Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence
- Benoît Remenant1,
- Bénédicte Coupat-Goutaland†2,
- Alice Guidot†3,
- Gilles Cellier1, 4,
- Emmanuel Wicker1,
- Caitilyn Allen5,
- Mark Fegan6,
- Olivier Pruvost1,
- Mounira Elbaz3,
- Alexandra Calteau7,
- Gregory Salvignol7,
- Damien Mornico7,
- Sophie Mangenot8,
- Valérie Barbe8,
- Claudine Médigue7 and
- Philippe Prior9Email author
© Remenant et al; licensee BioMed Central Ltd. 2010
Received: 27 February 2010
Accepted: 15 June 2010
Published: 15 June 2010
The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats.
The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole.
Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic distances between strains, in conjunction with CGH analysis of a larger set of strains, revealed differences great enough to consider reclassification of the R. solanacearum species complex into three species. The data are still too fragmentary to link genomic classification and phenotypes, but these new genome sequences identify a pan-genome more representative of the diversity in the R. solanancearum species complex.
The rapidly accumulating complete genomes in databases provide unique opportunities to study relationships among organisms. Since DNA sequences are conserved between closely related organisms, comparative genomic analyses are a powerful tool for understanding the complex evolutionary events in specific phylogenetic lineages.
R. solanacearum, formerly known as Pseudomonas solanacearum and Burkholderia solanacearum, is the causal agent of bacterial wilt . This soil-borne vascular pathogen is widely distributed in tropical and subtropical climates and affects an unusually broad range of crops, including both monocot and dicot plants [2–4]. Many affected hosts are critical for developing countries because of their strategic importance as cash crops or as subsistence foods like potato (Solanum tuberosum), tomato (S. lycopersicum), eggplant (S. melongena), cooking banana (Musa spp.) and peanut (Arachis hypogea). In the 1990s, potato brown rot strains of R. solanacearum historically known as race 3/biovar 2 (r3b2) were introduced in Europe and North America [5, 6]. Due to their adaptation to tropical highland climates, these strains, which are more virulent at cool temperatures (~20°C) than tropical strains , may pose major threats in temperate zones. Therefore, R. solanacearum was listed as a quarantine organism in Europe and Canada and as a Bioterrorism Select Agent in the U.S. .
R. solanacearum and the closely related species R. syzygii (a pathogen of clove in Indonesia) and the banana blood disease bacterium (BDB) form a complex in the R. picketii lineage [9–12]. This species complex includes thousands of genetically distinct strains that can differ from each other by more than 30%, and thus do not belong in the same species by conventional definition . This species complex includes strains with broad and narrow host ranges, which are ecologically different as well: potato strains are cold-tolerant and banana strains are insect-transmitted, and with different geographic origins. Because R. solanacearum strains have been isolated from virgin jungle soils in both Asia and the Americas, the origin of the species complex is believed to predate the geological separation of the continents . Based on analyses of the 16S-23S internal transcribed spacer (ITS) region, egl and hrpB genes and on comparative genomic hybridization (CGH), the R. solanacearum species complex is hierarchically classified in four phylogenetic groups called phylotypes, which reflect their origins as follows: Asia (phylotype I), the Americas (phylotype II), Africa (phylotype III) or Indonesia (phylotype IV, which includes R. syzygii and BDB) [15, 16]. Each phylotype can be further subdivided into sequevars, or sequence variants, which may contain isolates with similar virulence patterns or common geographic origin .
Despite their considerable phylogenetic diversity, R. solanacearum strains are unified by their common pathology. All cause bacterial wilt disease, which is characterized by bacterial colonization of the plant xylem vessels to very high cell densities (109 - 1010 CFU/ml xylem fluid), vascular browning, stunting, wilting, and often rapid death . The bacterium is transmitted by soil, surface water, and infected propagation materials like potato tubers or ornamental cuttings. It most commonly infects plants through the roots, but some strains are insect-transmitted . Bacterial wilt is difficult to control because the bacterium survives for years in infested soils and weed hosts. Breeding for host resistance, the best management strategy is complicated by the pathogen's high genetic diversity. For example, tomatoes resistant to R. solanacearum strains in one region are often susceptible to those in another .
Genome sequences of R. solanacearum strains can answer historic questions about what traits allow this bacterium to be such an aggressive and lethal pathogen of so many different plants, and to survive in such different habitats as soil, water, non-host plant rhizospheres and host xylem vessels. The bi-partite genome of R. solanacearum strain GMI1000 (phylotype I, sequevar 18) was sequenced and analyzed [19, 20] . The genome has two replicons, called the chromosome and the megaplasmid, with a mosaic structure that implies many rearrangements and horizontal gene transfers. Several factors have been shown to contribute to bacterial wilt virulence, especially the type III secretion system (TTSS) and associated effectors , and production of extracellular polysaccharides and enzymes . The GMI1000 genome encodes more than a hundred TTSS effectors or putative effectors . Draft genomes are available for two additional R. solanacearum strains: IPO1609/UW551, which cause potato brown rot disease in cool-temperate climates and Molk2, which causes Moko disease of banana and plantain [24, 25]. These respectively belong to phylotype IIB sequevar 1 (IIB-1) and sequevar 3 (IIB-3).
To better understand how this highly diverse and scattered species complex has evolved and diverged without losing its fundamental pathological qualities, we sequenced the genomes of three additional broad host range strains from other phylotypes, namely: American strain CFBP2957 (IIA-36), African strain CMR15 (III-29) and Indonesian strain PSI07 (IV-10). All were originally isolated from tomato. Their complete genomes were manually annotated and analyzed with the aim of investigating the conserved, variable, and specific gene repertoires of these strains and the three previously sequenced ones, with a special emphasis on genes involved in virulence and pathogenicity. In addition, the genomes of 51 R. solanacearum strains (including the six sequenced strains) were compared by CGH on a pan-genomic microarray. These comparative genomic approaches produced new insights into the evolution and taxonomy of the R. solanacearum species complex.
Results and Discussion
It has long been known that R. solanacearum is a highly heterogeneous group of strains and thus no one genome sequence could represent the entire species complex. Initial genomic studies revealed that R. solanacearum strains have a substantial backbone of common housekeeping and virulence functions, but also carry a divergent set of genomic modules that likely confer distinct ecological phenotypes and host specificity [19, 24]. Comparing multiple bacterial genomes distributed around the phylogenetic tree was therefore essential to understand the evolutionary driving forces and mechanisms that have produced such a phenotypically and genotypically diverse group. Four existing genome sequences represented phylotype I (tomato isolate GMI1000) and phylotype II (IPO1609/UW551 and Molk2, from potato and banana, respectively) [19, 24, 25]. The phylotype-sequevar subclassification system provided rational criteria for choosing more strains to sequence to cover significant additional diversity in the species complex. We therefore sequenced genomes of strains that cause bacterial wilt of tomato from Indonesia (PSI07, phylotype IV), Cameroon (CMR15, phylotype III), and the French West Indies (CFPPB2957, phylotype II).
Overview of genomes of CFBP2957, CMR15 and PSI07
The two-replicon (chromosome and megaplasmid) genome architecture of these three tomato strains was identical to that found in the three previously sequenced strains. As in the GMI1000 genome , most -but not all- housekeeping genes were carried on the chromosome. However, the capture or creation of the megaplasmid by Ralstonia spp. appears to be an ancient event, since all strains in the R. pickettii lineage studied to date have two replicons (Lucas et al, unpublished). Moreover, CGH microarray analyses by Guidot et al.  established that both replicons have a long history of coevolution within the R. solanacearum species complex.
General features of genomes of R. solanacearu m strains CFBP2957, CMR15, PSI07, GMI1000, IPO1609 and Molk2
French West Indies
Automatic re-annotation of the R. solanacearum sequences in the public domain identified 517 additional coding sequences (CDS) in GMI1000 (263 on the chromosome and 254 on the megaplasmid [Additional file 2]). These newly identified genes, encoding mostly proteins of unknown function, were encoded 'RALSO' to distinguish them from previously-annotated CDS ('RSc' and 'RSp').
The R. solanacearum pan-genome
Genome plasticity and genomic islands
Interestingly, the megaplasmids of CFBP2957 and PSI07 also harbor a genomic island containing the rhi operon. This operon was composed of 9 genes: rhiB (20274 pb), rhiC (97185 pb), rhiI (867 pb), rhiD (12468 pb), rhiH (1458 pb), rhiE (12657 pb), rhiF (7824 pb), rhiA (7158 pb) and rhiG (1983 pb). The rhi genes were previously unknown in Ralstonia strains, but are present in Pseudomonas fluorescens and in Burkholderia rhizoxinica where they encode a non-ribosomal peptide synthase (NRPS) that synthesizes the antimitotic toxin rhizoxin . Although this operon spans more than 80 kb, it could have been horizontally acquired since R. solanacearum can exchange and integrate DNA fragments of 30 to 90 kb by natural transformation . The insertion of such a toxin operon in the genome of R. solanacearum could provide a competitive advantage in the soil environment.
Comparison of some metabolic properties
Careful examination of the metabolic potential of the studied genomes has revealed some specificity capabilities for compound degradation. Although R. solanacearum is a highly adapted and ancient plant pathogen, this organism can also survive well in soil and rhizospheres and, consequently, its genome encodes ability to metabolize diverse organic compounds as energy sources. Pathway analyses indicate that this bacterium can utilize a wide range of substrates including amino acids, sugars, and fatty acids as well as glycerol, ethanol, methylglyoxal, and beta-ketoadipate ([Additional file 4, sheet B], also Genin and Boucher ). GMI1000 was predicted to grow on various aromatic compounds including ferulate, vanillate, and protocatechuate , which are released when lignin degrades in soil. However strains CMR15 and GMI1000 do not harbour the benABCD and catABC genes involved in benzoate and catechol degradation to beta-ketoadipate. Another contrasting example is D-galactonate degradation, which can serve as energy and sole carbon source for many enteric bacteria . In soil, bacteria such as Azotobacter vinelandii and Sinorhizobium meliloti convert D-galactose to D-galactonate via the De Ley-Doudoroff pathway, and finally to D-glyceraldehyde-3-phosphate and pyruvate. Our data indicate that D-galactonate degradation is functional only in strains GMI1000, CMR15 and PSI07 [Additional file 4, sheet B]. However, this substrate must be taken up whole from the environment because no degradation enzymes upstream in the pathway were apparent in these genomes. Curiously, it seems that the D-galactonate transport system is different in CMR15, which harbors only one likely transport gene in the corresponding synton, and in GMI1000 and PSI07, which each have 3 genes highly similar to the L-arabinose ABC transporter (annotated as araFGH).
Urease, which is necessary for utilization of urea as sole nitrogen source, has three main subunits and five accessory proteins. Genetic determinants for this enzymatic activity were found in all strains except Molk2, the only banana wilt pathogen sequenced to date. All six strains appear able to metabolize inorganic nutrients such as sulfate and nitrate, consistent with experimental data . However, the denitrification pathway was complete only in strains GMI1000 and CMR15, because the nosZ gene encoding nitrous oxide reductase was absent from the four other strains; this heterogeneity was previously noted . Genes for periplasmic nitrate reductase (nap), nitrate reductase (nar), nitric oxide reductase (nor) and nitrite reductase (nir) were present on the megaplasmids of all six sequenced R. solanacearum strains.
Finally, all six genomes contain genes involved in the detoxification of noxious compounds and in metal resistance, which likely support colonization and survival in specific ecological niches. An interesting example is arsenic resistance which in bacteria is mediated in part by ars genes. Among these, arsC encodes an arsenate reductase [As(V)→As(III)], arsA and arsB encode an arsenite efflux pump, and arsR encodes a transcriptional regulator . As(III) is known to induce oxidative stress, to cause DNA damage, and to inhibit the DNA repair system . It is generally further oxidized by the arsenite oxidase [As(III)→As(V)] encoded by aoxAB genes. The arsC gene is the sole gene in this pathway present in all six strains, with two tandem copies in PSI07. In addition, only PSI07 can oxidize arsenite: on the PSI07 megaplasmid is a cluster containing two arsC genes, aoxAB, and arsR. The annotation of the arsC-like gene is probably erroneous in the other Ralstonia species (and in many other bacterial genomes as well).
Many traits contribute to virulence of R. solanacearum strains. The best known are the type III secreted effectors, well described in this bacterium and in other plant pathogens . However, other traits, such as production of EPS and cell wall-degrading enzymes, are also important for wilt disease development. Based on the literature, we created an inventory of 128 genes involved in virulence from the six sequenced R. solanacearum genomes [Additional file 5: Supplemental Table S5]. Some genes are involved in swimming motility, twitching motility and chemotaxis. Table S5 gives a representative pair of genes for each of those functions. Virulence genes were subdivided into 5 categories: type III effectors (TTE) and putative effectors, the exopolysaccharide (EPS) biosynthetic genes, the cell wall-degrading enzyme (CWDE) genes, response to host defence genes and key virulence regulators. Scrutiny of the genomes shows that all six strains have all genes needed for functional type II and III secretion systems. Similarity distances between each sequenced strain were computed on the basis of gene presence/absence data for these 128 virulence genes (data not shown). Phylogenetic analysis constructed on the basis of: 1) all known or putative Type 3 effector genes in the pan-genome, and 2) all known virulence factor genes of all kinds in the pan-genome, resulted in trees that were significantly different from each other, and significantly different from trees based on well-conserved genes like mutS and egl sequences, or on the entire genome sequences (data not shown). This result suggests that virulence factor genes have evolved or been lost or added at substantially different rates than R. solanacearum genes as a whole. A more fine-scale case-by-case analysis will likely be needed to trace the evolutionary history of individual virulence traits. Analysis of strains hosted by plants phylogenetically distant from tomato (a common host for all strains analyzed here except Molk2) may elucidate roles of individual virulence factors in determining host range.
Type III-secreted effectors are an important potential source of host range variability in R. solanacearum strains; these have mainly been described in GMI1000 and UW551 to date [23, 25]. Specific effectors that are important in CMR15, CFBP2957 and PSI07 are unknown. We attempted to detect new type III effectors with the Effective software , but this did not work well for R. solanacearum strains, giving about 50% false negative on previously annotated effectors (data not shown).
Plasmids in Ralstonia solanacearum strains
Genin and Boucher  suggested that the presence of small plasmids in R. solanacearum cells, initially described by Morales and Sequeira , was more an exception than a rule. However, we found small plasmids (<50 kb) in the African and Indonesian strains. These plasmids were named pRSC35 (35 kb, GC% = 61.3) in strain CMR15 and pRSI13 (12.8 kb, GC% = 61.0) in strain PSI07. The presence of small plasmids is therefore maybe less rare in R. solanacearum strains than previously thought. These small plasmids may have remained undetected until now because their very low copy number makes them difficult to purify (unpublished results).
Despite their low copy numbers, the stability of these plasmids is apparently ensured by two different toxin/antitoxin (TA) systems. On pRSC35, two CDS had a limited homology with zeta-toxin and epsilon-antitoxin (<40%), which form a post-segregational mechanism for plasmid maintenance in bacteria [40, 41]. The regulator ω was not detected in the CMR15 genome. This zeta/epsilon TA system is well described and a similar system confers a bactericidal effect on Bacillus subtilis, and bacteriostatic effects on E. coli.
The unexpected Type IV Secretion System is unique among R. solanacearum strains studied to date and could play diverse important roles in virulence and adaptation. The CMR15 Type IV secretion system genes, which are clustered together with the virB operon, have nearly the same organization as on pXAC64 of Xanthomonas citri pv citri. The type IV secretion system is a bacterial conjugation apparatus  and the DNA thus efficiently imported through the cell envelope can directly increase the fitness or virulence of bacteria by mediating the acquisition of new traits like effectors or antibiotic resistance genes. Type IV secretion systems can also be directly involved in virulence via direct injection of effectors or DNA into plant cells [49, 50]. No obvious type IV effectors were found on pRSC35 or in the complete genome of CMR15, but some proteins of unknown function could be Type IV effectors. Additional experiments are needed to investigate (i) the distribution of this plasmid in African phylotype III strains, (ii) the ecological and pathogenic role of this plasmid in the phenotype of phylotype III strain CMR15, and (iii) the occurrence of such plasmids in strains belonging to other phylotypes.
A second low-copy number plasmid, pRSI13, was present in PSI07. It was syntenic with a plasmid found in Nitrobacter hamburgensis X14 (34% of CDS in synteny), Burkholderia pseudomallei 9 and 91 (30 and 26% respectively), Parvibaculum lavamentivorans DS-1 (26%), Acidovorax sp. JS42 (26%) and E. coli pOLA52 (26%). pRSI13 contained 23 CDS, 16 of which encoded for proteins of unknown function and one for a putative transcriptional regulator. Other pRSI13 CDS coded for proteins putatively involved in DNA metabolism or conjugation (figure 6B). Thus, the functional annotation reveals no obvious role for this plasmid in either the ecology of the bacteria (saprophytic life in the soil) or during pathogenesis. The maintenance of this plasmid seems likely due to the TA system rather than to increased fitness.
New insight into the phylogeny of the R. solanacearum species complex
We compared the genome organizations and gene repertoires from six phylogenetically distant and phenotypically divergent strains from the R. solanacearum species complex. These strains shared many structural and genotypic traits observed in the first sequenced strain of R. solanacearum, GMI1000. Our results significantly expanded the known R. solanacearum pan-genome, identifying thousands of genes that more sharply define the common core, the dispensable and the specific (strain-unique) genomes. However, the genomes differed quite significantly. Our ANI and other analyses suggest that this large and heterogeneous species complex spans enough variation that it could be reclassified into at least three distinct taxonomic groups, which each have the equivalent of more than 30% divergence from the other two at the DNA-DNA hybridization level. Post-genomic mining of this pan-genome can combine comparative tools like CGH microarrays with phenotypic experiments to determine the distribution and the biological functions of likely traits identified with in silico analyses. Such combined approaches will increase our understanding of the evolutionary past, the phylogeography, and the biological specialization of R. solanacearum species complex strains. Although four of the six strains sequenced to date were isolated from tomato plants, our analysis did not identify any variations in previously known virulence factors that were unique to tomato pathogens. This could be explained by either 1) an insufficiently large sample of non-tomato pathogen genomes or 2) a biological unity in the core mechanisms of bacterial wilt across all R. solanacearum species complex members, with host specificity and ecological adaptations conferred by traits that remain to be identified. Sequencing of additional species complex members that infect highly divergent plant hosts such as clove trees and plantains will offer additional insights into the traits that confer host specificity on bacterial wilt pathogens.
The three sequenced strains were isolated from infected tomato plants (Solanum lycopersicum) in different geographic locations: CFBP2957 (phylotype IIA-36) was isolated in the French West Indies [Syn. MT5, ], CMR15 (phylotype III-29) in Cameroon  and PSI07 (phylotype IV-10) in Indonesia . Bacteria were grown at 28°C in B liquid medium . Strains CFBP2957, PSI07 (CFBP7288) and CMR15 (CFBP6941) were deposited at CFBP  (Collection Française de Bactéries Phytopathogènes, Angers, France). Table S6 [Additional file 6] provides a list of the 51 R. solanacearum strains used in microarray experiments, with their geographical origin and host of origin.
Sequencing and assembly
Genomic DNA was purified from overnight liquid cultures of each strain using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer's recommendations. Sequencing of the R. solanacearum strains CMR15, CFBP2957 and PSI07 was performed using the strategy described by Aury et al. . Around 20× coverage of 454 GSflx reads were mixed with 1× coverage Sanger reads for the scaffolding, which was derived from a 10 kb insert fragment size library. Each library was constructed after mechanical shearing of genomic DNA and cloning of generated inserts into plasmid pCNS (pSU18-derived). Plasmid DNAs were purified and end-sequenced (11520 R. solanacearum PIII, 14592 R. solanacearum PIIA and 7680 R. solanacearum PI) by dye-terminator chemistry with ABI3730 sequencers (Applied Biosystems, Foster City, USA) leading to an approximately 1-fold coverage. The sequences were assembled using Newbler (Roche Diagnostics) and validated via the Consed interface . For the finishing phases, we used primer walking of clones and/or PCRs and transposon bombs Template Generation System™ II Kit (TGS™ II Kit) (Finnzyme), Kan3 as well as around 60× coverage using Solexa reads GAI to polish the genome draft.
Automatic and expert annotation of the Ralstonia genomes
Coding sequences (CDS) were predicted using AMIGene (Annotation of Microbial Genomes) software . Each predicted CDS was assigned a unique identifier prefixed with "CMR15_", "CMR15_mp" and "pCMR15_", for R. solanacearum CMR15 (respectively the chromosome, megaplasmid, and plasmid), with "PSI07_", "PSI07_mp" and "pPSI07" for R. solanacearum PSI07 (respectively the chromosome, megaplasmid, and plasmid), and with "RCFBP_", "RCFBP_mp" for R. solanacearum CFBP2957 (respectively the chromosome and megaplasmid). The set of predicted genes were submitted to automatic functional annotation using the tools listed in Vallenet et al. . Apart from the plasmid-encoded genes, the functional assignment was first based on the reference genome of Cupriavidus taiwanensis annotations  for strong orthologs i.e., 85% identity over at least 80% of the length of the smallest protein. All these data (syntactic and functional annotations, and results of comparative analysis, see below) are stored in a relational database, called RalstoniaScope. Manual validation of the automatic annotation was performed using the web-based MaGe  (Magnifying Genomes) interface, which allows graphic visualization of the annotations enhanced by a synchronized representation of synteny groups in other genomes chosen for comparison. As described by Vallenet et al.  the system also offers several functions to guide accurate manual expert annotation. We performed a complete manual annotation of the CMR15 genome and then used it to automatically annotate strong orthologs in PSI07 and CFBP2957. Only 'specific' regions of these two strains, i.e. those containing genes not orthologous to ones in CMR15, were manually annotated. Finally, this expert work was used to update the annotation of GMI1000, which was published in 2002, and to automatically annotate the two other sequenced (but not finished) strains, Molk2 and IPO1609 . Using the available contigs of Molk2 and IPO1909, we were not able to properly organize the corresponding sequences using GMI1000 as a reference genome. Genomes of these two R. solanacearum strains are thus not correctly assembled in MaGe and some analyses remain impossible with these genomes.
Complete sequence data for CFBP2957, CMR15, PSI07, GMI1000, Molk2 and IPO1609 are publicly available via the MaGe interface (RalstoniaScope ). Sequences and annotations data of R. solanacearum CFBP2957, CMR15 and PSI07 have also been deposited at the EMBL database  [EMBL:FP885897 and EMBL:FP885907 (chromosome and megaplasmid), EMBL:FP885895 and EMBL:FP885896, and EMBL:FP885906 and EMBL:FP885891 respectively. Sequences of plasmids pRSC35 and pRSI13 are available using accession numbers EMBL:FP885893 and EMBL:FP885890].
Genomic Island Identification
We used the RGPfinder tool in the MaGe annotation platform (Roche et al., in preparation) to investigate Regions of Genomic Plasticity (RGPs) in the whole genome sequences of R. solanacearum GMI1000, CMR15, PSIO7, CFBP2957, Molk2 and IPO1609. RGPs are defined as regions of at least 5 kb that are missing in at least one of the genomes compared. This definition makes no assumption about the evolutionary origin or genetic basis of these variable chromosomal segments. RGPfinder searches for synteny breaks between a target genome and a set of closely related bacteria (generally other strains) to define RGPs. It also provides information about composition abnormalities (%G+C deviation, Codon Adaptation Index) and RGP flanking features such as tRNA, IS and repeats, which are common characteristics of genomic islands (GI). Moreover the tool integrates the results of Alien Hunter  a method that analyses compositional biases to detect atypical sequences (i.e., sequences potentially acquired by horizontal gene transfer).
Synteny group computation
Sequence data for comparative analyses were obtained from the NCBI database (RefSeq section ). Putative orthology relationships between two genomes were defined by gene pairs satisfying either the BBH criterion or an alignment threshold (at least 40% sequence identity over at least 80% of the length of the smallest protein). These relationships were subsequently used to search for synteny groups (i.e., conservation of the chromosomal co-localisation between pairs of orthologous genes from different genomes) among several bacterial genomes using an algorithm based on an exact graph-theoretical approach . This method allowed for multiple correspondences between genes, detection of paralogy relationships, gene fusions, and chromosomal rearrangements (inversion, insertion/deletion). The 'gap' parameter, representing the maximum number of consecutive genes that are not involved in a synteny group, was set to five.
Average nucleotide identity calculation
The average nucleotide identity (ANI) was calculated according to Konstantinidis and Tiedje . Pairwise comparisons between sequences were done separately for chromosomes and megaplasmids (GMI1000, CFBP2957, CMR15 and PSI07), and then for entire genomes (GMI1000, Molk2, IPO1609, CFBP2957, CMR15 and PSI07). Similarity between strains (using Euclidian distance) and dendrogram computation were conducted with the R statistical environment , using ape and ade4 libraries [72, 73].
Metabolic network comparison
The metabolic network of each genome was predicted by the "Pathway Tools" software  using MetaCyc  as a reference pathway database (version 12.0). Starting with the functional annotation performed in MaGe, this software applies selection rules to infer possible metabolic pathways and builds a special database called a PGDB (Pathway/Genome Database). These metabolic networks for each R. solanacearum genome are directly available in the MaGe graphical interface.From those data, a two-dimensional matrix was built, wherein each line represents a Ralstonia genome, and each column a specific pathway measure (according to the Metacyc metabolic classification). Each value corresponds to a pathway completion measure (defined as the number of enzymatic reactions which have been found in a given pathway divided by the total number of reactions in this pathway in Metacyc). This data matrix is the starting point of Principal Component Analysis, which highlights possible metabolic similarities and specificities between the genomes.
All R. solanacearum genomes were considered in this statistical analysis, but only pathways with non-constant completion could be analyzed. After examination of the amount of inertia captured by the method's resulting factors, 2 axes were kept for further analysis (they represent more than 67% of the total dispersion). For graphical representations, the variable (pathways) plot and the individual (genomes) plot were combined, restricting plotted variables to those with a quality of representation greater than 0.75, in order to conserve interpretability. Solely as an additionnal aid to interpretation and to listing readability, for each factorial plane, the pathway variables were hierarchically clustered according to the angles between their vectors. The clustering method used a Euclidean distance and ward's criterion; the number of classes was chosen after manual examination of the cluster tree, and led to 8 classes for the first factorial plane (see also Table S4-A).
CGH microarray experiments
The DNA microarray used in these experiments was generated by C. Boucher and collaborators (INRA-CNRS, Toulouse, France). This spotted microarray consists of 6,516 65-mer and 70-mer oligonucleotides representative of the genes identified from the genomes of R. solanacearum GMI1000 [EMBL:AL646052.1 and EMBL:AL646053.1], IPO1609 [GenBank:NW_002196569.1] and Molk2 [GenBank: NW_002196564.1]. Each gene was represented by a single oligonucleotide except for 115 effector genes, which were represented by two to six oligonucleotides to distinguish allelic forms of a given gene. A limited number of oligonucleotides representative of particular intergenic regions were also included on the microarray. This microarray also includes a set of appropriate negative controls. Each oligonucleotide was spotted twice on the microarray. DNA extraction and labelling, and microarray hybridization were performed as described by Guidot et al . Standard control DNA used for all genome hybridization experiments consisted of an equimolar combination of the genomic DNA from the three sequenced strains GMI1000, IPO1609 and Molk2. Analysis was conducted as previously described using ImaGene and GeneShight (BioDiscovery) softwares . A gene was considered as absent from the tested strain when the base 2 logarithm of the ratio of the normalized hybridization signal of the tested strain over the normalized hybridization signal with the control DNA was lower than the cutoff value of -1 .
Average Nucleotide Identity
Bidirectional Best Hit
Blood Disease Bacterium
Collection Française de Bactéries Phytopathogènes
Colony Forming Unit
Comparative Genomic Hybridization
Cell Wall Degrading Enzymes
Internal Transcribed Spacer
Non-Ribosomal Peptide Synthase
Region of Genomic Plsaticity
Type Three Effectors
Type Three Secretion System.
We thank S. Genin for manuscript review prior to submission, and I. Robène-Soustrade, N. Becker, L. Gagnevin, C. Vernière and L. Costet for assistance in manual annotation. We thank also K. Konstantinidis for sharing perl scripts for ANI calculation, and X. Nesme, S. Brisse and J.P. Euzéby for helpful discussion about phylogeny and taxonomy. DNA sequencing was funded by Genoscope (grant No 133/AP2007-2008) under the authority of Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant, Institut de Génomique. This work was funded by the Fédération Nationale des Producteurs de Plants de Pommes de Terre, Mission-DAR, Grant-7124 of the French Ministry of Food, Agriculture and Fisheries. The European Regional Development Fund (ERDF) of the European Union, Conseil Régional de La Réunion also provided financial support as part of Biorisk programme. This work was supported by the "Agence Nationale de la Recherche" ANR PFTV MicroScope and the GIS IBiSA.
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