The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome
© Saillard et al; licensee BioMed Central Ltd. 2008
Received: 13 February 2008
Accepted: 28 April 2008
Published: 28 April 2008
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.
S. citri was originally cultured from leaves of sweet orange trees affected with stubborn disease [1, 2]. This phloem-restricted mollicute was later confirmed as the aetiological agent of stubborn disease of citrus and brittle root disease of horse radish [3, 4]. S. citri is naturally transmitted to a wide range of host plants in a propagative manner by sap-feeding leafhopper vectors . The successful transmission of S. citri by its leafhopper vector relies on the ability of the spiroplasmas to cross the gut epithelium and the salivary gland barriers [6–9]. During the insect invasion process, spiroplasmal surface proteins play a key role. Proteolysis of the S. citri membrane protein P89 was associated with a decrease of the adhesion of spiroplasmas to the insect vector cells . Spiralin, the major lipoprotein of S. citri membranes, which functions as a lectin that interacts with insect glycoproteins, was shown to be required for efficient insect transmission [11, 12]. Inability to achieve the complete transmission cycle within the insect vector has also been reported for some S. citri strains propagated in planta or in vitro [13, 14]. Proteomes of such S. citri strains, specifically lack the membrane associated, hydrophilic P32- and P89-related proteins . Protein P89, later named SARP1, is encoded by the arp 1 gene located on the pBJS-O plasmid of S. citri BR3-3X [15, 16]. An homologous protein SkARP1 is encoded by plasmid pSKU146 in S. kunkelii .
The genetic bases of the interaction of S. citri with its plant and insect hosts have been investigated with the molecular tools available for S. citri transformation, mutagenesis and complementation [18–22]. Using random and directed mutagenesis, fructose import was identified as one of the major determinants of S. citri pathogenicity [23, 24]. In contrast, S. citri mutants unable to import glucose through the phosphotransferase system were not affected in insect transmission nor in multiplication and symptoms induction in plants [25, 26]. Mutants deficient in insect transmissibility were also produced by transposon mediated mutagenesis. Disruption of the P-type ATPase resulted in a non-vectored phenotype , whereas disruption of the solute binding protein of a putative glucose ABC-transporter led to a reduced level of transmission . To allow comprehensive, functional studies of the interaction between S. citri and its hosts, the genome of the insect-transmissible and triply cloned S. citri strain GII3-3X is currently being deciphered. Since many S. citri strains have been shown to carry native plasmids [28–30], we first looked for the presence of extrachromosomal molecules in genome assembly data.
Plasmid assembly and general properties of plasmids
pSci plasmids properties and number of copies deduced from library coverage
Shotgun reads per kbp
Copies per chromosome
Total DNA (kbp)
Total pScis (% of total)
Genetic content of the pSci plasmids
The ScARP protein family
Sequence similarity between ScARP proteins
amino acids identity (%)
Primer sequences that define fragments amplified by PCR and used as hybridization probes.
Nucleotide sequence (5'-3')
As shown in Fig. 3D, probe A designed on pSciA, hybridized with several DNA bands from the total undigested genomic DNA (track A). The fastest and slowest migrating bands should be the covalently circular and the open circular forms of pSciA (7.8 kbp). Many pSciA supercoiled forms displaying intermediate migration were also detected by the A probe, indicating that pSciA is a highly supercoiled plasmid.
Secondary structures and GC skew of pSci plasmids
Plasmids were first detected and purified in the early eighties, from S. citri strains isolated from naturally infected citrus, periwinkle and insect vectors collected in the South of the Mediterranean basin and south-western USA [28–30]. A small plasmid of 7.8 kbp was consistently detected in these studies. In the present work, pSciA of 7.8 kbp was characterized from strain GII3-3X and its restriction map based on nucleotide sequence data is identical to the restriction maps of the two previously reported plasmids pIJ2000 and pM41 [29, 30]. Its genetic organization is described in this study, but except for the CDS encoding a Soj-like protein, no link to a specific biological function arose from the characterization of genetic content of pSciA. The functions conferred by pSciA-type plasmids therefore remain essentially unknown. The presence of plasmids larger than 10 kbp is common in S. citri strains. Archer and colleagues  isolated from S. citri strain SP4.6as two large plasmids of 14.8 and 35.4 kbp, similar in size to those of pSci2 (14.4 kbp) and pSci6 (35.3 kbp). It was recently shown that S. citri BR3-3X harbours the 13.3 kbp pBJS-O , the genetic map of which is highly similar to pSci2. Ranhand and colleagues  detected 4 different plasmids with sizes ranging from 4.1 to 26.1 MDa (6.5 to 41 kbp approximately) in all S. citri strains analyzed. They estimated that these extrachromosomal elements accounted for 12% of the total spiroplasma DNA. In the triply cloned S. citri strain GII3-3X, we estimated the plasmid content to represent nearly 50% of the total DNA, and to consist of 7 different plasmids. The presence of 7 plasmids in GII3-3X, a triply cloned strain, is consistent with the co-existence of these 7 plasmids in each spiroplasma cell. The copy numbers of plasmids pSci1-6 are not correlated with plasmid size and appear to be kept in the range of 10–14 copies per cell in the spiroplasma population. The copy numbers of pSciA could have been underestimated as the intensity of hybridization signal seems to indicate it is more abundant than calculated from sequence coverage. Two properties might explain a reduced occurrence of pSciA inserts in plasmid libraries: the very low G+C content of pSciA (21.3%) and a lower sensitivity to mechanical shearing due to its high degree of supercoiling. High copy numbers and stable maintenance of the pSci plasmids suggest efficient mechanisms for plasmid replication and partitioning. It also means that pSci plasmids are compatible and that the mechanisms of their partitioning in daughter cells are specific enough to prevent them from interfering with each other. Every pSci plasmid possesses at least one copy of soj, a gene encoding a partitioning protein of the parA/soj protein family, usually involved in the active segregation of low-copy-number plasmids . The copy number of the pSci plasmids estimated in this study could have increased artificially due to in vitro cultivation; under natural conditions, it could be different.
In addition to being vertically inherited, pSci1-6 plasmids might be transferred between cells by conjugation, since they encode TrsE, TraG and Mob corresponding to VirB4/D4 components of the type IV secretion pathway, known to drive translocation of DNA through cytoplasmic membranes . In walled bacteria, conjugation systems involve many other components that were not detected in the pSci plasmids. However, conjugation in wall-less bacteria might require only a reduced set of protein components. It should nevertheless involve other pSci CDS products, and especially relaxase, which is necessary for plasmid DNA binding and cleavage, and a polytopic membrane protein equivalent to VirB6, which is necessary to help VirB4/D4 ATPases to translocate the DNA through the cytoplasmic membrane [41, 42]. Proteins PD, encoded by a conserved CDS on pSci1-4 and predicted to contains 7 transmembrane segments, could play such a role. None of the pSci CDS was found to share similarities with relaxase. However, the implication of a chromosomally encoded relaxase cannot be excluded. Such a situation has been described for the conjugative plasmid RP4 of Helicobacter pylori . As reported for the S. citri pBJS-O , pSci plasmids lack the putative oriT region predicted in the S. kunkelii pSKU146 . The possibility that the conserved IRR regions found in all pSci plasmids act as transfer origins for conjugation should be considered. Although former studies have reported the existence of genetic exchanges via a conjugation-like process in S. citri , conjugal transfer of pSci plasmids remains to be documented.
The mechanism of pSci replication is not known. Circular plasmids replicate by 3 general mechanisms, namely (i) unidirectional or bi-directional theta type, (ii) bi-directional strand displacement, and (iii) unidirectional rolling circle (RC) . For pSci plasmids, the lack of switch in cumulative GC skew is indicative of an unidirectional replication mechanism. RC replication of pScis is unlikely because pScis are much larger than rolling circle replicating plasmids, which also are usually less than 10 kbp in size . In addition, no CDS with similarities to rep protein of RC replication has been identified in pSci plasmids. The theta replication mode has been described in gram-negative as well as in gram-positive bacteria . In Gram positive bacteria, from which Mollicutes originate, unidirectional theta replication has been reported for plasmids of the streptococcal/enterococcal Inc18 group such as the 26.5 kbp pAMbeta1 . Their replication requires a plasmid-encoded Rep protein and the host DNA polymerase I, and is initiated from an origin located downstream of the Rep protein gene [47–49]. Such an origin of replication as well as a Rep protein have not yet been identified in pSci plasmids.
The regulatory mechanism that maintains multiple copies of pScis extrachromosomally, could help plasmid encoded determinants to better escape the S. citri chromosome instability known to occur during prolonged propagation in host plants [50, 51]. We recently showed that ScARPs and P32 were absent from non-transmissible strains of S. citri such as strain 44, which also lack all pSci plasmids [14, 52]. Transfer of pSci6 into S. citri strain 44 confers to the spiroplasmas the ability to cross the salivary gland barrier, a necessary step for insect transmission . Plasmid pSci6 does not encode full length ScARPs. ScARPs, which are expected to be involved in S. citri adhesion to insect cells [10, 16], could participate at a different stage of the insect colonization . The high number of scarp genes raise the question of their function. The high diversity of ScARPs certainly reflects an important diversifying selection pressure exerted on these proteins. Are these different ScARPs required for S. citri interaction with different insect vector cell types? Or do the different ScARPs confer the ability to interact with the three leafhopper species that are known S. citri vectors in the Mediterranean region [54–56], where the GII3 strain of S. citri was originally isolated? It is interesting to notice that the Mediterranean GII3-3X strain possesses more ScARPs than does the BR3-3X strain  which was isolated in USA, where only Circulifer tenellus is known to be present. Even though diversity and abundance of S. citri plasmids certainly reflect its adaptation to its complex life cycle and ecological niche, the biological role of pSci plasmids remains to be further established.
Spiroplasma strain and cultivation
S. citri strain GII3 was originally isolated from the leafhopper Circulifer haematoceps collected in Morocco in 1980 . A triply cloned strain was further produced by plating on SP4 medium and one of the clones was further propagated as GII3-3X. Spiroplasmas were grown at 32°C in SP4 medium .
Sequencing and assembly
Sequencing data were produced following a chromosome map-based approach and classical shotgun strategy completed by end sequencing of inserts from a miniBAC library. Ten libraries were produced from Apa I and Bss HII overlapping large DNA fragments covering the spiroplasma chromosome. Fragments were separated by PFGE according to standard procedure, eluted from agarose gels, agarase treated and ethanol precipitated. Chromosome specific libraries were constructed in pBluescript (Stratagene, La Jolla, California, USA) after partial Sau 3A digestion of purified chromosomal fragments. About 6,000 reads produced on ABI-prism 377XL were obtained from this map-based phase of the project. S. citri total DNA was purified according to Marmur's method  and mechanically sheared to construct two plasmid libraries. A first pSMART library of 4,000 clones with 3–4 kbp inserts (prepared by Amplicon Express, Pullman, Washington, USA) and a second pBluescript library of 2,400 clones with 1–3 kbp inserts were produced. A miniBAC library with inserts of 15–25 kbp was prepared by cloning Sau 3A partially digested total DNA of S. citri in pECBAC1. Inserts were end-sequenced on ABI-prism and MEGABACE capillary sequencers.
Assembly and editing of 20,000 sequencing reads were performed with the phred-phrap-consed package [60–62]. Incorrect assembly of repeated sequences were detected by phrap, due to abnormally long distance between insert extremities, for instance, those exceeding 4 kbp for plasmid inserts. These DNA regions were assembled separately and completed by primer walking. The resulting consensus sequences were introduced back in the general assembly to resolve repeated regions and restore a normal scaffold. This strategy allowed us to resolve misassemblies due to the highly similar regions occurring in plasmids pSci1-6. The physical maps of pSci1-6 circular contigs were verified by digesting overlapping miniBAC inserts with Eco R1, Eco RV, Hinc II, Hind III and Hpa I. Sequences were deposited under accession numbers EMBL:AJ966734, EMBL:AJ969069, EMBL:AJ969070, EMBL:AJ969071, EMBL:AJ969072, EMBL:AJ969075, EMBL:AJ969076.
Annotation and bioinformatic analyses
Sequence analysis and annotation were managed with the iANT (integrated Annotation Tool) web-based annotation environment developed for Ralstonia solanacearum genome annotation . Protein-coding genes were predicted by using the FrameD program  trained on known S. citri genes and the NCBI-BLASTX program . Protein motifs prediction included TMPRED for membrane spanning domains, ProDom and Prosite for conserved protein domains [66, 67]. Signal peptides were predicted using SignalP 3.0  and transmembrane topology predicted by TMHMM . ScARPs were aligned using ClustalW , nucleic acid secondary structures were predicted at Mfold web server  and cumulative GC skew (G-C/G+C)  was calculated at (see Availability and requirements section for URL).
Southern blot hybridization
S. citri genomic DNA was isolated using the Wizard Genomic DNA purification Kit (Promega). Whole genomic DNA was submitted during 60 h to a 0.5% agarose gel electrophoresis (0.2 volts/cm). The gel was then blotted to a positively charged nylon membranes by the alkali transfer procedure. Hybridizations with appropriate digoxigenin-11-dUTP-labelled DNA probes were carried out by using the standard method . Detection of hybridized probes was achieved using anti-digoxigenin antibodies coupled to alkaline phosphatase and the fluorescent substrate HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) (Roche Molecular Biochemicals). Chemifluorescence was detected by using a high-resolution camera (fluor-S Multimager, Bio-Rad) and Quantity One, a dedicated software for image acquisition (Bio-Rad). Probes specific to each ScARP, probes U and pSciA were produced by PCR amplification of genomic DNA with primer pairs indicated in Table 3. Probe U consisted of a 191-bp sequence present in all S. citri GII3-3X plasmids pSci1-6. Probe pSciA was specific to the pSciA plasmid.
Availability and requirements
This work was supported by grants from the INRA department of Plant Health and Environment (INRA-SPE), Regional Council of Aquitaine and FUNDECITRUS. NK was supported by the Egyptian Ministry of Higher Education and INRA. We thank M. P. Dubrana for her help in designing hairpin representation and A.S. Javaugue for excellent technical assistance. We are grateful to professor A. Purcell for editing language.
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