75b74913-1f1f-411c-934f-b9a2deaa1a81
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.
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.