Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H2 can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.
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The complete and annotated genome of Trichlorobacter ammonificans G1 is deposited at the European Nucleotide Archive under project PRJEB49551. The mass spectrometry proteomics raw data have been deposited in the ProteomeXchange consortium database with the dataset identifier PXD031212.
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The authors thank Ben Abbas for technical support, Geert Cremers for assistance in genome assembly and closure, and Berhard Schink for nomenclatural advise. DYS and MCMvL were supported by the Gravitation Program of the Dutch Ministry of Education, Culture and Science (SIAM grant 024.002.002); TVT, NID, AYS and VOP by the Russian Science Foundation (grant 23-74-30004); HK and SL by the Netherlands Organization for Scientific Research (grants VI.Veni.192.086 and 016.Vidi.189.050, respectively). In addition, Russian authors also had support from the Russian Ministry of Science and Higher Education. The LABGeM (CEA/Genoscope & CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of Investissement d'Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform.
The authors declare no competing interests.
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Sorokin, D.Y., Tikhonova, T.V., Koch, H. et al. Trichlorobacter ammonificans, a dedicated acetate-dependent ammonifier with a novel module for dissimilatory nitrate reduction to ammonia. ISME J 17, 1639–1648 (2023). https://doi.org/10.1038/s41396-023-01473-2