Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle?

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TRENDS in Microbiology

Vol.14 No.5 May 2006

Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle? Graeme W. Nicol1 and Christa Schleper2 1 2

School of Biological Sciences, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen, UK, AB24 3UU Department of Biology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway

Cultivation-independent molecular surveys show that members of the kingdom Crenarchaeota within the domain Archaea represent a substantial component of microbial communities in aquatic and terrestrial environments. Recently, metagenomic studies have revealed that such Crenarchaeota contain and express genes related to those of bacterial ammonia monooxygenases. Furthermore, a marine chemolithoautotrophic strain was isolated that uses ammonia as a sole energy source. Considering the ubiquity and abundance of Crenarchaeota, these findings considerably challenge the accepted view of the microbial communities involved in global nitrogen cycling. However, the quantitative contribution of Archaea to nitrification in marine and terrestrial environments still remains to be elucidated. Crenarchaeota in moderate environments With many novel and considerably abundant microbial lineages predicted in molecular surveys, it is not surprising that even fundamental biogeochemical cycles must be constantly revised as the metabolisms of newly predicted organisms are identified. Recent examples of such discoveries include the detection of anaerobic methane oxidation by Archaea in the deep sea and the identification of planctomycetes that perform anaerobic oxidation of ammonia with nitrite (anammox) [1]. The detection of ammonia oxidation by non-thermophilic Crenarchaeota could similarly challenge current perspectives on global microbial nitrogen cycling, at least with respect to the nature of the microorganisms that are major players in this process. Crenarchaeota are distributed globally and constitute a considerable proportion of prokaryotic biomass in mesophilic marine and terrestrial environments. Since the initial discovery of Crenarchaeota in the marine picoplankton [2,3], 16S rRNA gene surveys have recovered crenarchaeal sequences from all major moderate environments including forest, agricultural, grassland and alpine soils [4–8], freshwater [9], sediments [10,11] and tissues and digestive tracts of terrestrial and aquatic animals [12–14]. Until recently, none of these Archaea had been obtained in laboratory culture and the closest cultivated relatives were exclusively (hyper)thermophiles from terrestrial or marine hot springs. However, one species Corresponding author: Schleper, C. ([email protected]).

in this group, Cenarchaeum symbiosum, was quasicultivable in laboratory aquaria at only 108C as a symbiont of the marine sponge Axinella mexicana [12]. Phylogenetic analyses revealed distinct lineages within the non-thermophilic Crenarchaeota that reflect some level of ecological differentiation (Figure 1). Most of the planktonic marine sequences are placed within the group 1.1a lineage. This group was estimated to represent w20% of all planktonic prokaryotes in different oceans and to dominate specifically in deeper regions [15–18]. In most soil samples analysed, group 1.1b dominate [19] and represent w1–5% of prokaryotic 16S rRNA genes [19], hybridised cells [20] or extracted rRNA [6]. Although quantitative extrapolations are difficult to make for these vast ecosystems, based on the existing studies, at least 2.3!1028 cells of the estimated 3.7!1029 microbial cells in aquatic and soil environments (see [21]) might represent Crenarchaeota (i.e. w6% of prokaryotes in these environments, if not more). The two abundant groups of soil and marine Crenarchaeota (Figure 1) share R80% 16S rRNA gene identity. Within these two groups, genes that potentially encode ammonia monooxygenase, a key enzyme in nitrification, have recently been discovered [22–25]. In addition, a marine ammonia-oxidising crenarchaeote has been cultivated [23]. Here, we summarise these novel findings that suggest a role for marine and terrestrial Crenarchaeota in ammonia oxidation and speculate on their potential contribution to global nitrogen cycling. Microbial nitrification Nitrification is central to the global nitrogen cycle and involves the oxidation of ammonia to nitrate (through nitrite) by two physiologically distinct groups of organisms: autotrophic ammonia- and nitrite-oxidisers (Figure 2). Alternatively, organic and inorganic nitrogen can be oxidised by heterotrophic bacteria and fungi (‘heterotrophic nitrification’) [26,27]. The oxidation of ammonia is considered to be the rate-limiting step of nitrification. In the soil environment, it can lead to substantial amounts of net nitrogen loss through subsequent denitrification or leaching of nitrate. In the marine environment, ammonia oxidation is an important component of nitrogen mineralisation from organic sources and the removal of anthropogenic nitrogen inputs in coastal waters. Ammonia oxidation is also a key step in the removal of nitrogen during wastewater treatment

www.sciencedirect.com 0966-842X/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.03.004

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Group 1.3 Flooded soils, sediments, freshwater and other environments AS01-06

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Group 1.2 Sediments Kazan-3A-30

BBA6

VAL159

Marine benthic group B

E_D10

ARR11

NANK-A102 P4b-Ar-26

Marine benthic group C

Amsterdam-1A-17 100

100

JTB173 CRA9-27cm NANK-A126 MA-C1-3

CRA8-27cm 100

100

Desulfurococcus mobilis

57

Sulfolobus acidocaldarius

100

FFSB6

Group 1.1c/FFS Grassland and coniferous or GFS6-9500i boreal forest soils FFSB7

91 SAGMA-D SAGMA-10

92

67

87 90

FFSC3

84

SAGMCG-1 Subsurface, plant roots

LMA226 SBAR5 ANTARCTIC12

GFS4-135i SCA1173 GRU16 29i4 TRC23-30

APA3-0cm

0.05

Nitrosopumilus maritimus

Cenarchaeum symbiosum

54d9 SCA1154

Group 1.1b Ubiquitous in most soils and other environments

Cultured (hyper) thermophiles Terrestrial hot springs and hydrothermal vents

4B7

99 GFS1-9500i

Acidianus brierleyi

ZmrA42

Group 1.1a Marine water and sediments TRENDS in Microbiology

Figure 1. Phylogenetic tree describing major non-thermophilic and cultivated (hyper)thermophilic lineages within the kingdom Crenarchaeota. Lineage descriptions follow those of Schleper et al. [25]. Two lineages with representatives that have crenarchaeal AMO genes are highlighted from marine (blue) and soil (brown) environments. Pairwise distances (with LogDet–Paralinear correction) of unambiguously aligned positions were calculated using variable sites only (estimated from a maximum-likelihood model). Bootstrap support was calculated using maximum likelihood, distance and parsimony methods (100, 1000 and 1000 replicates, respectively) with values at major nodes representing the most conservative value from all three methods (expressed as a percentage). Multifurcation indicates where the relative branching order of major lineages could not be determined in the majority of bootstrap replicates with all methods. The scale bar represents an estimated 0.05 changes per nucleotide position.

[26,27]. However, ammonia oxidisers also have a further substantial environmental impact as contributors to greenhouse gas emissions through nitrifier–denitrification mechanisms [28].

Until recently, ammonia oxidation was considered to be performed largely by autotrophic ammonia-oxidising bacteria (AOB) that form two distinct monophyletic groups within the g- and b- proteobacteria. To date, most

Organic sources Nitrogen fixation Atmospheric sources NH3 NO2–

Organic assimilation Denitrification

AMO

NH2OH

HAO

Autotrophic aerobic ammonia oxidisers

NO2–

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Figure 2. Autotrophic ammonia oxidation during nitrification. Ammonia-oxidising organisms convert ammonia to nitrite through hydroxylamine using ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). Autotrophic nitrite oxidisers subsequently use the enzyme nitrite oxidoreductase (NOR) to convert nitrite to nitrate, which can be assimilated or subjected to denitrification processes. In anaerobic environments, ammonia can be converted to molecular nitrogen by the ‘anammox’ process by several enzymatic steps (represented by dashed arrows). www.sciencedirect.com

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cultured strains belong to the b-subgroup [27] and b-proteobacterial AOB are believed to be the dominant ammonia oxidisers in most environments. Similar to Crenarchaeota, they are widely distributed in terrestrial and aquatic environments with distinct lineages that exhibit ecological differentiation [29]. Ammonia monooxygenase (AMO) is the key functional enzyme responsible for the conversion of ammonia to hydroxylamine (which is subsequently converted to nitrite by hydroxylamine oxidoreductase; Figure 2). The AMO enzyme is composed of three subunits (gene products of amoA, amoB and amoC) and is evolutionarily and functionally related to particulate methane monooxygenase (pMMO) enzymes of methane-oxidising bacteria [30].

Discovery of crenarchaeal genes encoding potential ammonia monooxygenases Autotrophic growth by mesophilic marine Crenarchaeota had been inferred earlier in a series of isotopic studies from different laboratories. Signatures in ancient crenarchaeal lipids, incorporation of 14C-depleted inorganic carbon and incorporation of H13 2 CO3 into growing crenarchaeal populations all indicated autotrophic carbon fixation [31–33]. Recent autoradiographic studies performed at the single-cell level in combination with phylogenetic stains have also confirmed inorganic carbon incorporation by Crenarchaeota [18]. However, these studies did not indicate a specific energy source for Crenarchaeota. The first link between amo-like genes and mesophilic Crenarchaeota was found through analysis of a 43 kbp metagenomic fragment from a soil library (‘54d9’)*. It contained a 16S rRNA gene of group 1.1b Crenarchaeota (Figure 1) alongside genes encoding potential homologues of bacterial AMO A and B subunits (Figure 3) and led to the hypothesis that non-thermophilic Crenarchaeota of soil could be ammonia oxidisers [25]. In a metagenomic study of the Sargasso Sea, Venter et al. [22] noted the presence of an ammonia monooxygenase gene on an archaeal-associated scaffold and indicated the potential role of Archaea in nitrification processes of the ocean. Indeed, a search for homologues of the amoA and amoBlike genes of the soil clone revealed several highly similar genes in the Sargasso Sea dataset, which enabled these marine genes to be assigned also to Crenarchaeota [25]. In addition, a neighbouring gene encoding a potential amoC was identified in this dataset [25]. In another metagenomic study from soil [34], a further amoC homologue can be dissected (G. Nicol and C. Schleper, unpublished; Figure 3). Transcription of the potential crenarchaeal amoA gene was demonstrated in situ (i.e. in soil) by reverse transcription PCR studies that demonstrated an intact and active gene [24]. Furthermore, Treusch et al. [24] demonstrated that incubation of soil in the presence of ammonia resulted in a significant increase of crenarchaeal amoA expression compared with controls incubated without ammonia. These experiments suggested that the identified genes encoded an ammonia monooxygenase * GenBank entry AJ627422, submitted 11 February 2004 by A.H. Treusch et al. (see Ref. [24]). www.sciencedirect.com

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rather than a particulate methane monooxygenase. The ultimate confirmation of ammonia-oxidising Archaea (AOA) activity was achieved by cultivation of a mesophilic crenarchaeote from a marine aquarium in Seattle, USA [23]. Nitrosopumilus maritimus is phylogenetically placed within the ‘marine’ group 1.1a lineage (Figure 1). It grows chemolithoautotrophically, using ammonia as a sole energy source, and seems to grow at similar rates and densities as cultured ammonia-oxidising bacteria (AOB) with near-stoichiometric conversion of ammonia to nitrite [23]. With primers designed from the amo gene sequences obtained in the metagenomic studies of Treusch et al. [24] and Venter et al. [22], the authors amplified highly similar amoABC-like genes from N. maritimus, which again suggested that these genes encode the key metabolic enzyme of AOA (Figure 3). Other putative archaeal amo gene clusters have been isolated from the sponge symbiont C. symbiosum and from a variety of marine environmental libraries [35]. The identification of genes encoding potential ammonia permease, urease and urea transporters in C. symbiosum adds further support to the use of reduced nitrogen compounds as an energy source by Crenarchaeota [35]. Archaeal and bacterial amo genes Phylogenetic analysis of both bacterial and archaeal amoA–pmoA shows that crenarchaeal genes are comparatively distant to their bacterial homologues (Figure 4). No significant homology is apparent at the DNA level between AOA and AOB amo-like sequences, whereas a substantial amount of sequence identity exists between all amo–pmo sequences from Proteobacteria. However, w25% sequence identity and 40% sequence similarity can be found at the protein level between archaeal and bacterial variants with conserved amino acid residues that coordinate potential metal centres. This indicates that these enzymes share an evolutionary history and belong to the same protein family (for alignment see Ref. [24]). Comparison of amo gene clusters in soil and marine Crenarchaeota with those of Proteobacteria reveals several differences. The individual proteobacterial genes are larger with a conserved amoCAB operon arrangement, whereas the gene order in crenarchaeal amo clusters varies. For example, amoC is not linked to amoA and amoB in soil Crenarchaeota (Figure 3), although Proteobacteria do have an additional isolated amoC gene that is not associated with the amoCAB operon [36]. In addition to the genes for the amoA, -B, and -C subunits, the crenarchaeal gene clusters contain a gene between amoA and -B that encodes a protein of unknown function with no known bacterial homologue (Figure 3). But similarly, both b- and g-proteobacterial amoCAB operons also precede an additional conserved open reading frame that encodes a membrane-bound protein of unknown function [36]. Initial retrieval of crenarchaeal amoA-like gene sequences from soil and marine samples reflected two ecologically distinct lineages and mirrored 16S rRNA gene phylogenies of group 1.1a and 1.1b Crenarchaeota [25]. In a recent survey of marine, estuarine, freshwater, sediment and soil samples, Francis et al. [37] further demonstrated

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54d9 Soil genomic fragment AACY01007942 and AACY01075167/8 Sargasso Sea genomic fragments

Mesophilic Crenarchaeota

Nitrosopumilus maritimus AAFX01011825 Soil genomic fragment

Nitrosospira sp. NpAV Proteobacteria

Methylococcus capsulatus TRENDS in Microbiology

Figure 3. Schematic representation of ammonia monooxygenase and particulate methane monooxygenase gene arrangements of Crenarchaeota and Proteobacteria (drawn to scale). Key to colours: amoA–pmoA genes (orange); amoB–pmoB genes (red); amoC–pmoC genes (blue); conserved hypothetical genes (grey).

that potential crenarchaeal amoA genes are ubiquitous in distribution, similar to surveys of crenarchaeal 16S rRNA genes. Phylogenetic analysis of translated protein sequences from these and other studies reveal at least five distinct crenarchaeal amo lineages (G. Nicol and C. Schleper, unpublished). It is worth noting that all archaeal amoA genes amplified to date show a high degree of sequence similarity (O73% on the amino acid level, O66%

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on the DNA level). This gene, therefore, seems to be an ideal biomarker for detecting ammonia-oxidising Archaea in environmental samples. Archaeal versus bacterial nitrification The major question that arises from these recent discoveries is how important are Crenarchaeota in nitrification? In most environments, autotrophic AOB are considered to be the most important contributors to ammonia oxidation. However, in most (if not all) mesophilic environments, Crenarchaeota are considerably more abundant than characterised AOB populations. For example, soil typically contains 104–106 b-proteobacterial AOB cells gK1 [38–41]. By contrast, extrapolating from 16S rRNA and whole cell probe data, soil might contain 107 Crenarchaeota gK1 or even more [6,20]. It remains to be shown, however, if all Crenarchaeota detected in soil derive their energy primarily by ammonia oxidation. Studies that have specifically compared AOB nitrification kinetics in the environment with growth in laboratory culture can show good correlation [40] and do not indicate the presence of a ‘missing’ nitrifier group. However, inhibition experiments have also indicated that AOB are not the major contributors to nitrification processes in certain habitats [42,43], where it has been speculated that undefined (heterotrophic) nitrifiers are primarily α (pmo)

Nitrosococcus oceanus

γ (amo/pmo)

Methylocystis parvus Methylosinus trichosporium Nitrosospira briensis 100/100 Nitrosospira sp. NpAV

Methylococcus capsulatus 56/–

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Methylomicrobium album

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Nitrosomonas europaea

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RA21 1 GP5

Unknown amo/pmo

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54d9 Austria glacier soil Scotland pasture

Sargasso Sea AACY01075168 Norway coastal water Deep Atlantic Scotland coastal water Nitrosopumilus maritimus ES HI 5 MX 6 3

Crenarchaeota (amo) TRENDS in Microbiology

Figure 4. Phylogenetic tree showing the relationship of amoA–pmoA in a-, b-, and g-proteobacterial sub-divisions to crenarchaeal amoA sequences derived from marine water (blue), sediment (purple) and soil (brown). Maximum likelihood distances were calculated from 137 aligned amino acid positions using the Jones, Taylor and Thornton (JTT) substitution model with site variation (invariable sites and eight variable gamma rates). Bootstrap support values at major nodes O50% are shown for distance/parsimony methods (expressed as a percentage of 1000 replicates). Only a few amoA and pmoA sequences of each group are represented to highlight the overall phylogeny of this protein family. For more amoA sequences of Archaea, see Refs [25,35,37]. www.sciencedirect.com

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responsible for ammonia oxidation. It will, therefore, be important to determine whether crenarchaeal AMO (and AOA nitrification) is also sensitive to AOB inhibitory compounds such as acetylene, and whether nitrification processes can be attributed specifically to AOA populations. Although cultivation approaches involve recognised selective biases, if AOA and AOB have similar properties as the recent cultivation of N. maritimus suggests, it is somewhat surprising that numerically dominant AOA have not been cultured earlier. In most cases, culture media for ammonia oxidisers consist of an inorganic salts medium containing ammonia [26] and are not tailored specifically for bacteria per se. However, there might be important physiological reasons why Crenarchaeota have not been isolated and AOA media could require particular additives such as vitamins and trace elements [23]. Genomic and biochemical data will be required to further elucidate this novel metabolism of Archaea. For example, a crenarchaeal variant of hydroxylamine oxidoreductase, which performs the second chemical transformation in nitrification, has yet to be found (Figure 2). No homologue can be dissected in the Sargasso Sea dataset (G. Nicol and C. Schleper, unpublished) or in the genome of the marine sponge symbiont C. symbiosum [35]. Furthermore, it will be of interest to study the pathway of carbon assimilation. The genome data of C. symbiosum, which show high similarities to those of marine planktonic Crenarchaeota, suggest that either a 3-hydroxypropionate pathway or a reductive tricarboxylic acid cycle might be used for autotrophic carbon assimilation, both of which are found in hyperthermophilic Crenarchaeota [35]. Are all non-thermophilic Crenarchaeota ammonia oxidisers? Extrapolating from the limited 16S rRNA gene diversity of Crenarchaeota in marine and terrestrial environments, one might speculate that most or all of group 1.1a and 1.1b Crenarchaeota are ammonia oxidisers. Given the high abundance of Crenarchaeota in the marine picoplankton [15–18] of up to 40% of total prokaryotic cell numbers in some regions, the amount of ammonia oxidation would have to be unexpectedly high in the ocean. It might well be possible that some or all of these organisms are capable of mixotrophic growth under certain conditions or that only some lineages within this group contain ammonia oxidisers. Although the first cultivated marine isolate was reported to grow exclusively on inorganic carbon [23], uptake of amino acids by marine planktonic Archaea has been demonstrated [18,44], which indicates that some Crenarchaeota can grow heterotrophically or mixotrophically. From the genome data of C. symbiosum, potential mixotrophic growth can also be predicted with the identification of a near-complete oxidative tricarboxylic cycle [35]. Concluding remarks and future perspectives Metagenomic and cultivation studies have been instrumental in elucidating the potential role of Crenarchaeota in nitrification processes. Molecular biological tools will continue to be essential in identifying the distribution and abundance of ammonia-oxidising Archaea. The novel www.sciencedirect.com

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cultivated isolate will help in the establishment of cultivation conditions for other mesophilic Crenarchaeota and in elucidating the biochemistry of this novel archaeal metabolism. Although many questions remain regarding the ecophysiology, metabolism and biochemistry of the organisms, these recent studies demonstrate the huge potential of combining metagenomics and cultivation. One of the major and, perhaps, most difficult tasks in the future will be to elucidate the ecological impact of archaeal ammonia oxidisers. Although many data might suggest at present that marine and terrestrial Crenarchaeota are major contributors to the biogeochemical transformation of nitrogen and carbon, this still remains to be shown. Furthermore, it will be interesting to explore if nitrifying organisms are found in more phylogenetic lineages of the Archaea and if this metabolism originated in hot environments. Acknowledgements We thank Professor Jim Prosser (University of Aberdeen) and the four reviewers of this article for helpful comments on the manuscript. C.S. thanks all past and present researchers of her laboratory for their excellent intellectual and technical contributions to the study of Crenarchaeota in soil.

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