Nitrate reduction to ammonium in Nautilia profundicola via a proposed reverse HURM pathway
University of Delaware
Nautilia profundicola is an epsilonproteobacterium that inhabits hydrothermal vents on the dorsal epithelium of Alvinella pompejana or as a free-living bacterium. N. profundicola was isolated with polysulfide as the electron acceptor for respiratory growth, but is also able to utilize nitrate as an electron acceptor for growth. Interestingly, the N. profundicola genome lacks homologs of known enzymes used in nitrite reduction. Therefore, the pathway of nitrate reduction is likely to be novel. Genome annotation suggested that the reverse hydroxylamine ubiquinone redox module (rHURM) may be used for anaerobic cellular respiration in N. profundicola when nitrate is the terminal electron acceptor. The proposed rHURM pathway includes rHaoA and hcp/har which catalyze the reduction of nitrite to hydroxylamine, as an intermediate, and hydroxylamine to ammonium, respectively. In this study, cell growth was monitored in batch cultures under varied concentrations of sulfide, nitrate, formate, and H2. Optimal concentrations were used for culturing cells in continuous culture and in batch culture for substrate addition assays. The four nitrogen compounds in the proposed pathway were measured in the substrate addition assays—nitrate, nitrite, hydroxylamine, and ammonium. Hydroxylamine was present in small concentrations during nitrate reduction to ammonium. When given as a substrate, hydroxylamine was directly and entirely reduced to ammonium (up to 5 mM). An anaerobic continuous culture apparatus for N. profundicola was developed to grow cells in steady state with different electron acceptors to eliminate growth rate variations that have affected prior gene expression measurements. The results presented here are consistent with hydroxylamine as a free intermediate in nitrate reduction to ammonium in N. profundicola. Additional experiments including in vitro assays of enzymatic activity and gene disruption will be required to fully address the role of the proposed rHURM pathway in nitrate reduction.
Nautilia profundicola, Reverse hydroxylamine:ubiquinone redox module, Dissimilatory nitrate reduction to ammonium, Epsilonproteobacteria from hydrothermal vent