Comparative Analysis of Ralstonia solanacearum Methylomes

Abstract

Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants.

Keywords: Ralstonia; comparative genomics; epigenomics; genome; methylome; nucleotide modification; transcriptional regulation; transposon.

Figure 1

Fraction of modification marks mapping to upstream regions of annotated genes across a panel of 210 methylomes. The fraction of upstream marks is relative to the sum of marks mapping to upstream, downstream, and intragenic regions of annotated genes in each genome. The boxplot columns designate different datasets: non-motif associated modification marks, motif-associated modification marks, %GC-controlled (28–38% GC) motif-associated modification marks, and marks associated with the widely distributed GTNAC and GTAC motifs. For each column, the bracketed numbers in the abscissa legend indicate the number of unique motifs in the dataset, the number of instances of those motifs identified in the complete set of methylomes and the number of organisms on which such instances were detected. The data points corresponding to the R. solanacearum GMI1000 and R. solanacearum UY031 GTWWAC motifs are boxed.

Figure 2

Distribution of GTWWAC methylation marks conserved in R. solanacearum GMI1000 and R. solanacearum UY031 as a function of their location relative to annotated genes. The plot shows the number of GTWWAC methylation marks conserved in each location category. Non-conserved marks are distinguished from those mapping to genes lacking an identifiable ortholog in either strain. The relative conservation of GTWWAC methylation marks in each region (excluding marks mapping to genes lacking orthologs) is indicated on top of the bars. (A) GTWWAC methylation marks. (B) Non-GTWWAC methylation marks.

Figure 3

Conservation of GTWWAC and non-GTWWAC associated marks across a panel of 12 reference complete Ralstonia genomes. The plot shows the average number of genomes in which the R. solanacearum GMI1000 context of a methylation mark is considered to be conserved (alignment identities above 70%) for different regions (upstream, intragenic, and downstream) relative to genes with orthologs in R. solanacearum GMI1000 and R. solanacearum UY031. Vertical bars indicate the standard error of the mean. The p-values of a two-tailed Mann Whitney U-test between GTWWAC and non-GTWWAC associated marks are provided on top of the bars.

Figure 4

Positional distribution of nucleotide changes with respect to R. solanacearum GMI1000 in gapless alignments of conserved modification marks. The plots show the fraction of alignments containing mismatches at each alignment position for marks conserved in R. solanacearum GMI1000 and UY031 strains located in upstream, downstream and intragenic regions. The fraction is computed based on cumulative alignment mismatch counts for full gapless BLAST alignments (100% coverage) against a panel of 12 complete Ralstonia genomes. The number of conserved marks in each region, and the number of full alignments used to tally mismatches are provided. Mismatches on the first and last two positions of the alignment are not expected due to the greedy nature of the BLAST hit extension process.

Figure 5

Schematic representation of the upstream region for conserved loci enriched in methylated, non-methylated and highly-conserved GTWWAC sites. Accessions, locus tags and coordinates are provided for the R. solanacearum GMI1000 genome. A mapping to old GMI1000 locus tag identifiers is provided in Supplementary Table 2. When not annotated in R. solanacearum GMI1000, gene acronyms are derived from homology searches against the E. coli genome or from representative domains (uppercase). GTWWAC sites are denoted by boxes, with their methylation state in R. solanacearum GMI1000 indicated by solid/dotted outlines and their methylation state in R. solanacearum UY031 indicated by white/shaded fillings. Triangles denote ParA boxes annotated in the R. solanacearum GMI1000 genome. Arrows indicate directional −35 and −10 promoter elements predicted by Phi-Site, BPROM, and PePPER. When predictions overlap, the results are shown using the following coloring precedence: Phi-Site, BPROM, and PePPER.

Figure 6

Association of normalized modification mark density with transposable elements. The plot shows the genomic distribution of normalized modification mark density using a 1,000 bp window with a 100 bp step size on the R. solanacearum UY031 chromosome and megaplasmid. The presence of transposable elements within the sliding window is indicated by light blue bars. The point-biserial correlation coefficient and its p-value are provided for each replicon. A green horizontal line indicates the threshold for high modification density (three standard deviations above the mean normalized modification density).

Figure 7

Distribution of modification marks along the ISRso3 transposase [WP_003261205.1; ENOG4105F2I] of R. solanacearum UY031. The plot shows aggregated modification mark counts in upstream, intragenic and downstream regions of the 86 genes coding for WP_003261205.1 in R. solanacearum UY031. Mark counts were computed on 5 bp bins. Upstream, intragenic and downstream regions are delineated by shading color. Red arrows designate the location of the inverted repeats (IR) targeted by the ISrso3 transposase.