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.
Less...