Genomic structure and phylogeny of the plant pathogen Ralstonia solanacearum inferred from gene distribution analysis

Abstract

In the present study, we investigated the gene distribution among strains of the highly polymorphic plant pathogenic beta-proteobacterium Ralstonia solanacearum, paying particular attention to the status of known or candidate pathogenicity genes. Based on the use of comparative genomic hybridization on a pangenomic microarray for the GMI1000 reference strain, we have defined the conditions that allowed comparison of the repertoires of genes among a collection of 18 strains that are representative of the biodiversity of the R. solanacearum species. This identified a list of 2,690 core genes present in all tested strains. As a corollary, a list of 2,338 variable genes within the R. solanacearum species has been defined. The hierarchical clustering based on the distribution of variable genes is fully consistent with the phylotype classification that was previously defined from the nucleotide sequence analysis of four genes. The presence of numerous pathogenicity-related genes in the core genome indicates that R. solanacearum is an ancestral pathogen. The results establish the long coevolution of the two replicons that constitute the bacterial genome. We also demonstrate the clustering of variable genes in genomic islands. Most genomic islands are included in regions with an alternative codon usage, suggesting that they originate from acquisition of foreign genes through lateral gene transfers. Other genomic islands correspond to genes that have the same base composition as core genes, suggesting that they either might be ancestral genes lost by deletion in certain strains or might originate from horizontal gene transfers.

FIG. 1.

Clustering of the GMI1000 reference genes and of R. solanacearum strains based on gene distribution among the different strains, as determined from CGH experiments. For each individual strain, the presence of a particular gene is represented by a black box, whereas absence of the gene corresponds to a gray box. White boxes correspond to missing data. The percentage of GMI1000 genes that are categorized as conserved in each individual strain is also given, together with the strain phylotype.

FIG. 2.

Distribution of core and variable genes within the different functional categories defined in the annotation of the genome of R. solanacearum strain GMI1000. Pathogenic regulon, set of genes that belong to the hrpB and hrpG pathogenicity regulon.

FIG. 3.

Spatial distribution of core and variable genes on the chromosome (A) and on the megaplasmid (B) of R. solanacearum strain GMI1000. Each individual gene is represented by a vertical bar on lines 1. Core genes are represented in red, and variable genes are in blue. The positions of the bars above or below the middle line depend on the orientation of the corresponding open reading frames. Lines 2 represent the positions of insertion sequence elements and prophages, lines 3 the position of ACURs, and lines 4 the percent GC variation along each replicon (using a sliding windows of 5,000 nucleotides and a 500-nucleotides step). Major islands of variable genes are boxed in brown when they are included in ACURs or in green when the corresponding genes use the standard codon usage.

FIG. 4.

Distribution of core (gray bars) and variable (black bars) genes from GMI1000 according to the GC composition of the corresponding sequences. The inset is a magnification of the histogram for low-GC content genes.

FIG. 5.

Phylogenetic trees within the R. solanacearum species complex based upon analysis of partial mutS gene sequences and of the presence or absence of the GMI1000 genes. (a) mutS gene; (b) all GMI1000 genes; (c) chromosomal genes; (d) megaplasmid genes; (e) genes within ACURs; (f) mobile element genes; (g) effector genes; (h) hrpB- and hrpG-regulated genes. Triangles represent strains from phylotype I; squares, phylotype II; circles, phylotype III; stars, phylotype IV. Bootstrap values are indicated when they are below 85. The scale bar in panel a represents 1 nucleotide substitution per 10 nucleotides. The scale bars in panels b, c, d, e, f, g, and h represent a Sokal-Michener distance value of 0.1.