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

Abstract

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 (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB 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.

FIG 1

During tomato infection, Ralstonia solanacearum strain GMI1000 has high levels of expression of a complete nitrate respiration and denitrification pathway, including a nitric oxide detoxification system. (A) Inorganic N metabolic pathway with relevant enzyme shown above each reaction. The numbers below each reaction arrow indicate fold induction of the corresponding gene during plant infection relative to expression levels in culture (17). (B) Cellular context of NO₃⁻ respiration and denitrification. Following glycolysis and the citric acid cycle, electrons move through the electron transport chain to a terminal electron acceptor (TEA). In R. solanacearum, nitrate (NO₃⁻), nitrite (NO₂⁻), nitric oxide (NO^.), and nitrous oxide (N₂O) can potentially serve as TEAs. Data associated with each enzyme and its corresponding mutant are color coded consistently in all figures.

FIG 2

Nitrate respiration facilitates Ralstonia solanacearum growth at oxygen concentrations found in planta. (A) Culture densities (OD₆₀₀) of wild-type (WT), ΔnarG, and complemented ΔnarG strains of R. solanacearum following 24 h of incubation in VDM medium with 10 mM NO₃⁻ at a range of oxygen concentrations. The WT strain grew better at 15% and 10% O₂ than at 20.9% O₂ (P = 0.0166 and 0.0206, respectively). Each bar represents the mean result from 3 biological replicates, and error bars indicate standard errors of the means. *, significantly different from the result for the WT at the same O₂ concentration (P < 0.05, t test). (B) A Unisense multimeter microsensor was used to measure O₂ concentrations in xylem sap from wilt-susceptible tomato plants following soil soak inoculation with either water or wild-type R. solanacearum. Each point represents data collected from a single plant. Ten plants per treatment were sampled at the time of symptom onset. Horizontal bars indicate median values. The gray dotted line indicates O₂ saturation under these conditions. O₂ concentrations in sap from healthy and infected plants were different (P = 0.0003, t test).

FIG 3

Nitrate, nitrite, and nitric oxide respiration contribute to R. solanacearum’s growth under anaerobic conditions. Growth of R. solanacearum strains was measured as the OD₆₀₀ following 24 h of anaerobic (A) and aerobic (B) incubation in VDM medium with 10 mM NO₃⁻. Bars show mean results for growth of 3 biological replicates, each containing 3 technical replicates. Error bars represent standard errors. *, significantly different from the result for the WT (P < 0.05, t test). Complementation did not fully restore the ability of all mutants to grow under these conditions, but each complemented strain grew better than its parent mutant (P < 0.05, t test).

FIG 4

Nitrate directly supports the growth of R. solanacearum at biologically relevant concentrations. (A) Growth of wild-type R. solanacearum in 0.1% O₂ in VDM medium with various NO₃⁻ concentrations. Data are mean results from 3 biological replicates. Growth in 0 mM NO₃⁻ was significantly different from growth in 1 mM, 10 mM, 25 mM, 50 mM, and 100 mM NO₃⁻ (P < 0.05, repeated measures ANOVA). Growth in 50 mM NO₃⁻ (not shown) was indistinguishable from growth in 25 mM NO₃⁻. Error bars represent standard errors. (B) Twenty-one-day-old wilt-susceptible tomato plants were inoculated with either water or wild-type R. solanacearum. At symptom onset, plant xylem sap was collected, and NO₃⁻ was quantified in each sample using Unisense multimeter NO_x and NO₂⁻ probes. Horizontal bars indicate mean values. Symbols indicate values from four plants inoculated with water and 12 plants inoculated with wild-type R. solanacearum.

FIG 5

Nitrite inhibits R. solanacearum’s growth under low-oxygen conditions and is produced and consumed in planta via nitrate respiration and denitrification. (A) Growth of wild-type R. solanacearum in 0.1% O₂ in VDM medium with various NO₂⁻ concentrations. Data are mean results from 3 biological replicates. Growth with 0 mM NO₂⁻ was significantly different from growth with 10 mM and 100 mM NO₂⁻ (P < 0.05, repeated measures ANOVA). Error bars represent standard errors. (B) NO₂⁻ concentrations in xylem sap from tomato plants infected with wild-type (WT), ΔnarG, or ΔaniA R. solanacearum or water-inoculated controls. At symptom onset, xylem sap was harvested and NO₂⁻ concentrations were determined using the Griess reaction. Horizontal bars indicate mean values, and each symbol represents the NO₂⁻ level in an individual plant. Xylem sap from plants infected with the ΔaniA mutant contained more NO₂⁻ than sap from plants infected with the WT (P = 0.0337, t test).

FIG 6

Relative energy charge levels indicate that R. solanacearum uses nitrate directly as a terminal electron acceptor. Cellular adenylate energy charge of wild-type and denitrification mutants incubated anaerobically in VDM medium with 10 mM NO₃⁻ for 20 h at 28°C was determined as described previously (28). Mean energy charges for each strain are shown as percentage of wild-type levels; data reflect 3 biological replicates, each with 3 technical replicates. Error bars show standard errors. *, significantly different from the WT result (P < 0.01, unpaired t test with Welch’s correction).

FIG 7

R. solanacearum detoxifies nitric oxide by denitrification under low-oxygen conditions and by HmpX under high-oxygen conditions. Overnight bacterial cultures in VDM medium without NO₃⁻ were pelleted, resuspended in fresh VDM medium without NO₃⁻, and incubated for 4 h. Cultures were then diluted 1:10 in VDM medium with 10 mM NO₃⁻ and incubated statically for 2 h to induce the expression of denitrifying enzymes. One millimolar NO in the form of spermine NONOate was added to half of the wells, and the OD₆₀₀ was read over 72 h. The growth of each strain with and without NO is shown as a ratio for lag time (A) and doubling time (B). *, significantly different from the WT result (P < 0.05, t test). The experiment was replicated 3 times with 3 technical replicates for each treatment; data from a representative assay are shown. Error bars indicate standard errors. (C) Oxidase inhibition assay. Overnight cultures were resuspended to uniform OD₆₀₀ in fresh VDM medium with 10 mM NO₃⁻ and shaken aerobically for 3 h. One millimolar NO in the form of spermine NONOate was spiked into cultures, and O₂ consumption was tracked until concentrations reached 0 or plateaued. Data are means of results from 3 biological replicates.

FIG 8

Respiratory nitrogen metabolism and nitric oxide degradation contribute to bacterial wilt virulence and support pathogen growth in planta. Twenty-one-day-old tomato plants were inoculated with 500 CFU of the specified R. solanacearum strains through a cut leaf petiole. (A) Bacterial growth in planta. At 3 days postinoculation (DPI), the pathogen population size in each plant was determined by grinding and dilution plating a 0.1-g stem section centered at the site of inoculation. Ten plants were sampled per strain; each symbol represents the bacterial population size in a single plant, and the horizontal bars indicate the median population size for each strain. The gray dotted line indicates the limit of detection; samples containing no detectable bacteria were given a value of 1. The wild-type strain grew significantly better in planta than all mutants tested; complementation restored growth of mutants, although not always to wild-type levels. Compared to the WT result, the P values for each strain’s growth were as follows: ΔnarG, P < 0.0001; Δnarg + comp (complemented Δnarg mutant), P = 0.6371; ΔaniA, P < 0.0001; Δania + comp, P < 0.0001; ΔnorB, P < 0.0001; Δnorb + comp, P < 0.0001; ΔnosZ, P = 0.0150; ΔhmpX, P = 0.0602; and Δhmpx + comp, P = 0.411. (B) Virulence assay. Plants were rated daily for 14 days using a disease index of 0 to 4. Data presented are mean results from 3 to 4 independent assays, each containing 10 plants per strain. Error bars indicate standard errors of the means. Disease progress curves of the ΔaniA and ΔhmpX mutants were significantly different from those of the WT strain (P < 0.001, repeated measures ANOVA).