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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Genetic content of S. citri strain GII3-3X: pScis and related plasmids pBJS-O and pSKU146. Circular plasmids are represented by linear maps to facilitate comparisons. Conserved CDS are represented in coloured boxes. Letters A to N indicate the PA to PN paralog families. TraG, TrsE and Soj belong to TraG-TraD, TraE and parA protein families respectively. Arrows indicate the positions of IRR shown on Fig. 4 and Fig. 5.

Figure 2

Comparison of ScARPs domains organization. S and TM represent putative signal peptides and hydrophobic transmembrane domains, CR and VR mean conserved and variable regions, KR and KDE are Lysine-Arginine and Lysine-Aspartate-Glutamate rich domains respectively. The amino acid sequence of the repetitive element A (sarpin repeat) and the variations observed are given above the figure.

Figure 3

Southern blotting hybridization of undigested S. citri GII3-3X total DNA. Plasmids were separated from chromosomal DNA by long electrophoresis on 0.5% agarose gels (A, B, C) or classical 1% agarose gel electrophoresis (D) and stained with ethidium bromide (A, D track 1). Probes used for Southern blotting hybridization (C and D track A) are indicated above tracks. U, pSci1-6 universal probe; A, pSciA specific probe; M, DNA ladder. Membrane strips of a preparative gel were used for Fig. 3C hybridizations.

Figure 4

Comparison of hairpin secondary structures and organization of inverted repeats on pScis. Hairpins structures in IRR of pSci1-4 (A), S. kunkelii pSKU146 (B). Organization and distribution of IRR in pSci1-6 (C). Such sequences marked as arrows in figure 1 could act as signals for replication or conjugation of the plasmid. Numbers below sequences (A and B) indicate positions in plasmids.

Figure 5

Hairpin secondary structures and organization of inverted and direct repeats on pSciA. This sequence marked as an arrow in figure 1 could act as signals for replication or conjugation of the plasmid. Numbers below sequences indicate positions in pSciA.

Figure 6

Cumulative GC skew along pSci molecules and plasmid replicated via theta or rolling circle mechanisms. Arrows indicate the positions of IRR shown on Fig. 4 and Fig. 5.