The physiological potential of anammox bacteria as revealed by their core genome structure

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although NO2- reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. K. stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologs. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least 5 paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH2OH by self-production for anammox metabolism.

Genomic and morphologic characterization of a planktonic Thiovulum (Campylobacterota) dominating the surface waters of the sulfidic Movile Cave, Romania

ABSTRACTLife in Movile Cave (Romania) relies entirely on primary carbon fixation by bacteria oxidizing sulfide, methane and ammonia with oxygen, nitrate, sulfate, and ferric iron. There, large spherical-ovoid bacteria (12-16 μm diameter), rich in intracellular sulfur globules, dominate the stable microbial community in the surface water of a hypoxic Air Bell. These were identified as Thiovulum sp. (Campylobacterota). We obtained a closed genome of this Thiovulum and compared it to that of Thiovulum ES. The genes for oxidizing sulfide to sulfate are absent, therefore, Thiovulum likely avoids constant accumulation of elemental sulfur either by oxidizing sulfide to sulfite which is then excreted, or via dissimilatory nitrate reduction to ammonia using the formate-dependent nitrite reductase or hydroxylamine oxidoreductase. Thus, Thiovulum, found also in other caves, is likely important to both S and N cycles in subterranean aquatic ecosystems. Additionally, using electron microscopy, we suggest that in absence of motor-like structures along the membrane, the peritrichous flagella-like structures are type IV pili, for which genes were found in both Thiovulum genomes. These pili may play a role in veil formation, connecting adjacent cells. The force exerted by coordinated movement of such pili may partly explain the exceptionally fast swimming of these bacteria.

Multiheme hydroxylamine oxidoreductases produce NO during ammonia oxidation in methanotrophs

Aerobic and nitrite-dependent methanotrophs make a living from oxidizing methane via methanol to carbon dioxide. In addition, these microorganisms cometabolize ammonia due to its structural similarities to methane. The first step in both of these processes is catalyzed by methane monooxygenase, which converts methane or ammonia into methanol or hydroxylamine, respectively. Methanotrophs use methanol for energy conservation, whereas toxic hydroxylamine is a potent inhibitor that needs to be rapidly removed. It is suggested that many methanotrophs encode a hydroxylamine oxidoreductase (mHAO) in their genome to remove hydroxylamine, although biochemical evidence for this is lacking. HAOs also play a crucial role in the metabolism of aerobic and anaerobic ammonia oxidizers by converting hydroxylamine to nitric oxide (NO). Here, we purified an HAO from the thermophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV and characterized its kinetic properties. This mHAO possesses the characteristic P460 chromophore and is active up to at least 80 °C. It catalyzes the rapid oxidation of hydroxylamine to NO. In methanotrophs, mHAO efficiently removes hydroxylamine, which severely inhibits calcium-dependent, and as we show here, lanthanide-dependent methanol dehydrogenases, which are more prevalent in the environment. Our results indicate that mHAO allows methanotrophs to thrive under high ammonia concentrations in natural and engineered ecosystems, such as those observed in rice paddy fields, landfills, or volcanic mud pots, by preventing the accumulation of inhibitory hydroxylamine. Under oxic conditions, methanotrophs mainly oxidize ammonia to nitrite, whereas in hypoxic and anoxic environments reduction of both ammonia-derived nitrite and NO could lead to nitrous oxide (N2O) production.

Genomic Insights into the Carbon and Energy Metabolism of a Thermophilic Deep-Sea Bacterium Deferribacter autotrophicus Revealed New Metabolic Traits in the Phylum Deferribacteres

Information on the biochemical pathways of carbon and energy metabolism in representatives of the deep lineage bacterial phylum Deferribacteres are scarce. Here, we report the results of the sequencing and analysis of the high-quality draft genome of the thermophilic chemolithoautotrophic anaerobe Deferribacter autotrophicus. Genomic data suggest that CO2 assimilation is carried out by recently proposed reversible tricarboxylic acid cycle (“roTCA cycle”). The predicted genomic ability of D. autotrophicus to grow due to the oxidation of carbon monoxide was experimentally proven. CO oxidation was coupled with the reduction of nitrate to ammonium. Utilization of CO most likely involves anaerobic [Ni, Fe]-containing CO dehydrogenase. This is the first evidence of CO oxidation in the phylum Deferribacteres. The genome of D. autotrophicus encodes a Nap-type complex of nitrate reduction. However, the conversion of produced nitrite to ammonium proceeds via a non-canonical pathway with the participation of hydroxylamine oxidoreductase (Hao) and hydroxylamine reductase. The genome contains 17 genes of putative multiheme c-type cytochromes and “e-pilin” genes, some of which are probably involved in Fe(III) reduction. Genomic analysis indicates that the roTCA cycle of CO2 fixation and putative Hao-enabled ammonification may occur in several members of the phylum Deferribacteres.

Insights into the Variations of Hao-Dependent Nitrifying and Nir-Dependent Denitrifying Microbial Communities in Ammonium-Graduated Lake Environments

Biological nitrification and denitrification play significant roles in nitrogen-associated biogeochemical cycles. However, our understanding of the spatial scales at which microbial communities act and vary is limited. We used gene-specific metagenomic PCR to explore changes in nitrifying and denitrifying microbial communities within pristine lake and its branches, where the ammonium and dissolved organic carbon (DOC) concentrations form a gradient. The biomarkers hydroxylamine oxidoreductase and nitrite reductase genes indicated that strong relationships exist between the diversities and community structures of denitrifiers and ammonium gradients. It showed that the Nitrosomonas oligotropha cluster dominates the nitrifying bacteria in low-nutrition environments, while a new Nitrosomonas ureae cluster accounted for nearly 80% of the nitrifying bacteria in high-nitrogen environments. The distribution and diversity of nirS/K-dependent denitrifiers in the various habitats were similar, but predominantly affiliated with unknown clusters. Moreover, the abundance of all the hao genes dramatically outnumbered that of nir genes. The relative abundance of hao was clearly higher during eutrophication (13.60%) than during oligotrophy (5.23%), whereas that of nirS showed opposite tendencies. Overall, this study provides valuable comparative insights into the shifts in nitrifying and denitrifying microbial populations in lake environments with ammonium gradients, suggesting that unique dominant denitrifiers probably play an important role in the nitrogen cycle.

Genomic profiling ofNitrospiraspecies reveals ecological success of comammoxNitrospira

Nitrification was long thought to consist in the stepwise oxidation of ammonia to nitrite and of nitrite to nitrate by ammonia oxidizing and nitrite oxidizing microorganisms, respectively. Recently, single microorganisms capable of complete ammonia to nitrate oxidation (comammox) were identified in theNitrospiragenus. This genus, previously considered to only contain canonical nitrite oxidizers is diverse and has a broad environmental distribution. Yet, a global insight into the abundance, niche preference, and genomic diversity ofNitrospirais missing. Here, we established the largestNitrospiragenome database to date, revealing 68 putative species, most without cultivated representatives. We performed a global survey through read recruitment of metagenomic data from various environments against this database that identified that environmental filtering structures species distribution, without large scale biogeographical signal. The ecological success of comammoxNitrospirais evident as they outnumber and are more diverse than canonicalNitrospirain communities from all environments but wastewater treatment plants. We detect a phylogenetic signal inNitrospiraspecies habitat preference, that is strongest for canonicalNitrospiraspecies. ComammoxNitrospiraeco-evolutionary history is more complex with subclades achieving rapid niche divergence via horizontal transfer of genes, including that encoding the hydroxylamine oxidoreductase, one of the key enzymes involved in nitrification.

A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway

The hydroxylamine oxidoreductase/hydrazine dehydrogenase (HAO/HDH) protein family constitutes an important group of octaheme cytochromes c (OCCs). The majority of these proteins form homotrimers, with their subunits being covalently attached to each other via a rare cross-link between the catalytic heme moiety and a conserved tyrosine residue in an adjacent subunit. This covalent cross-link has been proposed to modulate the active-site heme towards oxidative catalysis by distorting the heme plane. In this study, the crystal structure of a stable complex of an HAO homologue (KsHAOr) with its diheme cytochrome c redox partner (KsDH) from the anammox bacterium Kuenenia stuttgartiensis was determined. KsHAOr lacks the tyrosine cross-link and is therefore tuned to reductive catalysis. The molecular model of the KsHAOr–KsDH complex at 2.6 Å resolution shows a heterododecameric (α6β6) assembly, which was also shown to be the oligomeric state in solution by analytical ultracentrifugation and multi-angle static light scattering. The 60-heme-containing protein complex reveals a unique extended electron transfer pathway and provides deeper insights into catalysis and electron transfer in reductive OCCs.