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logeography, 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 IU1 analysis did not identify IU1 any variations in previously known virulence fac tors 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. solan acearum species complex members, with host specificity and ecological TCID adaptations conferred by traits that remain to be identified. Sequencing of additional species com plex members that infect highly divergent plant hosts GMI100 0 such as clove trees and plantains will offer additional insights into the traits that confer host specificity on bac terial wilt pathogens.
Methods Strains The three sequenced strains were isolated from infected tomato plants in different geo graphic locations. CFBP2957 was iso lated in the French West Indies ], CMR15 in Cameroon and PSI07 in Indonesia, Bacteria were grown at 28 C in B liquid medium, Strains CFBP2957, PSI07 and CMR15 Resonance (chemistry) were deposited at CFBP, Table S6 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 cul tures of each strain using a DNeasy Blood Tissue Kit, according to the manufac turers recommendations. Sequencing of the R.
solan acearum 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 AZ20 into plasmid pCNS, Plasmid DNAs were purified and end sequenced by dye terminator chemistry with ABI3730 sequenc ers leading to an approximately 1 fold coverage. The sequences were assembled using Newbler and vali dated 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, Kan3 as well as IU1 around 60× coverage using Solexa reads GAI to polish the genome draft.
Automatic and expert AZ20 annotation of the Ralstonia genomes Coding sequences were predicted using AMIGene software, Each predicted CDS was assigned a unique identifier prefixed with CMR15, CMR15 mp and pCMR15, for R. solanacearum CMR15, with PSI07, PSI07 mp and pPSI07 for R. solanacearum PSI07, and with RCFBP, RCFBP mp for R. solanacearum CFBP2957, The set of predicted IU1 genes were submitted to automatic func tional annotation using the tools listed in Vallenet et al, Apart from the plasmid encoded genes, the func tional 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 are stored in a relational database, called RalstoniaScope.
Manual validation of the auto matic annotation was performed using the web based MaGe interface, which allows graphic visualization of the annotations AZ20 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 per formed 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 anno tated. 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 strains, Molk2 and IPO1609, Using the available contigs of Molk2 an