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Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. Hydroxylamine is an intermediate in both AOB and AOA, but homologues of hydroxylamine dehydrogenase (HAO), catalyzing bacterial hydroxylamine oxidation, are absent in AOA. Hydrazine is a substrate for bacterial HAO, while phenylhydrazine is a suicide inhibitor of HAO. Here, we examine the effect of hydrazines in AOA to gain insights into the archaeal ammonia oxidation pathway. We show that hydrazine is both a substrate and an inhibitor for AOA and that phenylhydrazine irreversibly inhibits archaeal hydroxylamine oxidation. Both hydrazine and phenylhydrazine interfered with ammonia and hydroxylamine oxidation in AOA. Furthermore, the AOA " Candidatus Nitrosocosmicus franklandus" C13 oxidized hydrazine into dinitrogen (N 2 ), coupling this reaction to ATP production and O 2 uptake. This study expands the known substrates of AOA and suggests that despite differences in enzymology, the ammonia oxidation pathways of AOB and AOA are functionally surprisingly similar. These results demonstrate that hydrazines are valuable tools for studying the archaeal ammonia oxidation pathway. IMPORTANCE Ammonia-oxidizing archaea (AOA) are among the most numerous living organisms on Earth, and they play a pivotal role in the global biogeochemical nitrogen cycle. Despite this, little is known about the physiology and metabolism of AOA. We demonstrate in this study that hydrazines are inhibitors of AOA. Furthermore, we demonstrate that the model soil AOA " Ca. Nitrosocosmicus franklandus" C13 oxidizes hydrazine to dinitrogen gas, and this reaction yields ATP. This provides an important advance in our understanding of the metabolism of AOA and expands the short list of energy-yielding compounds that AOA can use. This study also provides evidence that hydrazines can be useful tools for studying the metabolism of AOA, as they have been for the bacterial ammonia oxidizers.

Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C 2 to C 8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, " Candidatus Nitrosocosmicus franklandus" C13 and " Candidatus Nitrosotalea sinensis" Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C 5 ) appeared to be conserved across AOA lineages. Similarities in C 2 to C 8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from " Ca Nitrosocosmicus franklandus" and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits " Ca Nitrosocosmicus franklandus" and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from " Ca Nitrosocosmicus franklandus," and it does not compete with NH 3 for binding at the active site. IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase., (Copyright © 2020 Wright et al.)

" Candidatus Nitrosocosmicus franklandus" C13 is an ammonia-oxidizing archaeon (AOA) isolated from soil. Its complete genome is 2.84 Mb and possesses predicted AOA metabolic pathways for energy generation and carbon dioxide fixation but no typical surface layer (S-layer) proteins, only one ammonium transporter, and divergent A-type ATP synthase genes., (Copyright © 2019 Nicol et al.)

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization., (Copyright © 2016 Lehtovirta-Morley et al.)