [19] and S. citri GII3-3X [18,20], we did not recover any plasmid-like contig in our draft assembly of S. melliferum IPMB4A genome (Table 1). This lack of plasmid-like contig is consistent with the negative result from our plasmid extraction tests. "
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    ABSTRACT: Background The genus Spiroplasma contains a group of helical, motile, and wall-less bacteria in the class Mollicutes. Similar to other members of this class, such as the animal-pathogenic Mycoplasma and the plant-pathogenic ‘Candidatus Phytoplasma’, all characterized Spiroplasma species were found to be associated with eukaryotic hosts. While most of the Spiroplasma species appeared to be harmless commensals of insects, a small number of species have evolved pathogenicity toward various arthropods and plants. In this study, we isolated a novel strain of honeybee-associated S. melliferum and investigated its genetic composition and evolutionary history by whole-genome shotgun sequencing and comparative analysis with other Mollicutes genomes. Results The whole-genome shotgun sequencing of S. melliferum IPMB4A produced a draft assembly that was ~1.1 Mb in size and covered ~80% of the chromosome. Similar to other Spiroplasma genomes that have been studied to date, we found that this genome contains abundant repetitive sequences that originated from plectrovirus insertions. These phage fragments represented a major obstacle in obtaining a complete genome sequence of Spiroplasma with the current sequencing technology. Comparative analysis of S. melliferum IPMB4A with other Spiroplasma genomes revealed that these phages may have facilitated extensive genome rearrangements in these bacteria and contributed to horizontal gene transfers that led to species-specific adaptation to different eukaryotic hosts. In addition, comparison of gene content with other Mollicutes suggested that the common ancestor of the SEM (Spiroplasma, Entomoplasma, and Mycoplasma) clade may have had a relatively large genome and flexible metabolic capacity; the extremely reduced genomes of present day Mycoplasma and ‘Candidatus Phytoplasma’ species are likely to be the result of independent gene losses in these lineages. Conclusions The findings in this study highlighted the significance of phage insertions and horizontal gene transfer in the evolution of bacterial genomes and acquisition of pathogenicity. Furthermore, the inclusion of Spiroplasma in comparative analysis has improved our understanding of genome evolution in Mollicutes. Future improvements in the taxon sampling of available genome sequences in this group are required to provide further insights into the evolution of these important pathogens of humans, animals, and plants.
    BMC Genomics 01/2013; 14(1):22. DOI:10.1186/1471-2164-14-22 · 3.99 Impact Factor
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    • "The DNA fragment (912 bp long) corresponding to the ScARP3d repeat region (aa 24–328) was PCR amplified from the S. citri GII3 plasmid pSci2 [14] with primers Rep3dXbaF (5′-ATG CAT TCT AGA AAC ACA CCC GCA ACA CGC ACC-3′) and Rep3dBamR (5′-TAC TAG GAT CCT TAA ACT AAG TTA AAC TTA CTT TGT G-3′); the introduced restriction sites are in bold. The expected 912-bp DNA fragment was digested by XbaI and BamHI and ligated to the pET28a(+) vector (Novagen) digested by NheI+BamHI. "
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    ABSTRACT: Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.
    PLoS ONE 10/2012; 7(10):e48606. DOI:10.1371/journal.pone.0048606 · 3.23 Impact Factor
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    • "We designed the primer pair Scif⁄Scir based on the pE gene of pSci plasmids (Breton et al. 2008). Seven circular multicopied plasmids have been reported in S. citri (Saillard et al. 2008), and CDS pE region is common to all six of the pSci plasmids (Breton et al. 2008). From 43 symptomatic plants examined in this research, 79% were positive by primer pair pScif⁄pScir, while nested PCR of the spiralin gene based on primer pairs D⁄D¢ (Foissac et al. 1996) followed by F1⁄R1 (Khanchezar et al. 2010) in the second round resulted in detection of only 36.6% infection in symptomatic samples. "
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    ABSTRACT: Spiroplasma citri was associated with a disease of safflower characterized by stunting, yellowing, phloem discoloration and local or general necrosis in the Fars province of Iran. It was identified by ELISA using a locally produced polyclonal antiserum, by PCR with specific primers and isolation in culture medium. The 16S rDNA restriction fragment length polymorphism patterns of safflower isolates were identical with those of the other S. citri isolates. A known isolate of S. citri from periwinkle induced stunting, yellowing, phloem discoloration and wilting in safflower seedlings when transmitted by dodder under greenhouse conditions. A primer pair designed on the basis of S. citri plasmid was more sensitive than those based on spiralin gene or 16S rDNA for the detection of S. citri. Based on the sequence of the spiralin gene, S. citri isolates from safflower as well as other Iranian isolates were variable and grouped into two genetic clusters with 91.9–92.9% identity between groups. This is the first report of association of S. citri with a safflower disease.
    Journal of Phytopathology 08/2012; 160:331-336. DOI:10.1111/j.1439-0434.2012.01896.x · 0.82 Impact Factor
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