Nitrate reduction salvage pathway in Methanococcales

Nitrate is the most oxidized form of nitrogen and an essential nutrient for many living organisms. Its utilization was considered impossible in methanogenic archaea, since nitrate reduction inherently generates nitrite, a potent oxidant that can disrupt their catabolism. Yet, our study demonstrates that the hyperthermophile Methanocaldococcus infernus defies this rule by growing on nitrate as its sole nitrogen source. Comparative analyses revealed genes encoding a putative nitrate transporter and a nitrate reductase in M. infernus, as well as in Methanothermococcus thermolithotrophicus, which was first discovered to consume nitrate. The minimal operon is detected in many bacterial species inhabiting similar niches, supporting horizontal gene transfer acquisition. Based on in silico investigations, we propose that the transporter is a symporter that would rely on an ion gradient. We also predict that the putative nitrate reductase contains all molecular determinants for its activity. The observed nitrate-dependent growth in the absence of molybdenum would imply a tungsten-dependent nitrate reductase. The last reaction of the pathway is catalyzed by a F₄₂₀H₂-dependent sulfite reductase. The structure obtained at atomic resolution reveals an endogenous mixture of nitrite and sulfite bound to the siroheme catalyst, underscoring the enzyme's dual function previously demonstrated in vitro. Our results led us to a metabolic model in which the nitrate-assimilation pathway would be indirectly powered by methanogenesis and H₂-oxidation. This adaptation is another remarkable example of how Methanococcales extend their assimilation capabilities by hijacking bacterial systems and repurposing their F₄₂₀H₂-sulfite reductase to prevent oxidative damage.