Ralstonia solanacearum Uses Inorganic Nitrogen Metabolism for Virulence, ATP Production, and Detoxification in the Oxygen-Limited Host Xylem Environment

Abstract

ABSTRACT Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO 3 − ), nitrite (NO 2 − ), nitric oxide (NO), and nitrous oxide (N 2 O) 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 NO 3 − , corresponding to R. solanacearum's optimal NO 3 − 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 (Δ narG ), denitrification (Δ aniA , Δ norB , and Δ nosZ ), or NO detoxification (Δ hmpX ). The ΔnarG , ΔaniA , and ΔnorB mutants grew poorly on NO 3 − compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO 3 − respiration directly enhanced growth, AniA-dependent NO 2 − reduction did not. NO 2 − and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO 3 − acts as a TEA, but the resulting NO 2 − and NO likely do not. None of the mutants grew as well as the wild type in planta , and strains lacking AniA (NO 2 − reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO 3 − to respire, grow, and cause disease. Degradation of NO 2 − 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.