Ammonia-oxidizing archaea (AOA) are ubiquitous and abundant and contribute significantly to the carbon and nitrogen cycles in the ocean. In this study, we assembled AOA draft genomes from two deep marine sediments from Donghae, South Korea, and Svalbard, Arctic region, by sequencing the enriched metagenomes. Three major microorganism clusters belonging to Thaumarchaeota, Epsilonproteobacteria, and Gammaproteobacteria were deduced from their 16S rRNA genes, GC contents, and oligonucleotide frequencies. Three archaeal genomes were identified, two of which were distinct and were designated Ca. “Nitrosopumilus koreensis” AR1 and “Nitrosopumilus sediminis” AR2. AR1 and AR2 exhibited average nucleotide identities of 85.2% and 79.5% to N. maritimus, respectively. The AR1 and AR2 genomes contained genes pertaining to energy metabolism and carbon fixation as conserved in other AOA, but, conversely, had fewer heme-containing proteins and more copper-containing proteins than other AOA. Most of the distinctive AR1 and AR2 genes were located in genomic islands (GIs) that were not present in other AOA genomes or in a reference water-column metagenome from the Sargasso Sea. A putative gene cluster involved in urea utilization was found in the AR2 genome, but not the AR1 genome, suggesting niche specialization in marine AOA. Co-cultured bacterial genome analysis suggested that bacterial sulfur and nitrogen metabolism could be involved in interactions with AOA. Our results provide fundamental information concerning the metabolic potential of deep marine sedimentary AOA.
Citation: Park S-J, Ghai R, Martín-Cuadrado A-B, Rodríguez-Valera F, Chung W-H, Kwon K, et al. (2014) Genomes of Two New Ammonia-Oxidizing Archaea Enriched from Deep Marine Sediments. PLoS ONE 9(5): e96449. https://doi.org/10.1371/journal.pone.0096449
Editor: Celine Brochier-Armanet, Université Claude Bernard - Lyon 1, France
Received: August 8, 2013; Accepted: April 9, 2014; Published: May 5, 2014
Copyright: © 2014 Park et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Basic Science Research Program (2012R1A1A2A10039384) through the National Research Foundation of MEST (Ministry of Education, Science, and Technology), Marine and Extreme Genome Research Center Program of the MLTM (Ministry of Land, Transport, and Maritime Affairs), and the Energy Efficiency & Resources Core Technology Program (20132020000170) of the KETEP (Korea Institute of Energy Technology Evaluation and Planning) granted financial resource from the MTIE (Ministry of Trade, Industry & Energy), Republic of Korea. ELM was supported by NSF grant #DEB-0841999. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Aerobic nitrification is a key process in the nitrogen cycle that converts ammonia to nitrate via nitrite and is catalyzed by aerobic autotrophic ammonia-oxidizing and nitrite-oxidizing microorganisms. The first step in autotrophic nitrification, the oxidation of ammonia, was long thought to be exclusive to Proteobacteria in the domain Bacteria ; however, more recently, metagenomic analyses of terrestrial  and marine environments  revealed that ammonia oxidation is also associated with Archaea. Moreover, critical evidence for the existence of autotrophic ammonia-oxidizing archaea (AOA) was obtained through characterization of the first ammonia-oxidizing archaeon, Nitrosopumilus maritimus SCM1, which was isolated from a marine aquarium . This discovery was followed by the successful cultivation of diverse AOA of Thaumarchaeota ,  from marine (group I.1a) , ,  and soil (group I.1a and I.1b) – environments. Furthermore, molecular ecological studies indicate that AOA often predominate over ammonia-oxidizing bacteria in marine environments such as the North Sea and coastal sediments , .
The seafloor comprises approximately two-thirds of the Earth’s surface and is therefore one of the most extensive of all microbial habitats. Quantitative assessments of subsurface microbial populations indicate that prokaryotes constitute a large portion of the Earth’s overall biomass, and that marine sediment processes may therefore substantially contribute to the global nitrogen budget. Research into nitrification, a key step in the nitrogen cycle, has focused on water-column, and studies regarding marine sediment nitrification are minimal. Investigations into the metabolic properties and nitrification potential of sedimentary AOA are therefore necessary to understand the nitrogen cycle in marine environments.
Fundamental information about microorganisms and their metabolic features can be revealed via metagenomic and genomic techniques. Analysis of the genome sequence of an amoA-encoding archaeon Ca. “Cenarchaum symbiosum” from a marine sponge ,  and a marine ammonia-oxidizing archaeon N. maritimus  provided valuable insights into the evolution of nitrogen and carbon metabolism in marine AOA of the Nitrosopumilus lineage (also called group I.1a). Comparative analyses of group I.1a AOA genome sequences from low-salinity aquifers and terrestrial environments have revealed several genetic traits likely to be adaptations to such habitats, such as motility and protection from osmotic stress , . AOA metagenomic information obtained from the water column of the Gulf of Maine has shed light on the metabolic potential of planktonic AOA . Although the genomes of two AOA enriched from low-salinity sediments have been sequenced , , genomic data from deep marine sedimentary AOA are not yet available.
AOA are widespread and dominant ammonia-oxidizers in marine sediment . One of the main difficulties in obtaining axenic AOA cultures is their dependence on co-cultured bacteria, as described in AOA characterization reports , , , . Sedimentary AOA were, however, successfully enriched when co-cultured with sulfur-oxidizing bacteria (SOB) in a technique that facilitated characterization of the AOA . Here, we analyzed metagenomes from enrichment cultures and were able to assemble the genomes of two deep marine sedimentary AOA. The aims of this study were to investigate the genomic features of deep marine sedimentary AOA through comparisons with the genomes of other AOA and to assess possible microbial interactions between deep marine sedimentary AOA and co-cultured bacteria.
Results and Discussion
Metagenome analysis, assembly, and binning
We obtained 536.8 Mb and 308.2 Mb of metagenomic sequences from two independently enriched ammonia-oxidizing cultures containing thaumarchaeotal group I.1a archaeal strains, named AR (from Svalbard, Arctic region) and SJ (from Donghae, South Korea), respectively. General features of the metagenome datasets are as indicated in Table S1. The GC% profiles of the raw reads from the two enrichment metagenomes were very similar to one another (Figure S1).
Single reads of 16S rRNA genes recovered from the metagenome dataset (n = 1,100 in AR and n = 908 in SJ cultures) were used to analyze the compositions of the microbial communities that were enriched in the two cultures (Figure S2). The most frequently recovered 16S rRNA gene sequences were affiliated to Epsilonproteobacteria (60–62%), Thaumarchaeota (13–17%), and Gammaproteobacteria (10–18%), with the proportions of these three taxa being similar in the two cultures (Figure S2). Most of the 16S rRNA gene sequences of Epsilonproteobacteria were affiliated with the sulfur-oxidizing genus Sulfurovum. More than 10% of the 16S rRNA gene reads from each metagenome were affiliated with Thaumarchaeota, and, specifically, the genus Nitrosopumilus. Gammaproteobacteria sequences were related to those of diverse Gammaproteobacteria (e.g., Marinobacter, Marinobacterium, and Neptuniibacter). Overall, this analysis suggested that the proportion of 16S rRNA genes from archaea was approximately 20%, which was lower than the proportion of archaea observed by previous fluorescence in situ hybridization analysis of the SJ and AR cultures . This discrepancy could have arisen due to the presence of multiple rRNA operons in bacterial genomes  by contrast with the single rRNA operon in the genome of N. maritimus (Thaumarchaeota) . Indeed, Nakagawa et al.  reported that the genome of Sulfurovum sp. NBC37-1 (Epsilonproteobacteria), a close relative of the dominant bacterium in the SJ and AR cultures, has three copies of the rRNA operon. Data obtained from 16S rRNA gene reads were complemented by comparing the entire metagenome dataset of functional genes to homologous genes of known microbial genomes using the MG-RAST server (Figure S3).
Assembly of the metagenomic data produced 15,155 and 2,595 contigs from the AR and SJ metagenomic sequences, respectively (Table S1). We filtered the contigs, selecting only those that were ≥ 5 Kb in length (n = 118 for AR and n = 91 for SJ) and which yielded consistent hits to a single high-level taxon (e.g., Thaumarchaeota, Epsilonproteobacteria and Gammaproteobacteria). An examination of GC% versus length in the selected contigs indicated they comprised three clusters (Figure S4). Moreover, principal component analysis of the oligonucleotide frequencies also revealed three distinct clusters in each enriched sample (Figure 1). Based on BLAST analysis of the genes, we assigned clusters 1, 2, and 3 to Thaumarchaeota, Epsilonproteobacteria, and Gammaproteobacteria, respectively, which was consistent with results obtained from the 16S rRNA analysis (Figure S2). The GC% range in cluster 1 (Thaumarchaeota) (Figure 1) was similar in both the AR and SJ assemblies (27–37% in AR and 32–35% in SJ). With the exception of Ca. “C. symbiosum” (57%)  and Ca. “Nitrososphaera gargensis” (48%) , all other previously analyzed AOA, including N. maritimus, had GC contents of 32–34% –. The amounts of sequence obtained for cluster 1 differed between the two clusters: 3.44 Mb in AR and 1.65 Mb in SJ. Considering the size of the N. maritimus genome (1.64 Mb), the 1.65 Mb size of the archaeal cluster from the SJ metagenome assembly potentially represented a draft genome of a single AOA. However, the 3.44 Mb of contigs in cluster 1 of the AR metagenome suggested that two putative archaeal draft genomes had been assembled.
(A) AR culture, and (B) SJ culture. Reference genomes are shown as larger circles. The total number of contigs for each group (Gammaproteobacteria, Epsilonproteobacteria, and Thaumarchaeota), total length, mean length, and GC content range are also indicated. The contig types and published genomes are as follows: orange, Gammaproteobacteria; yellow, Thaumarchaeota; green, Epsilonproteobacteria; light green, assembled contigs including viral coding sequences; gray, not identified; red, Ca. “Cenarchaum symbiosum” A (CsymA); fuchsia, Ca. “C. symbiosum” B (CsymB); lime, Nitrosopumilus maritimus SCM1 (Nmar); blue, Ca. “Nitrosoarchaeum koreensis” MY1 (MY1); cyan, Ca. “Nitrosoarchaeum limnia” (Nlim); violet, Ca. “Nitrososphaera gargensis” (Ngar); teal, Sulfurovum sp. NBC37-1 (Sul); and purple, Thiomicrospira crunogena XCL-2 (Tcr).
The GC content of cluster 2 was approximately 43%, which corresponded to that of Sulfurovum sp. NBC37-1 (43.8%) . The expected genome size of cluster 2 (2.12 Mb) was slightly smaller than that of Sulfurovum sp. NBC37-1 (2.56 Mb). We were unable to detect the 16S rRNA gene within cluster 3, which contained the gammaproteobacterial contigs, and so were unable to definitively determine phylogenetic position. BLAST analysis indicated that cluster 3 contig genes were most similar to genes in Gammaproteobacteria genomes such as Oceanospirillum. The Average Nucleotide Identity (ANI)  of the gamma- and epsilonproteobacterial clusters in the two metagenome sets indicated that they were nearly identical (∼99%). Some features of the binned contigs from both metagenomic datasets are summarized in Table 1.
Establishing draft genome assemblies for three deep marine sedimentary archaea and defining their unique characteristics
The binning and assembly procedures described above were used to define three AOA draft genomes. We hypothesized that the cluster 1 (thaumarchaeotal) sequences from culture AR (3.44 Mb) represented two genomes, henceforth termed AR1 and AR2. Cluster 1 sequences from culture SJ (1.65 Mb) appeared to represent a single genome.
Genomic diversity in a microbial population can be determined by analyzing sequence variations in metagenome reads. We used the Strainer program (http://www.bioinformatics.org/strainer/wiki/) to assess variation in the archaeal populations of the metagenome datasets. Archaeal diversity in the AR and SJ cultures was assessed by analyzing the ammonia monooxygenase gene (ammonia monooxygenase alpha subunit, amoA), which is involved in ammonia oxidation, and the 16S-23S rRNA intergenic spacer (ITS) region. The amoA and ITS sequences were examined in raw reads (data not shown), and the results fully supported the above hypothesis that the metagenomic data captured a single draft archaeal genome in the SJ culture and two draft archaeal genomes in the AR culture. Archaeal contigs in the AR culture clearly separated into two distinct groups based on contig alignment with N. maritimus using Mauve  and ANI analysis with N. maritimus.
We propose that our assembled genomes warrant draft genome status for the following reasons: (i) Each draft genome features 97–98% of the archaeal genes used by the NIH Human Microbiome Project as criteria for complete draft genomes (http://hmpdacc.org/tools_protocols/tools_protocols.php) . These archaeal genes are known to be highly conserved between the genomes of free-living Archaea and comprise 104 core gene groups. Additionally, the majority of the core archaeal genes are found in the complete or nearly complete genomes of several published AOA (Ca. “C. symbiosum”, 92%; Ca. “Na. koreensis”, 98%; N. maritimus, 100%; and one exception, Ca. “N. gargensis”,74%); (ii) The two draft genomes of SJ and AR1 were independently sequenced and assembled but were nearly identical to one other, as recognized by gene content and synteny comparisons; (iii) A high degree of genomic similarity was observed between the three draft archaeal genomes and the completed N. maritimus genome. Furthermore, the number of tRNAs (n = 44) was identical in the draft genomes of SJ, AR1, and AR2, and the complete genome of N. maritimus.
The two AR1 and AR2 archaeal genomes exhibited approximately 80% ANI with each other and ANIs of 85.2% and 79.5% with N. maritimus, respectively. The ANI of the AR1 archaeal bins with those of the SJ culture was ∼99%; no significant differences were observed between the SJ and AR1 archaeal contigs with respect to gene content or local synteny. On the basis of these results, we concluded that the SJ and AR1 assembled archaeal genomes were indistinguishable and might have originated from very closely related microorganisms. Therefore, our further analyses focused on two of the three archaeal genomes: AR1 (synonymous with the archaeon from culture SJ) and AR2.
Despite the strong similarities (>99.5%) between the 16S rRNA gene sequences in N. maritimus and in the AOA obtained from our enrichments (Table S2 and Figure S5), the low ANI (<85%) indicates high genomic variation within this cluster of marine AOA. The proposed cutoff for defining separate species is 94% ANI between two genome sequences . This criterion suggests that each archaeal strain (AR1 and AR2) can be considered a separate species distinct from N. maritimus. We propose that these genomes represent two new marine AOA within the genus Nitrosopumilus, named Ca. “Nitrosopumilus koreensis” (AR1 and SJ)  and “Nitrosopumilus sediminis” (AR2) .
Genetic differences between AOA genomes and their adaptive implications
Most of the putative coding sequences (CDS) in the AR1 and AR2 genomes (71.9% and 65.1%, respectively) had homology to N. maritimus genes, and most of the genes were syntenic with those in the N. maritimus genome (Figure S6). However, 20.5% and 24.4% of the putative CDS of the AR1 and AR2 genomes, respectively, had no similarity to genes in other known organisms.
We hypothesized that the adaptive traits of deep sedimentary AOA in our enrichment cultures might contrast with those of water-column AOA. To address this, a recruitment analysis was performed in which nucleotide-sequence fragments from the planktonic Sargasso Sea metagenome dataset of the global ocean sampling (GOS) database  were mapped onto the AR genomes (Figure S7). Many of the genes that were present in the AR genomes but absent in the Sargasso Sea metagenome dataset were clustered in genomic islands (GIs) of >15 Kb (Figure S7, and Tables S3 and S5).
GIs were a major feature of the AR1 and AR2 genomes (Tables S3 and S5) and comprised approximately 15% of the total AR1 (six GIs) and AR2 genomes (12 GIs). Most of the GIs in the AR1 and AR2 genomes were different from one another and were absent from the N. maritimus genome, and gene functions can be putatively inferred for approximately half of the genes in the GIs. Most GI genes in both the AR1 and AR2 genomes were related to cell-wall biosynthesis, osmotic stress tolerance, antibiotic resistance, sensory signal transduction, and phage proteins. In addition, the GIs of both genomes comprised genes with high anomalies in codon usage, indicating that they might have been obtained via horizontal transfer events, as suggested by Rusch et al. .
The Clusters of Orthologous Genes (COG) classification of the GI genes from the two genomes indicated that genes belonging to COG class M (cell wall/membrane/envelope biogenesis), K (transcription), and T (signal transduction mechanisms) were abundant (Figure S8). This is in partial contrast to the COG classes found in the GIs of other archaeal genomes, which are predominantly M or Q (secondary metabolite biosynthesis, transport, and catabolism) . The proteinaceous surface layers of AOA have an abundance of reactive surface sites that are conceivably related to their oligotrophic adaptations . The frequent observation of COG class M genes in the GIs of the AR1 and AR2 genomes could contribute to variations in cell surface structure, which might be important factors for niche specialization in AOA ecotypes. Overall, the identified GIs might constitute strain-specific (hyper)variable regions or sedimentary AOA-specific regions.
Ammonia oxidation, electron transfer, and carbon fixation for the deep marine sedimentary AOA
Pathways for ammonia oxidation, electron transport, and carbon fixation were assembled from the AR1 and AR2 archaeal genomes and compared with other reference AOA genomes. The AR1 and AR2 archaeal strains held key metabolic traits in common with other AOA, including N. maritimus (Table S4).
Ammonia oxidation and electron transport chain.
All of the putative ammonia monooxygenase genes (amo; amoA, amoB, and amoC) were found in the AR1 and AR2 genomes. The gene arrangement [amoA-hypothetical gene (named amoX)-amoC-amoB] was similar to that in other AOA of the Nitrosopumilus cluster (e.g., N. maritimus) as well as into Ca. “N. devanaterra” , but differs from the gene arrangements in group I.1b AOA ,. For example, the amo genes in some group I.1a marine lineages and in most of the soil lineages (group I.1b) were not consecutive, but were interrupted by other genes. In most AOA, another small protein encoding a transmembrane protein and referred to as amoX was linked to the amoA gene .
Although AOA produce nitrite as the final product of ammonia oxidation, homologs of the heme-containing hydroxylamine oxidoreductase (hao) gene of ammonia-oxidizing bacteria (AOB) were absent from the AR1 and AR2 genomes, as in other AOA genomes ,,,. However, Vajrala et al.  observed hydroxylamine-induced oxygen consumption and ATP production in the marine ammonia-oxidizing archaeon N. maritimus. The number and sequences of six putative genes encoding copper-containing oxidases, which were suggested to function as possible hydroxylamine oxidoreductases (HAOs) , were conserved between N. maritimus and strains AR1 and AR2, encoding proteins with 88% amino acid identity on average. The number of putative genes encoding copper-containing oxidases found in the AOA genomes was six for Ca. “N. gargensis” and 3–4 for Ca. “Na. koreensis”, Ca. “Na. limnia”, and Ca. “C. symbiosum”. A putative gene for copper-containing oxidase was highly conserved (average 83% amino acid identity) between soil strain Ca. “Na. koreensis” (MY1_0289) and the marine AOA genomes (Nmar_1131, AR1_298, and AR2_318), and warrants further investigation as a possible HAO candidate. The other putative copper-containing oxidase gene, nirK, was highly conserved in all AOA, which might be involved in nitrifier denitrification . A TATA box and parts of a BR element (transcription factor B recognition element), 23 nt or 25 nt upstream of the nirK gene ( Figure S9), were observed as in the archaeal amo gene , suggesting that the nirK gene could be expressed independently under the control of its own promoter.
As in other AOA genomes, strains AR1 and AR2 appear to encode a complete respiratory chain with complexes I–V, which are used for energy generation and reverse electron transport. The components have ∼93% amino acid identity to those of N. maritimus. Complex V is an archaeal type ATPase that is known to use both Na+ and proton gradients to generate ATP . Na+ is frequently used instead of H+ in gradient formation during electron transport in oligotrophic or energy-stressed environments, since Na+ is usually less permeable to the cellular membrane.
Like other AOA genomes, the genomes of AR1 and AR2 lack homologs of cytochrome c proteins –,, and therefore blue copper-containing proteins (Table S6) might be involved in the transfer of electrons from complex III. Known homologs encoding essential genes for heme biosynthesis (ahb-nirJ1 and ahb-nirJ2) were missing  and putative genes for heme-containing proteins were rare in the AOA genomes. The only heme-containing gene detected in the AOA genomes (including AR1 and AR2) was that encoding the cytochrome b/b6 family protein of respiratory complex III. Since heme uptake by prokaryotes from the environment is not plausible , AOA genomes require further screening and analysis to characterize gene sets for heme biosynthesis. The variability in iron availability in marine and terrestrial environments suggests that the abundance of copper-containing oxidases for redox reactions in both soil (e.g., Ca. “Na. koreensis”) and marine AOA might be an evolutionary trait of Thaumarchaeota rather than a functional or environmental adaptation of the AOA. The high abundance of multicopper-containing proteins and blue copper-containing proteins in AOA, rather than heme-containing proteins, implies that ammonia oxidation pathways and respiratory chains in AOA groups I.1a and I.1b may be novel and conserved.
Most AOA characterized to date are able to grow chemolithotrophically using inorganic carbon (carbon dioxide and/or bicarbonate) as their sole carbon source ,,–,. By contrast with their bacterial counterparts, AOA genomes do not contain key genes for the Calvin-Bassham-Benson cycle ,, but might instead utilize the 3-hydroxtpropionate/4-hydroxybutyrate pathway. The genes encoding the three main proteins for this pathway, 4-hydroxybutyrate-CoA dehydratase, acetyl-CoA carboxylase, and methylmalonyl-CoA epimerase, were present in the AR1 and AR2 genomes and the putative proteins had 80–98% amino acid identity to the N. maritimus homologs.
Stable-isotopic and molecular studies raised questions regarding the mixotrophic nature of the marine lineage of group I.1a ,. Ammonia oxidation and growth of N. viennensis (a soil lineage of group I.1b) was supported by pyruvate and some pyruvate carbons were incorporated into archaeal cells . Genes encoding proteins that are possibly involved in the transport of organic compounds, such as carbohydrates, amino acids, oligo/dipeptides, and nucleosides, were evident in the AR1 and AR2 genomes and in other AOA genomes. However, there has been no direct biochemical and physiological evidence from cultivated AOA to support the hypothesis that the marine lineage of group I.1a is mixotrophic. The Ca. “N. gargensis” genome encodes alanine dehydrogenase and an array of pyruvate transformation genes , suggesting that Ca. “N. gargensis” might utilize pyruvate or alanine as an alternative carbon source, by contrast with other AOA. Pyruvate phosphate dikinase, which is involved in the transformation of pyruvate to phosphoenolpyruvate for gluconeogenesis, was encoded in the genomes of marine AOA, including the AR1 and AR2 strains.
Genomic traits of the deep marine sedimentary AOA
A complete set of genes involved in urea utilization was identified in the AR2 genome (Figure 2). This was absent from other marine (AR1 and N. maritimus) and soil/low-salinity AOA (Ca. “Na. koreensis” and Ca. “Na. limnia”) genomes. Urease operons were identified in the genomes of Ca. “C. symbiosum” , N. viennensis , Ca. “N. salaria”  and Ca. “N. gargensis” , and in a scaffold from a recent ocean metagenomic study , with 46–86% amino acid identities to the AR2 operon, respectively. Moreover, two copies of a urea transporter gene were identified in the AR2 genome that were 50–76% identical to the dur3 gene from Ca. “C. symbiosum”, Ca. “N. gargensis”, and to the dur3 gene from the Pacific Ocean metagenome recovered from a 4,000 m depth at station ALOHA . A recruitment analysis comparing the AR2 genome to a Sargasso Sea metagenome showed that the archaeal urease utilization trait was widespread in water-column archaea. Since urea comprises a significant proportion of the dissolved nitrogen compounds in the surface layer of marine sediment , the capacity for urea utilization within sedimentary AOA may confer a selective advantage within that niche. Moreover, Alonso-Sáez et al.  suggested that deep water Thaumarchaeota in the Arctic and Antarctic oceans use urea as an energy source in nitrification.
Ca. “N. sediminis” AR2 genome is central, with the Ca. “C. symbiosum”, Ca. “Nitrososphaera gargensis”, and environmental metagenomic regions above and below, respectively. Homologous genes are connected with shaded regions, and the shaded color indicates the percent identity as determined by TBLASTX.
Ectoine is a compatible solute that is found in a wide range of bacteria. The AR1 and AR2 genomes (as well as that of N. maritimus ) contained all four genes in the archaeal ectoine biosynthesis cluster (ectA, ectB, ectC, and ectD). In AR1 and AR2, the ectoine gene clusters were located in the centers of GI 6 and GI 3, respectively and the codon usage in these islands deviated markedly from the conserved core genes in the AR genome (Table S3). Recruitment analysis did not find ectoine biosynthesis genes in the Sargasso Sea metagenome or the Ca. “Na. limnia”, Ca. “Na. koreensis”, Ca. “N. gargensis”, or Ca. “C. symbiosum” genomes ,,,. Instead, Ca. “Na. limnia”, Ca. “Na. koreensis”, and Ca. “N. gargensis” employ mechanosensitive ion channels (MS channels; mscS and mscL genes) for regulating osmotic pressure. The AR1, AR2, and N. maritimus genomes also harbored genes for a small-conductance MS channel (mscS), but no large-conductance MS channel gene (mscL) was apparent; thus the ability to synthesize ectoine might be an important osmotic adaptation in members of the genus Nitrosopumilus.
Clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system.
The CRISPR/Cas system mediates resistance against phages, and is found in the majority of investigated Archaea genomes . Possible spacer-repeat arrays were identified in the AR1 (n = 3) and AR2 (n = 1) genomes, but only a single CDS exhibited similarity to a gene encoding a Cas protein (CAS1-like) (see GI 4 and 6, respectively, in Table S3). It is unclear whether the putative CRISPR spacers observed in AR1 and AR2 are artifacts or instead represent remnants of previous CRIPSR-loci. By contrast with the wide distribution of CRISPR in archaea, only one thaumarchaeon (Ca. “N. gargensis”) has so far been found to contain a CRISPR-locus and associated CAS-genes .
High-affinity phosphate uptake genes are often found in AOA, including the recently published Ca. “N. gargensis” genome , but we were unable to identify a high-affinity, high-activity phosphate uptake operon (pstSCAB) in either of the AR1 or AR2 genomes. The absence of these genes in the deep marine sedimentary AOA metagenome datasets may reflect habitat-specific circumstances. It is likely that sufficient phosphate is available in marine sediment as phosphate levels up to 100 µM were previously noted ; this is 50-fold higher than phosphate concentrations in the marine water column (∼2.0 µM) .
Perchlorate (ClO4−), chlorate (ClO3−) and chlorite (ClO2−) are important pollutants in groundwater, surface waters, and soils . Several (per)chlorate-reducing bacteria, including Dechloromonas aromatic, Idenella dechloratnas, and nitrite-oxidizing bacteria , contain a cld gene, which encodes enzymes that degrade chlorite (ClO2−) to chloride (Cl−) and oxygen (O2). Although cld genes are not present in AOB genomes, they are contained in all AOA genomes examined to date, including the AR1, AR2, N. maritimus, Ca. “Na. koreensis”, Ca. “Na. limnia”, Ca. “N. gargensis”, and Ca. “C. symbiosum” genomes. Cld proteins in AR1 and AR2 exhibited 35–68% and 50–87% identity, respectively, with those of other AOA. Cld in AOA may be necessary for chlorite detoxification, since chlorite is a selective inhibitor of ammonia oxidation . This concurs with our previous results , showing that group I.1a AOA tolerated higher concentrations of chlorite than Nitrosomonas europaea ,,.
Genomic features of co-cultured SOB
Successful cultivation of sedimentary AOA reportedly depends upon co-cultivation with SOBs . Epsilonproteobacterial and gammaproteobacterial genomes were major constituents of the AR and SJ culture sequences, as detailed herein. Because the metagenomic features of the Epsilonproteobacteria (cluster 2) and Gammaproteobacteria (cluster 3) from the AR and SJ cultures were nearly identical (reciprocal ANI 99%), we selected epsilonproteobacterial (cluster 2) and gammaproteobacterial (cluster 3) bins from the AR and SJ cultures, respectively, for further analysis. These are designated “EP_AR” and “GM_SJ”, and their metabolic capabilities as determined by genomic analysis are discussed below and summarized in Table S4.
Strain EP_AR was affiliated with chemolithoautotrophic SOB. Several key enzymes involved in sulfur oxidation (e.g., sulfur-compounds oxidation system, SOX) were encoded within the EP_AR genome  (Table S4). The putative SOX proteins had 55–92% amino acid identity to those of the close relatives Sulfurovum sp. NBC37-1  and Sulfurimonas denitrificans DSM 1251 . Strain GM_SJ resembled a typical marine heterotroph since no genes related to sulfur oxidation or carbon fixation were observed in the genome (Table S4).
Microbial interactions play a critical role in shaping niches for microorganisms in natural environments. Sedimentary AOA and SOB occupy similar niches in sediment redox gradients , since AOA and SOB at oxic-anoxic interfaces consume ammonia and sulfide, respectively, diffused from the anoxic layers of marine sediment. Joye and Hollibaugh  reported that sulfide (<100 µM) inhibits nitrification in marine sediments. The prevalence of AOA may therefore be assisted by SOB detoxification of sulfides. The unusually tight associations between AOA and SOB were described in a terrestrial cold sulfidic spring , and thaumarchaeotal strains were physically associated with SOB in sulfide-rich mangrove swamps . Sulfide-quinone reductase (sqr), sulfite:cytochrome c oxidoreductase (dsrAB), and the SOX system genes (soxYZABCFHL) in the EP_AR genome could mediate sulfide oxidation reactions . This suggests that strain EP_AR might be a natural co-habitant of sedimentary AOA, and, although we used thiosulfate instead of sulfide for enrichment in this study , interactions between SOB and AOA might be exploited for the successful enrichment of SJ and AR in the laboratory.
AOB have a low efficiency for N2O production during nitrifier denitrification and most NO is emitted to an extracellular environment ,. Excess NO is therefore potentially toxic to the nitrifier itself and to other bacteria. Nitric oxide is suggested as an intermediate during bacterial , and archaeal nitrification. Archaeal NO production was suggested by genomic analysis  in this study and by Walker et al.  and is supported by the inhibition of AOA by NO scavengers . N2O emissions during archaeal ammonia oxidation , provide indirect evidence of the involvement of NO in archaeal nitrifier denitrification ,. A putative gene encoding toxic NO-detoxifying flavohemoglobin [NO dioxygenase, NOD, 51.4% amino acid identity with that in Aquifex aeolicus VF5 ] was observed in strain EP_AR ( Figure S10), while no homolog was found in the genome of the closest relative, Sulfurovum sp. NBC37-1 ( Table S4). A gene-encoding phage integrase [48% amino acid identity with that in Sulfurimonas denitrificans ] located upstream of the NOD gene suggests that the NOD gene may have been acquired through horizontal gene transfer. Catalytic NO dioxygenation occurs most effectively via NOD under aerobic conditions , while nitric oxide reductase would be active under anoxic conditions . The NOD in co-cultured SOB might therefore play a role in stimulating AOA growth. Genomic analysis of co-cultured SOB suggested that sulfur and nitrogen metabolism might be involved in the interactions between sedimentary AOA and co-cultured bacteria. Further systematic investigations are warranted to determine the response of sedimentary AOA to nitric oxide scavengers and generators.
Metagenomic analyses enabled the assembly of two distinct deep marine sediment-derived AOA genomes, AR1 and AR2, and the determination of genetic similarities and differences between these organisms and previously sequenced AOA. Many key genomic features were conserved between AR1 and AR2 and other AOA, including genes pertaining to energy metabolism and carbon fixation. Nevertheless, genomic variations were also apparent, including: 1) Large GIs comprising ∼15% of the total genomes were found in AR1 and AR2; 2) Approximately 24% of CDS in AR1 and AR2 were unique; and 3) High-affinity phosphate uptake genes were absent in AR1 and AR2. In addition, a urease operon was found in the AR2 genome, but not the AR1 genome, suggesting potentially distinctive strategies for resource utilization between the two deep marine sedimentary AOA strains.
The availability of the genome sequences of deep marine sedimentary AOA will provide a foundation for evolutionary, biochemical, and ecophysiological studies that will contribute to the understanding of niche adaptations in marine AOA.
Materials and Methods
Cultivation of sediment microorganisms and preparation and sequencing of metagenomic DNA
Details of the enrichment and properties of the AOA used for this study were described previously . AOA were enriched from sediment samples collected from Donghae (128° 35_E, 38° 20_N; depth, 650 m) and Svalbard (Arctic region, 16° 28_E, 78 °21_N; depth, 78 m) and are referred to as SJ and AR cultures, respectively. The field studies did not involve endangered or protected species and no specific permits were required.
Ammonia (1 mM) and thiosulfate (0.1 mM) were used as energy sources and bicarbonate (3 mM) was used as a carbon source. The culture medium was supplemented with a trace element mixture and a vitamin solution. Ammonia consumption and nitrite production were monitored as described by Park et al. . After the ammonia was exhausted, cultures were transferred to fresh medium (inoculum comprising 10% of total medium volume) and cultivated at 25°C in the dark. The culture was maintained by transferring a 10% inoculum to fresh culture medium approximately every 2 weeks. After 50 months, cells from a 1 L culture were harvested using 0.22 µm pore size filters (Millipore, Billerica, MA) with a vacuum pump. The filters were placed in a sterile conical tube and stored at −70°C. Total DNA was extracted using a modified method based on that described by Park et al. . Briefly, filters were treated with DNA extraction buffer  at 60°C for 30 min, and nucleic acids were purified with phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol. Metagenomic DNA integrity was confirmed using 0.8% (w/v) agarose gel electrophoresis and DNA was quantified using a NanoDrop ND 1000 spectrophotometer. Total DNA (∼5 µg) was sequenced using single read and mate-paired (about 8 Kb insert library size) end sequencing methods using a 454 GS-FLX Titanium platform (Roche Applied Science, Indianapolis, IN). Sample sequencing and analytical data processing was performed at the National Instrumentation Center for Environmental Management, Seoul National University, South Korea. The average read length was approximately 291 bp for AR and 266 bp for SJ. Short sequences and sequences with a quality score <20 were removed to enhance metagenomic sequence quality.
rRNA gene analysis
rRNA genes were identified by comparing the obtained datasets to the RDP database . All reads that matched an rRNA sequence with an alignment length >100 bases and an e-value ≤ 0.001 were extracted. The best hit for each rRNA was used to assign a high taxonomic level (at or above class) to the sequence. Where possible, sequences were further assigned to a genus if they shared ≥ 95% rRNA sequence identity with rRNA from a known species.
Assembly, annotation, and functional classification
Assembly was performed using the Roche GS De Novo Assembler (Newbler assembler v. 2.3, >98% identity and >40 bp overlap length). After assembly, putative CDS were predicted using MetaGeneAnnotator . Protein sequences were annotated using the best BLAST hit against the NCBI NR database, and tRNAs were identified using tRNAscan-SE . Entire metagenome datasets were annotated using the MG-RAST server .
Assembled contigs that were <5 kb in length and those with fewer than three predicted genes were discarded. Contigs were only retained that yielded consistent hits to a single high-level taxon (e.g., Epsilonproteobacteria, Thaumarchaeota, and Gammaproteobacteria). Strict assembly requirements combined with a taxonomic uniformity condition imposed on the assembled sequences resulted in 118 (in AR culture) and 91 (in SJ culture) contigs that were >5 Kb in length, had a consistent phylogenetic profile, and were likely to originate from a single organism (e.g., Sulfurovum sp. NBC37-1 and N. maritimus). To test if the assembly strategy produced contigs that were “real,” we manually identified all contigs that belonged to the clades of Ca. “Nitrosopumilus” and Sulfurovum, which were abundant in both enrichment cultures. The criterion for assigning contigs to the clades of Ca. “Nitrosopumilus” and Sulfurovum was that all genes must provide best hits in these genomes. We identified 97 contigs (73 for Ca. “Nitrosopumilus” and 24 for Sulfurovum) in which all genes provided the best hit for N. maritimus and Sulfurovum sp. NBC37-1. To increase taxonomic uniformity, we directly compared the nucleotide sequence of these contigs to the reference genome, using BLASTN ,. Oligonucleotide frequencies of the assembled contigs were computed using the wordfreq program in the EMBOSS package , and principal component analysis was performed using the R package FactoMineR . All predicted CDS were also searched for similarity using RPSBLAST to predict clusters of orthologous group assignments (cutoff e-value of 10−5) . We used CUSP and CODCMP from the European Molecular Biology Open Software Suite package for codon usage analysis. The GC skew was calculated using the Oligoweb interface http://insilico.ehu.es/oligoweb/. CRISPRs were searched using CRISPR Finder .
Reciprocal BLASTN and TBLASTX searches between the metagenomes were used for comparative analyses, leading to the identification of regions of similarity, insertions, and/or rearrangements (e-value cutoff of 10−5). The Artemis Comparison Tool  was used to visualize comparisons of the genomic fragments. ANI was calculated as defined by Konstantinidis and Tiedje . Reciprocal BLASTCLUST was used to predict orthologous proteins between each contig (affiliated with Thaumarchaeota, Epsilonproteobacteria, and Gammaproteobacteria) and reference genome (e.g., N. maritimus and Sulfurovum sp. NBC37-1) using a minimum cutoff of 50% identity and 70% of the length of the query CDS. The JSpecies program  was used to confirm manual ANI analyses. A BLASTN  comparison (cutoff of 50% identity and 70% of the length of the query sequences) between the datasets formed by the two archaeal genomes and the metagenome dataset of the Sargasso Sea  was used for recruitment analysis.
GC content (%) of single and mate-paired reads of the AR and SJ metagenomes. The numbers of single reads of the AR and SJ metagenomes were about 727,301 and 631,686, and of mate-paired reads of AR and SJ metagenomes were 478,179 and 489,454, respectively.
Taxonomic profiles (at or above class level) using the 16S rRNA gene sequences of (A) AR (n = 1,100) and (B) SJ (n = 908) metagenome datasets.
Comparison of all sequence reads from the AR and SJ metagenome datasets with the M4N5 database using the MG-RAST server (BLASTX cutoff: e-value of 1e-5 and minimum alignment length of 50 bp).
GC% versus length of assembled contigs (≥5 Kb) from the AR (A) and SJ (B) metagenomes.
Phylogenetic analysis of the archaeal 16S rRNA gene sequences obtained from strain AR1 and AR2 indicated in boldface and published sequences. “ThAOA” indicates thermophilic AOA lineage. Cluster groups were denoted at the right of the figure based on the origin of reference sequences. Branching patterns supported by more than 50% bootstrap values (1,000 iterations) by means of neighbor-joining was denoted by their respective bootstrap values. The scale bar represents 2% estimated sequence divergence.
Dot plot representation of the pairwise alignments of the strain AR1 and SCM1 (A), AR2 and SCM1 (B), and AR1 and AR2 (C) genomes. Alignments were performed on the six-frame amino acid translation of the genome sequences using the program in the MUMmer 3.23 package. In all plots, a dot indicates a gene compared, with forward or reverse matches shown in red and blue, respectively.
Recruitment plots of the Sargasso Sea metagenome dataset of GOS to the draft genomes of (A) Ca. “Nitrosopumilus koreensis” AR1 and (B) Ca. “N. sediminis” AR2. (1) GC-content plotted with a sliding window of 25,000 nucleotides. Average percentage of GC (34.2% and 33.6%, respectively) is shown by red line. (2) GC skew of AR1 and AR2 draft genomes plotted with a sliding window of 25,000 nucleotides. (3) Mummerplot showing recruitment of the Sargasso Sea metagenome reads to the AR1 and AR2 draft genomes. Individual archaeal reads of the metagenome were blasted with the AR1 and AR2 draft genomes, respectively. Green boxes indicate genomic islands of the AR1 and AR2 draft genomes.
Distribution of COG functional classes. Percentage of COGs predicted in the Ca. “Nitrosopumilus koreensis” AR1 and Ca. “N. sediminis” AR2 genomes. All genes of both genomes (A) and genes found in genomic islands (B). COG; cluster of orthologous groups.
Alignment of start and upstream region of the nirK gene sequence from metagenome and cultivated marine ammonia-oxidizing archaea. The ATG start codon and TATAbox/Brelements are highlighted . NirK gene sequences are from Nitrosopumilus maritimus (nmar), N. koreensis (ar1), N. sediminis (ar2) and marine metagenome (Marine-met).
Phylogenetic analysis of the NO dioxygenase gene in strain EP_AR indicated in boldface and homolog enzymes based on amino acid sequences. “EUK” indicates Eukaryote domain. Branching patterns supported by more than 50% bootstrap values (1,000 iterations) by means of neighbor-joining was denoted by their respective bootstrap values. The scale bar represents 20% estimated sequence divergence.
General features of the metagenome datasets from the AR and SJ cultures.
Nucleotide (NT) and amino acid (AA) identities of rRNA and ammonia monooxygenase (amo) genes, respectively between archaeal genomes (AR1, AR2, and SJ) and Nitrosopumilus maritimus.
Characteristics of genomic islands of the draft genomes of Ca. “Nitrosopumilus koreensis” AR1 and Ca. “N. sediminis” AR2.
Summary of predicted metabolic capabilities of microorganisms based on draft genome sequences.
Putative coding sequences of the genomic islands of the AR1 and AR2 genomes.
Conceived and designed the experiments: SJP SKR. Analyzed the data: SJP RG A-BM-C FR-V WHC ELM SKR. Contributed reagents/materials/analysis tools: FR-V KKK JHL SKR. Wrote the paper: SJP SKR.
- 1. Bock E, Wagner M (2006) Oxidation of Inorganic Nitrogen Compounds as an Energy Source. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes: Springer New York. pp. 457–495.
- 2. Treusch AH, Leininger S, Kletzin A, Schuster SC, Klenk HP, et al. (2005) Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environ Microbiol 7: 1985–1995.
- 3. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, et al. (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66–74.
- 4. K?nneke M, Bernhard AE, de la TorreJR, Walker CB, Waterbury JB, et al. (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437: 543–546.
- 5. Brochier-Armanet C, Boussau B, Gribaldo S, Forterre P (2008) Mesophilic Crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota. Nat Rev Microbiol 6: 245–252.
- 6. Spang A, Hatzenpichler R, Brochier-Armanet C, Rattei T, Tischler P, et al. (2010) Distinct gene set in two different lineages of ammonia-oxidizing archaea supports the phylum Thaumarchaeota. Trends Microbiol 18: 331–340.
- 7. Park BJ, Park SJ, Yoon DN, Schouten S, Sinninghe Damste JS, et al. (2010) Cultivation of autotrophic ammonia-oxidizing archaea from marine sediments in coculture with sulfur-oxidizing bacteria. Appl Environ Microbiol 76: 7575–7587.
- 8. Wuchter C, Abbas B, Coolen MJ, Herfort L, van Bleijswijk J, et al. (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci U S A 103: 12317–12322.
- 9. Tourna M, Stieglmeier M, Spang A, K?nneke M, Schintlmeister A, et al. (2011) Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proc Natl Acad Sci U S A 108: 8420–8425.
- 10. Jung MY, Park SJ, Min D, Kim JS, Rijpstra WI, et al. (2011) Enrichment and characterization of an autotrophic ammonia-oxidizing archaeon of mesophilic crenarchaeal group I.1a from an agricultural soil. Appl Environ Microbiol 77: 8635–8647.
- 11. Kim JG, Jung MY, Park SJ, Rijpstra WIC, Damsté JSS, et al. (2012) Cultivation of a highly enriched ammonia-oxidizing archaeon of thaumarchaeotal group I.1b from an agricultural soil. Environ Microbiol 14: 1528–1543.
- 12. Park SJ, Park BJ, Rhee SK (2008) Comparative analysis of archaeal 16S rRNA and amoA genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments. Extremophiles 12: 605–615.
- 13. Hallam SJ, Konstantinidis KT, Putnam N, Schleper C, Watanabe Y, et al. (2006) Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum. Proc Natl Acad Sci U S A 103: 18296–18301.
- 14. Hallam SJ, Mincer TJ, Schleper C, Preston CM, Roberts K, et al. (2006) Pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine Crenarchaeota. PLoS Biol 4: e95.
- 15. Walker CB, de la TorreJR, Klotz MG, Urakawa H, Pinel N, et al. (2010) Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc Natl Acad Sci U S A 107: 8818–8823.
- 16. Blainey PC, Mosier AC, Potanina A, Francis CA, Quake SR (2011) Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis. PLoS One 6: e16626.
- 17. Kim BK, Jung MY, Yu DS, Park SJ, Oh TK, et al. (2011) Genome sequence of an ammonia-oxidizing soil archaeon, "Candidatus Nitrosoarchaeum koreensis" MY1. J Bacteriol 193: 5539–5540.
- 18. Tully BJ, Nelson WC, Heidelberg JF (2012) Metagenomic analysis of a complex marine planktonic thaumarchaeal community from the Gulf of Maine. Environ Microbiol 14: 254–267.
- 19. Mosier AC, Allen EE, Kim M, Ferriera S, Francis CA (2012) Genome sequence of "Candidatus Nitrosopumilus salaria" BD31, an ammonia-oxidizing archaeon from the San Francisco Bay estuary. J Bacteriol 194: 2121–2122.
- 20. Mosier AC, Allen EE, Kim M, Ferriera S, Francis CA (2012) Genome sequence of "Candidatus Nitrosoarchaeum limnia" BG20, a low-salinity ammonia-oxidizing archaeon from the San Francisco Bay estuary. J Bacteriol 194: 2119–2120.
- 21. de la TorreJR, Walker CB, Ingalls AE, K?nneke M, Stahl DA (2008) Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ Microbiol 10: 810–818.
- 22. Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, et al. (2008) A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. Proc Natl Acad Sci U S A 105: 2134–2139.
- 23. Fogel GB, Collins CR, Li J, Brunk CF (1999) Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population. Microb Ecol 38: 93–113.
- 24. Nakagawa S, Takaki Y, Shimamura S, Reysenbach A-L, Takai K, et al. (2007) Deep-sea vent ε-proteobacterial genomes provide insights into emergence of pathogens. Proc Natl Acad Sci 104: 12146–12150.
- 25. Spang A, Poehlein A, Offre P, Zumbragel S, Haider S, et al. (2012) The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations. Environ Microbiol 14: 3122–3145.
- 26. Konstantinidis KT, Tiedje JM (2005) Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci U S A 102: 2567–2572.
- 27. Darling ACE, Mau B, Blattner FR, Perna NT (2004) Mauve: Multiple Alignment of Conserved Genomic Sequence With Rearrangements. Genome Res 14: 1394–1403.
- 28. Human Microbiome Jumpstart Reference Strains C, Nelson KE, Weinstock GM, Highlander SK, Worley KC, et al. (2010) A catalog of reference genomes from the human microbiome. Science 328: 994–999.
- 29. Park SJ, Kim JG, Jung MY, Kim SJ, Cha IT, et al. (2012) Draft genome sequence of an ammonia-oxidizing archaeon, "Candidatus Nitrosopumilus koreensis" AR1, from marine sediment. J Bacteriol 194: 6940–6941.
- 30. Park SJ, Kim JG, Jung MY, Kim SJ, Cha IT, et al. (2012) Draft genome sequence of an ammonia-oxidizing archaeon, "Candidatus Nitrosopumilus sediminis" AR2, from Svalbard in the Arctic Circle. J Bacteriol 194: 6948–6949.
- 31. Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, et al. (2007) The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 5: e77.
- 32. Merkl R (2006) A comparative categorization of protein function encoded in bacterial or archeal genomic islands. J Mol Evol 62: 1–14.
- 33. Gorman-Lewis D, Martens-Habbena W, Stahl DA (2014) Thermodynamic characterization of proton-ionizable functional groups on the cell surfaces of ammonia-oxidizing bacteria and archaea. Geobiology 12: 157–171.
- 34. Lehtovirta-Morley LE, Stoecker K, Vilcinskas A, Prosser JI, Nicol GW (2011) Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil. Proc Natl Acad Sci U S A 108: 15892–15897.
- 35. Bartossek R, Spang A, Weidler G, Lanzen A, Schleper C (2012) Metagenomic analysis of ammonia-oxidizing archaea affiliated with the soil group. Front Microbiol 3: 208.
- 36. Vajrala N, Martens-Habbena W, Sayavedra-Soto LA, Schauer A, Bottomley PJ, et al. (2013) Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea. Proc Natl Acad Sci U S A 110: 1006–1011.
- 37. Jung MY, Well R, Min D, Giesemann A, Park SJ, et al.. (2013) Isotopic signatures of NO produced by ammonia-oxidizing archaea from soils. ISME J.
- 38. Schlegel K, Leone V, Faraldo-Gomez JD, Muller V (2012) Promiscuous archaeal ATP synthase concurrently coupled to Na+ and H+ translocation. Proc Natl Acad Sci U S A 109: 947–952.
- 39. Storbeck S, Rolfes S, Raux-Deery E, Warren MJ, Jahn D, et al. (2010) A novel pathway for the biosynthesis of heme in Archaea: genome-based bioinformatic predictions and experimental evidence. Archaea 2010: 175050.
- 40. Cavallaro G, Decaria L, Rosato A (2008) Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems. J Proteome Res 7: 4946–4954.
- 41. Stein LY, Arp DJ, Berube PM, Chain PS, Hauser L, et al. (2007) Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation. Environ Microbiol 9: 2993–3007.
- 42. Norton JM, Klotz MG, Stein LY, Arp DJ, Bottomley PJ, et al. (2008) Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment. Appl Environ Microbiol 74: 3559–3572.
- 43. Herndl GJ, Reinthaler T, Teira E, van Aken H, Veth C, et al. (2005) Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean. Appl Environ Microbiol 71: 2303–2309.
- 44. Ingalls AE, Shah SR, Hansman RL, Aluwihare LI, Santos GM, et al. (2006) Quantifying archaeal community autotrophy in the mesopelagic ocean using natural radiocarbon. Proc Natl Acad Sci U S A 103: 6442–6447.
- 45. Tourna M, Stieglmeier M, Spang A, Konneke M, Schintlmeister A, et al. (2011) Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proc Natl Acad Sci U S A 108: 8420–8425.
- 46. Konstantinidis KT, Braff J, Karl DM, DeLong EF (2009) Comparative metagenomic analysis of a microbial community residing at a depth of 4,000 meters at station ALOHA in the North Pacific subtropical gyre. Appl Environ Microbiol 75: 5345–5355.
- 47. Therkildsen MS, Lomstein BA (1994) Seasonal variation in sediment urea turnover in a shallow estuary. Mar Ecol Prog Ser 109: 77–82.
- 48. Alonso-Sáez L, Waller AS, Mende DR, Bakker K, Farnelid H, et al. (2012) Role for urea in nitrification by polar marine Archaea. Proc Natl Acad Sci U S A 109: 17989–17994.
- 49. Urakawa H, Martens-Habbena W, Stahl DA (2011) Physiology and genomics of ammonia-oxidizing archaea. In: Ward BB, Arp DJ, Klotz MG, editors. Nitrification. Washington, DC, USA: American Society for Microbiology. pp. 117–156.
- 50. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, et al. (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9: 467–477.
- 51. Sasaki K, Shinichiro N, Shizuo T (2001) Vertical distributions of interstitial phosphate and fluoride in anoxic sediment: Insight into the formation of an authigenic fluoro-phosphorus compound. Geochem J 35: 295–306.
- 52. Ghiglione JF, Murray AE (2011) Pronounced summer to winter differences and higher wintertime richness in coastal Antarctic marine bacterioplankton. Environ Microbiol.
- 53. Herman DC, Frankenberger WTJ (1998) Microbial-mediated reduction of perchlorate in groundwater. J Environ Qual 27: 750–754.
- 54. Maixner F, Wagner M, Lucker S, Pelletier E, Schmitz-Esser S, et al. (2008) Environmental genomics reveals a functional chlorite dismutase in the nitrite-oxidizing bacterium 'Candidatus Nitrospira defluvii'. Environ Microbiol 10: 3043–3056.
- 55. Bedard C, Knowles R (1989) Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers. Microbiol Rev 53: 68–84.
- 56. Park SJ, Ghai R, Martin-Cuadrado AB, Rodriguez-Valera F, Jung MY, et al. (2012) Draft genome sequence of the sulfur-oxidizing bacterium "Candidatus Sulfurovum sediminum" AR, which belongs to the Epsilonproteobacteria. J Bacteriol 194: 4128–4129.
- 57. Sievert SM, Scott KM, Klotz MG, Chain PS, Hauser LJ, et al. (2008) Genome of the epsilonproteobacterial chemolithoautotroph Sulfurimonas denitrificans. Appl Environ Microbiol 74: 1145–1156.
- 58. Yakimov MM, La Cono V, Denaro R, D'Auria G, Decembrini F, et al. (2007) Primary producing prokaryotic communities of brine, interface and seawater above the halocline of deep anoxic lake L'Atalante, Eastern Mediterranean Sea. ISME J 1: 743–755.
- 59. Joye SB, Hollibaugh JT (1995) Influence of sulfide inhibition of nitrification on nitrogen regeneration in sediments. Science 270: 623–625.
- 60. Koch M, Rudolph C, Moissl C, Huber R (2006) A cold-loving crenarchaeon is a substantial part of a novel microbial community in cold sulphidic marsh water. FEMS Microbiol Ecol 57: 55–66.
- 61. Muller F, Brissac T, Le Bris N, Felbeck H, Gros O (2010) First description of giant Archaea (Thaumarchaeota) associated with putative bacterial ectosymbionts in a sulfidic marine habitat. Environ Microbiol 12: 2371–2383.
- 62. Ghosh W, Dam B (2009) Biochemistry and molecular biology of lithotrophic sulfur oxidation by taxonomically and ecologically diverse bacteria and archaea. FEMS Microbiol Rev 33: 999–1043.
- 63. Anderson IC, Poth M, Homstead J, Burdige D (1993) A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis. Appl Environ Microbiol 59: 3525–3533.
- 64. Kester RA, De Boer W, Laanbroek HJ (1997) Production of NO and N2O by pure cultures of nitrifying and denitrifying bacteria during changes in aeration. Appl Environ Microbiol 63: 3872–3877.
- 65. Ritchie GA, Nicholas DJ (1972) Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea. Biochem J 126: 1181–1191.
- 66. Kampschreur MJ, Picioreanu C, Tan N, Kleerebezem R, Jetten MS, et al. (2007) Unraveling the source of nitric oxide emission during nitrification. Water Environ Res 79: 2499–2509.
- 67. Stahl DA, de la TorreJR (2012) Physiology and diversity of ammonia-oxidizing archaea. Annu Rev Microbiol 66: 83–101.
- 68. Yan J, Haaijer SC, Op den Camp HJ, van Niftrik L, Stahl DA, et al. (2012) Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory-scale model system. Environ Microbiol 14: 3146–3158.
- 69. Santoro AE, Buchwald C, McIlvin MR, Casciotti KL (2011) Isotopic signature of N2O produced by marine ammonia-oxidizing archaea. Science 333: 1282–1285.
- 70. Löscher CR, Kock A, Könneke M, LaRoche J, Bange HW, et al. (2012) Production of oceanic nitrous oxide by ammonia-oxidizing archaea. Biogeosciences 9: 2419–2429.
- 71. Deckert G, Warren PV, Gaasterland T, Young WG, Lenox AL, et al. (1998) The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392: 353–358.
- 72. Gardner AM, Gardner PR (2002) Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherichia coli. Evidence for a novel inducible anaerobic nitric oxide-scavenging activity. J Biol Chem 277: 8166–8171.
- 73. Moreno-Vivian C, Cabello P, Martinez-Luque M, Blasco R, Castillo F (1999) Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol 181: 6573–6584.
- 74. Park SJ, Kang CH, Chae JC, Rhee SK (2008) Metagenome microarray for screening of fosmid clones containing specific genes. FEMS Microbiol Lett 284: 28–34.
- 75. Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62: 316–322.
- 76. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, et al. (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37: D141–145.
- 77. Noguchi H, Taniguchi T, Itoh T (2008) MetaGeneAnnotator: detecting species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res 15: 387–396.
- 78. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964.
- 79. Meyer F, Paarmann D, D'Souza M, Olson R, Glass EM, et al. (2008) The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9: 386.
- 80. Ghai R, Hernandez CM, Picazo A, Mizuno CM, Ininbergs K, et al. (2012) Metagenomes of Mediterranean coastal lagoons. Sci Rep 2: 490.
- 81. Ghai R, Pasic L, Fernandez AB, Martin-Cuadrado AB, Mizuno CM, et al. (2011) New abundant microbial groups in aquatic hypersaline environments. Sci Rep 1: 135.
- 82. Rice P, Longden I, Bleasby A (2000) EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 16: 276–277.
- 83. Lê S, Josse J, F H (2008) FactoMineR: An R package for multivariate analysis. J Stat Soft 25: 1–18.
- 84. Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, et al. (2001) The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res 29: 22–28.
- 85. Grissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35: W52–57.
- 86. Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, et al. (2005) ACT: the Artemis Comparison Tool. Bioinformatics 21: 3422–3423.
- 87. Richter M, Rossello-Mora R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106: 19126–19131.
- 88. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.