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

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.

Fig. 1. Phylogenetic affiliation of T. ammonificans G1 and distribution of ONR compared to other N-transforming proteins in the order Geobacterales.

The maximum-likelihood tree is based on a concatenated alignment of 92 universal single-copy bacterial core genes. Two Desulfuromonas genomes served as outgroup. Bootstrap support values derived from 1000 ultrafast bootstrap iterations = 100% are represented by filled circles. The scale bar corresponds to 5% sequence divergence. The matrix on the right indicates presence and absence of ONR, NapA, NrfA and NarG (BLAST p ≥ 50% identity, ≥ 90% query coverage); white, no homologous protein; black, homologous protein to T. lovleyi. Coloured circles represent reductive nitrogen conversion potential; green, confirmed DNRA activity; yellow, confirmed nitrite reduction activity, but product not analyzed; blue, confirmed denitrification activity; red, no confirmed NO₃^- reduction. Except for T. ammonificans G1, the confirmed activity is based on previous studies. Accession numbers of included Geobacterales genomes are listed in Table S6. A phylogenomic tree including all Geobacterales genomes are shown in Fig. S2.

Fig. 2. Anaerobic growth dynamics of strain G1.

A Growth in nitrate-limited ammonifying batch culture in presence of 0.1 mM sulfide and 0.2 mM cysteine as reductants. B N₂O formation from nitrate and nitrite (1 mM additions) pulse-added to the nitrate-limited chemostat culture. Total N₂O emissions constituted 0.44% and 2.8% of the consumed nitrate and nitrite, respectively. C Use of H₂ (20 ml culture in 32 ml flasks, 15 ml addition to the gas phase after 67 h incubation is indicated by arrow) as an e-donor during ammonification in an acetate-limited batch culture of strain G1 with 5 mM nitrate. Black lines, growth (OD₆₀₀); blue lines, NO₂^- (mM); red lines, NH₄⁺ (mM); diamonds, 0.5 mM acetate; circles, 1 mM acetate; triangles, 2 mM acetate. Results in panels (A) and (C) are mean values from two parallel incubations.

Fig. 3. Organization of the novel DNRA module in T. ammonificans G1 based on genomic and biochemical analyses.

A Schematic representation of the Nap-ONR gene locus. B Scheme of the Nap-ONR module reducing nitrate to ammonium: NapAB reduces nitrate to nitrite, ONR catalyzes the dissimilatory reduction of nitrite to ammonium, and the ferredoxins NapGH are involved in electron transport from the menaquinone pool. NO and N₂O are minor side products. NO originates from partial nitrite reduction by ONR at high nitrite and suboptimal redox conditions, while N₂O is produced from NO in a detoxification mechanism due to the activity of hydroxylamine reductase (HCP) and a ferredoxin (Fd). NapD is a maturation chaperone for NapA, a role also speculated for NapF. Gene identifiers are indicated as numbers in the proteins and asterisks indicate low sequence similarity to characterized proteins. C Expression level (relative spectral abundance) of the Nar operon, the Nap-ONR module proteins, and HCP in strain G1 cells grown in a nitrate-limited chemostat culture with acetate as e-donor and C-source. The proteomics data is summarized in Fig. S6 and Table S4. HCP, hydroxylamine reductase; Fd, ferredoxin; MQ, menaquinone; MQH₂, menaquinol; Nap, periplasmic nitrate reductase; Nar, cytoplasmic nitrate reductase; ONR, octaheme nitrite reductase; TAT, twin-arginine translocation system; TR, transcriptional regulator.

Fig. 4. Phylogeny of the octaheme cytochrome c proteins potentially involved in nitrogen redox conversions.

The maximum likelihood tree was calculated using iqtree with 1000 ultrafast bootstrap iterations. Proteins with confirmed nitrite ammonifying activity mentioned in the main text are shown in bold; the T. ammonificans G1 ONR in red. Bootstrap support values ≥90% are indicated by filled circles. NrfA proteins in Geobacterales members and Anaeromyxobacter spp. were used to root the tree. The scale bar corresponds to 20% sequence divergence. ONR, octaheme nitrite reductase; HAO, hydroxylamine dehydrogenase; HZO, hydrazine dehydrogenase; eHAO, εHAO. An extended phylogenetic tree of the octaheme cytochrome c oxidoreductases is shown in Fig. S5.

Fig. 5. Functional properties of purified T. ammonificans G1 ONR (TaNiR).

A Visible spectra of oxidized and reduced forms of TaNiR. B Potentiometric titration of TaNiR and its homologue Tv. paradoxus ONR. C Screening of the TaNiR catalytic activities by oxidation of reduced TaNiR with potential substrates. 1.9 µM of EuCl₂-reduced TaNiR was incubated in 50 mM HEPES (pH 7.5) with 1.4 mM of the potential substrates NaNO₃, NaNO₂, NH₂OH, or Na₂SO₃. In the control experiment the corresponding volume of buffer was added.