Dataset Information

Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

ABSTRACT:

Unlabelled

Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3(-)), nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3(-), corresponding to R. solanacearum's optimal NO3(-) concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (?narG), denitrification (?aniA, ?norB, and ?nosZ), or NO detoxification (?hmpX). The ?narG, ?aniA, and ?norB mutants grew poorly on NO3(-) compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3(-) respiration directly enhanced growth, AniA-dependent NO2(-) reduction did not. NO2(-) and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3(-) acts as a TEA, but the resulting NO2(-) and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2(-) reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3(-) to respire, grow, and cause disease. Degradation of NO2(-) and NO is also important for successful infection and depends on denitrification and NO detoxification systems.

Importance

The plant-pathogenic bacterium Ralstonia solanacearum causes bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show that R. solanacearum rapidly depletes oxygen in host xylem but can then respire using host nitrate as a terminal electron acceptor. The microbe uses its denitrification pathway to detoxify the reactive nitrogen species nitrite (a product of nitrate respiration) and nitric oxide (a plant defense signal). Detoxification may play synergistic roles in bacterial wilt virulence by converting the host's chemical weapon into an energy source. Mutant bacterial strains lacking elements of the denitrification pathway could not grow as well as the wild type in tomato plants, and some mutants were also reduced in virulence. Our results show how a pathogen's metabolic activity can alter the host environment in ways that increase pathogen success.

SUBMITTER: Dalsing BL

PROVIDER: S-EPMC4453514 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

Dalsing Beth L BL Truchon Alicia N AN Gonzalez-Orta Enid T ET Milling Annett S AS Allen Caitilyn C

mBio 20150317 2

<h4>Unlabelled</h4>Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3(-)), nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. so ...[more]

PMID: 25784703

Similar Datasets

Project description:Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that R. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

Project description:BackgroundRalstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).ResultsFour hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.ConclusionsThe putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.

Project description:Strains of the Ralstonia solanacearum species complex in the phylotype IIB group are capable of causing Bacterial Wilt disease in potato and tomato at temperatures lower than 24°C. The capability of these strains to survive and to incite infection at temperatures colder than their normally tropical boundaries represents a threat to United States agriculture in temperate regions. In this work, we used a comparative genomics approach to identify orthologous genes linked to the lower temperature virulence phenotype. Six R. solanacearum cool virulent (CV) strains were compared to six strains non-pathogenic at low temperature (NPLT). CV strains can cause Bacterial Wilt symptoms at temperatures below 24°C, while NPLT cannot. Four R. solanacearum strains were sequenced for this work in order to complete the comparison. An orthologous genes comparison identified 44 genes present only in CV strains and 19 genes present only in NPLT strains. Gene annotation revealed a high percentage of genes compared with whole genomes in the transcriptional regulator and transport categories. A single nucleotide polymorphism (SNP) analysis identified 265 genes containing conserved non-synonymous SNPs in CV strains. Ten genes in the pathogenicity category were identified in this group. Comparisons of type 3 secretion system, type 6 secretion system (T6SS) clusters, and associated effectors did not indicate a correlation with the CV phenotype except for one T6SS VGR effector potentially associated with the CV phenotype. This is the first R. solanacearum genomic comparative analysis of multiple strains with different temperature related virulence. The candidate genes identified by this comparison are potential factors involved in virulence at low temperatures that need to be investigated. The high percentage of transcriptional regulators among the genes present only in CV strains supports the hypothesis that temperature dependent regulation of virulence genes explains the differential virulence phenotype at low temperatures. This comparison contributes to find new possible connections of temperature dependent virulence to the previously described complex regulatory system involving quorum-sensing, phenotype conversion (phcA), acyl-HSL production and responses to SA. It also added novel candidate T6SS effectors and useful detailed information about the T6SS in R. solanacearum.

Dataset Information

Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

Unlabelled

Importance

Publications

Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets