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

<p>Abstract</p> <p>Background</p> <p><it>Spiroplama citri</it>, the causal agent of citrus stubborn disease, is a bacterium of the class <it>Mollicutes </it>and is transmitted by phloem-feeding leafhopper vectors. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of <it>S. citri </it>strain GII3-3X is currently being deciphered.</p> <p>Results</p> <p>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 <it>S. citri </it>adhesion-related proteins (ScARPs) that are homologous to the previously described protein P89 and the <it>S. kunkelii </it>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.</p> <p>Conclusion</p> <p>The genome of <it>S. citri </it>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 <it>S. citri </it>plasmids may essentially aid the rapid adaptation of <it>S. citri </it>to more efficient transmission by different insect vectors and to various plant hosts.</p

Gene Disruption through Homologous Recombination in Spiroplasma citri: an scm1-Disrupted Motility Mutant Is Pathogenic

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein

The adhesin VmpA of the “flavescence dorée” phytoplasma binds insect cells in a lectin-like manner

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