Nitrification is a fundamental component of the global nitrogen cycle and leads to significant fertilizer loss and atmospheric and groundwater pollution. Nitrification rates in acidic soils (pH < 5.5), which comprise 30% of the world's soils, equal or exceed those of neutral soils. Paradoxically, autotrophic ammonia oxidizing bacteria and archaea, which perform the first stage in nitrification, demonstrate little or no growth in suspended liquid culture below pH 6.5, at which ammonia availability is reduced by ionization. Here we report the discovery and cultivation of a chemolithotrophic, obligately acidophilic thaumarchaeal ammonia oxidizer, “ Candidatus Nitrosotalea devanaterra,” from an acidic agricultural soil. Phylogenetic analysis places the organism within a previously uncultivated thaumarchaeal lineage that has been observed in acidic soils. Growth of the organism is optimal in the pH range 4 to 5 and is restricted to the pH range 4 to 5.5, unlike all previously cultivated ammonia oxidizers. Growth of this organism and associated ammonia oxidation and autotrophy also occur during nitrification in soil at pH 4.5. The discovery of Nitrosotalea devanaterra provides a previously unsuspected explanation for high rates of nitrification in acidic soils, and confirms the vital role that thaumarchaea play in terrestrial nitrogen cycling. Growth at extremely low ammonia concentration (0.18 nM) also challenges accepted views on ammonia uptake and metabolism and indicates novel mechanisms for ammonia oxidation at low pH.

Studies of the distribution of ammonia oxidising archaea (AOA) and bacteria (AOB) suggest distinct ecological niches characterised by ammonia concentration and pH, arising through differences in substrate affinity and ammonia tolerance. AOA form five distinct phylogenetic clades, one of which, the ‘Nitrososphaera sister cluster’, has no cultivated isolate. A representative of this cluster, named ‘Candidatus Nitrosocosmicus franklandus’, was isolated from a pH 7.5 arable soil and we propose a new cluster name: ‘Nitrosocosmicus’. While phylogenetic analysis of amoA genes indicates its association with the Nitrososphaera sister cluster, analysis of 16S rRNA genes provided no support for a relative branching that is consistent with a ‘sister cluster’, indicating placement within a lineage of the order Nitrososphaerales. ‘Ca. N. franklandus’ is capable of ureolytic growth and its tolerances to nitrite and ammonia are higher than in other AOA and similar to those of typical soil AOB. Similarity of other growth characteristics of ‘Ca. N. franklandus’ with those of typical soil AOB isolates reduces support for niche differentiation between soil AOA and AOB and suggests that AOA have a wider physiological diversity than previously suspected. In particular, the high ammonia tolerance of ‘Ca. N. franklandus’ suggests potential contributions to nitrification in fertilised soils.

Ammonia oxidation is a fundamental core process in the global biogeochemical nitrogen cycle. Oxidation of ammonia (NH3) to nitrite (NO2 -) is the first and rate-limiting step in nitrification and is carried out by distinct groups of microorganisms. Ammonia oxidation is essential for nutrient turnover in most terrestrial, aquatic and engineered ecosystems and plays a major role, both directly and indirectly, in greenhouse gas production and environmental damage. Although ammonia oxidation has been studied for over a century, this research field has been galvanised in the past decade by the surprising discoveries of novel ammonia oxidising microorganisms. This review reflects on the ammonia oxidation research to date and discusses the major gaps remaining in our knowledge of the biology of ammonia oxidation.

Autotrophic ammonia oxidation is performed by two distinct groups of microorganisms: ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB). AOA outnumber their bacterial counterparts in many soils, at times by several orders of magnitude, but relatively little is known of their physiology due to the lack of cultivated isolates. Although a number of AOA have been cultivated from soil, Nitrososphaera viennensis was the sole terrestrial AOA in pure culture and requires pyruvate for growth in the laboratory. Here, we describe isolation in pure culture and characterisation of two acidophilic terrestrial AOA representing the Candidatus genus Nitrosotalea and their responses to organic acids. Interestingly, despite their close phylogenetic relatedness, the two Nitrosotalea strains exhibited differences in physiological features, including specific growth rate, temperature preference and to an extent, response to organic compounds. In contrast to N. viennensis, both Nitrosotalea isolates were inhibited by pyruvate but their growth yield increased in the presence of oxaloacetate. This study demonstrates physiological diversity within AOA species and between different AOA genera. Different preferences for organic compounds potentially influence the favoured localisation of ammonia oxidisers within the soil and the structure of ammonia-oxidising communities in terrestrial ecosystems.

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.

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 C2 to C8 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 (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 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 NH3 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.

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Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.