Insect Gut Isolate Pseudomonas sp. Nvir Degrades the Toxic Plant Metabolite Nitropropionic Acid

Nitropropionic acid (NPA) is a widely distributed naturally occurring nitroaliphatic toxin produced by leguminous plants and fungi. It was found to be exceedingly poisonous to humans and animals leading to a number of outbreaks caused by ingestion of NPA-contaminated products all over the world. The Southern green shield bug feeds on leguminous plants and shows no symptoms of intoxication. Likewise, its gut-associated microorganisms are subjected to high levels of this toxic compound. In this study, using an NPA-based enrichment strategy, we isolated a bacterium from an insect's gut system that was highly resistant to NPA and was fully degrading it to inorganic nitrogen compounds and carbon dioxide. The isolate, classified as Pseudomonas sp. Nvir was using NPA as, carbon, nitrogen, and energy source. To elucidate the pathway of NPA degradation and to determine the metabolic fate of the carbon and nitrogen atoms in the NPA molecule, we performed isotopologue tracer experiments. Labelling experiment with 15N-NPA revealed that NPA was degraded to nitrite and nitrate which were transformed to ammonia with subsequent canonical nitrogen assimilation via amino acids, shown by the steady incorporation of 15N into glutamate and glutamine. Moreover, with 1-13C-NPA we determined that CO2 was another by-product of NPA degradation since a significant production of 13CO2 took place over 24 hours. Along, we aimed to identify the remaining NPA degradation intermediates and to determine their fate in the Pseudomonas sp. Nvir metabolism. An experiment with 13C-isotopically labelled biomass revealed the incorporation of 12C atoms from U-12C-NPA into the tricarboxylic acid cycle metabolites succinate, malate, aspartate, and glutamate, by that illustrating that NPA was used in central carbon metabolism. In order to understand the NPA degradation pathway in more detail, we performed genomics and transcriptomics. With these analyses we determined the majority of the genes encoding enzymes involved in the NPA detoxification. Besides this, we discovered a novel pnmR gene that possibly encodes an uncharacterized propionate-3-nitronate monooxygenase which is likely responsible for the first step in the NPA degradation. In the end, based on genomic, transcriptomic, and metabolic evidence we propose a new NPA detoxification pathway originating from a gut bacterium of the Southern green shield bug. It is tempting to speculate that the shield bug may benefit from the metabolic detoxification abilities of Pseudomonas sp. Nvir via host-microbe interactions, and hence may act as a symbiotic microorganism. Finally, we advocate that this NPA-detoxifying isolate might find application in the agriculture and food industry by dealing with postharvest NPA-contaminations in economically important crops.