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

Transmission de Spiroplasma citri par la cicadelle Circulifer haematoceps (implication de la protéine SC76)

Spiroplasma citri est l'agent causal de la maladie du "stubborn" des agrumes. Cette bactérie phytopathogène sans paroi de la classe des Molicutes est localisée exclusivement dans le phloème des plantes. S citri est transmis de plante à plante par des insectes vecteurs piqueurs-suceurs de sève élaborée, des cicadelles. Dans le but d'étudier les mécanismes régissant la transmission de S. citri par son ibsecte vecteur Circulifer haematoceps, des expériences de mutagenèse par transposition de la souche sauvage FII3 avec le Tn4001 ont été réalisées et le mutant G76 déficient dans sa transmission a sélectionné. Ce mutant est transmis à la plante par l'insecte de manière moins efficace que la souche sauvage entraînant ainsi un retard d'apparition des symptomes sur les plantes. Ceci, car il s'accumule en quantité moins importante dans les glandes salivaires de l'insecte. Le gène interrompu par le transposon code une lipoprotéine qui pourrait être une putative Solute Binding Protein d'un ABC transporteur de glucose. Cette protéine pourrait être impliquée dans la multiplication de S. citri dans les cellules des glandes salivaires des cicadelles ou bien dans l'attachement du spiroplasme à l'épithélium des glandes salivaires des insectes. Une lignée de la souche GII3, a également été étudiée. Cette lignée a été maintenue pendant une centaine de passages en milieu acellulaire. Ceci conduit à une forte diminution de la transmission de cette lignée aux plantes par l'insecte vecteur induisant un retard d'apparition des symptomes sur les plantes. Cette lignée possède une quantité de spiraline, protéine majeure de S. citri, plus faible par rapport à la souche sauvage FII3. Cette protéine pourrait donc être impliquée dans la transmission du spiroplasme par son insecte vecteur. La lignée GII.-CJ possède également de l'ADN extra-chromosmique qui code une protéine potentiellement impliquée dans la conjugaison bactérienne.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Spiroplasma Citri et son insecte vecteur Circulifer Haematoceps (interactions protéine marqueur de la transmission)

Des expériences de Far-western ont permis de préciser les interactions moléculaires entre le mollicute phytopathogène Spiroplasma citri et son vecteur la cicadelle Circulifer haematoceps. Deux glycoprotéines d'insectes de 50 et 60 kDa présentent une affinité pour la spiraline, lipoprotéine majeure de la membrane de S. citri. La comparaison des cartes peptidiques 2D de différentes souches de S. citri montre qu'une protéine de 32kDa (P32) est présente uniquement chez les souches transmissibles. Cette protéine identifiée par MALDI-TOF est composée de 238 acides aminés et ne présente aucune homologie avec des protéines connues. Le gène p32, localisé sur un grand plasmide, est présent uniquement chez les souches transmissibles. Cette protéine marqueur de la transmission peut jouer un rôle dans l'adhésion du spiroplasme aux cellules d'insectes.Far-western experiments allowed to study the specific molecular interactions between the phytopathogenic mollicute, Spiroplasma citri and its vector the leafhopper, Circulifer haematoceps. Two insect glycoproteins of 50 and 60 kDa showed affinity for the spiralin, the most abundant S. citri membrane lipoprotéine. The comparison between the 2-D peptide maps of transmissible and non transmissible strains showed that, a 32 kDa protein (P32) is present only in transmissible strains. This protein identified by MALDI-TOF is composed of 238 amino acids and has no homology with known proteins. The p32 gene, carried by a large plasmid is present only in transmissible strains. This protein could play a role in the adhesion of S. citri to insect cells.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Identification of a Spiroplasma citri hydrophilic protein associated with insect transmissibility

International audienceWith the aim of identifying Spiroplasma citri proteins involved in transmission by the leafhopper Circulifer haematoceps, protein maps of four transmissible and four non-transmissible strains were compared. Total cell lysates of strains were analysed by two-dimensional gel electrophoresis using commercially available immobilized pH gradients (IPGs) covering a pH range of 4–7. Approximately 530 protein spots were visualized by silver staining and the resulting protein spot patterns for the eight strains were found to be highly similar. However, comparison using PDQuest 2-D analysis software revealed two trains of protein spots that were present only in the four transmissible strains. Using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and a nearly complete S. citri protein database, established during the still-ongoing S. citri GII-3-3X genome project, the sequences of both proteins were deduced. One of these proteins was identified in the general databases as adhesion-related protein (P89) involved in the attachment of S. citri to gut cells of the insect vector. The second protein, with an apparent molecular mass of 32 kDa deduced from the electrophoretic mobility, could not be assigned to a known protein and was named P32. The P32-encoding gene (714 bp) was carried by a large plasmid of 35·3 kbp present in transmissible strains and missing in non-transmissible strains. PCR products with primers designed from the p32 gene were obtained only with genomic DNA isolated from transmissible strains. Therefore, P32 has a putative role in the transmission process and it could be considered as a marker for S. citri leafhopper transmissibility. Functional complementation of a non-transmissible strain with the p32 gene did not restore the transmissible phenotype, despite the expression of P32 in the complemented strain. Electron microscopic observations of salivary glands of leafhoppers infected with the complemented strain revealed a close contact between spiroplasmas and the plasmalemma of the insect cells. This further suggests that P32 protein contributes to the association of S. citri with host membranes

Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed