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Trichlorobacter ammonificans, a dedicated acetate-dependent ammonifier with a novel module for dissimilatory nitrate reduction to ammonia

The ISME Journal volume 17, pages 1639–1648 (2023)Cite this article

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Abstract

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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Fig. 1: Phylogenetic affiliation of T. ammonificans G1 and distribution of ONR compared to other N-transforming proteins in the order Geobacterales.
Fig. 2: Anaerobic growth dynamics of strain G1.
Fig. 3: Organization of the novel DNRA module in T. ammonificans G1 based on genomic and biochemical analyses.
Fig. 4: Phylogeny of the octaheme cytochrome c proteins potentially involved in nitrogen redox conversions.
Fig. 5: Functional properties of purified T. ammonificans G1 ONR (TaNiR).

Data availability

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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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

    Dimitry Y. Sorokin, Eveline M. van den Berg, Renske S. Hinderks, Martin Pabst, Gijs J. Kuenen & Mark C. M. van Loosdrecht

  2. Winogradsky Institute of Microbiology, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, Russia

    Dimitry Y. Sorokin

  3. Bach Institute of Biochemistry, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, Russia

    Tamara V. Tikhonova, Natalia I. Dergousova, Anastasia Y. Soloveva & Vladimir O. Popov

  4. Department of Microbiology, Radboud Institute for Biological and Environmental Sciences, Radboud University, Nijmegen, The Netherlands

    Hanna Koch & Sebastian Lücker

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  1. Dimitry Y. Sorokin
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Contributions

DYS, TVT, GJK, and MCMvL conceived the study and designed the research. GJK, VOP, MCMvL, and SL supervised the project. DYS, TVT, EMvdB, RSH, NID, and AYS performed experiments and data analysis. HK, MP, and SL performed proteomic and bioinformatic analyses. DYS, HK, and SL wrote the manuscript with input from all authors. All authors discussed results and commented on the manuscript.

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Correspondence to Dimitry Y. Sorokin or Sebastian Lücker.

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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

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