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 (NO 3 -), nitrite (NO 2 -), 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 ~25mMNO 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 (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). 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.