Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer

Ralstonia solanacearum Dps Contributes to Oxidative Stress Tolerance and to Colonization of and Virulence on Tomato Plants▿

Ralstonia solanacearum, an economically important soilborne plant pathogen, infects host roots to cause bacterial wilt disease. However, little is known about this pathogen's behavior in the rhizosphere and early in pathogenesis. In response to root exudates from tomato, R. solanacearum strain UW551 upregulated a gene resembling Dps, a nonspecific DNA binding protein from starved cells that is critical for stress survival in other bacteria. An R. solanacearum dps mutant had increased hydrogen peroxide sensitivity and mutation rate under starvation. Furthermore, dps expression was positively regulated by the oxidative stress response regulator OxyR. These functional results are consistent with a Dps annotation. The dps mutant caused slightly delayed bacterial wilt disease in tomato after a naturalistic soil soak inoculation. However, the dps mutant had a more pronounced reduction in virulence when bacteria were inoculated directly into host stems, suggesting that Dps helps R. solanacearum adapt to conditions inside plants. Passage through a tomato plant conferred transient increased hydrogen peroxide tolerance on both wild-type and dps mutant strains, demonstrating that R. solanacearum acquires Dps-independent oxidative stress tolerance during adaptation to the host environment. The dps mutant strain was also reduced in adhesion to tomato roots and tomato stem colonization. These results indicate that Dps is important when cells are starved or in stationary phase and that Dps contributes quantitatively to host plant colonization and bacterial wilt virulence. They further suggest that R. solanacearum must overcome oxidative stress during the bacterial wilt disease cycle

Necessity of OxyR for the Hydrogen Peroxide Stress Response and Full Virulence in Ralstonia solanacearum ▿ †

The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence