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Appl Environ Microbiol. Jun 2009; 75(12): 4216–4220.
Published online Apr 24, 2009. doi:  10.1128/AEM.01761-08
PMCID: PMC2698363

Diversity and Abundance of Ammonia-Oxidizing Archaea in Hydrothermal Vent Chimneys of the Juan de Fuca Ridge[down-pointing small open triangle]


The abundance and diversity of archaeal ammonia monooxygenase subunit A (amoA) genes from hydrothermal vent chimneys at the Juan de Fuca Ridge were investigated. The majority of the retrieved archaeal amoA sequences exhibited identities of less than 95% to those in the GenBank database. Novel ammonia-oxidizing archaea may exist in the hydrothermal vent environments.

Ammonia-oxidizing archaea (AOA) may play important roles in carbon and nitrogen cycles in various temperate environments (5, 7, 10, 12, 16). The frequent detection (23, 24) and successful enrichment (2, 6) of thermophilic AOA from terrestrial hot springs suggested a wide distribution of thermophilic AOA in geothermal environments. High concentrations of NH4+ (1, 9, 11) and high rates of ammonia oxidation (9, 22) have been observed at the Juan de Fuca Ridge. However, the presence of AOA in this deep-sea hydrothermal system has not been reported. Here, the abundance and diversity of AOA in three hydrothermal vent chimneys in the Endeavor segment of the Juan de Fuca Ridge were investigated by targeting the conserved amoA genes. This is also the first report on AOA from deep-sea hydrothermal vent chimneys.

These vent chimneys were sulfide structures obtained in the fall of 2005 using the submersible Alvin on board the research vessel Atlantis (dive numbers 4143, 4136, and 4148). Chimney 4148 was an active black smoker venting at around 310°C in the Main Endeavor field (47°56.876′N, 129°5.915′W; depth, 2,192 m). Chimney 4143-1 was an active black smoker venting at 316°C in the Mothra field (47°55.424′N, 129°6.533′W; depth, 2,267 m). The outer layers (samples 4148-1A and 4143-1A) of these chimneys were used in this study. The sample from chimney 4136-1 was from a diffusive field (Clambed field) (47°57.909′N, 129°5.443′W; depth, 2,200 m), where the in situ temperature was measured as 29.2°C. The chimney samples were stored at −20°C on board, transported to the home laboratory on dry ice, and stored at −80°C until analyses were performed.

Chimney samples were frozen in liquid nitrogen and milled upon thawing. This procedure was repeated three times to break down the solid sample into small particles, which were then mixed with DNA extraction buffer for DNA isolation as described before (25). The obtained crude DNA was purified by an E-Z N.A. Cycle-Pure kit (Omega Bio-Tek Inc., Norcross, GA). PCR amplifications for the archaeal 16S rRNA gene, the crenarchaeal marine group I (MGI) 16S rRNA gene, the archaeal amoA gene, and the bacterial amoA gene followed procedures previously described (Table (Table1)1) (3, 5, 10, 14). Quantitative PCR (Q-PCR) was performed using a model 7500 real-time system (Applied Biosystems, United Kingdom) and a 20-μl reaction mixture that consisted of 1 μl (1 to 10 ng) of DNA as the template, a 0.15 μM concentration of each primer, and 10 μl of Power SYBR green PCR master mix (Applied Biosystems, United Kingdom) with ROX and SYBR green I. The inserted PCR fragments of clones 4143-1A-71 (from the amoA gene library) and 4136-1-4 (from the archaeal 16S rRNA gene library) were amplified and purified to generate standard DNAs for amoA or archaeal 16S rRNA gene quantification. A serial dilution of standard DNAs was performed to generate calibration curves for sample quantification. A melting curve analysis was performed after amplification, and the cycle threshold was set automatically using system 7500 software, version 1.3.

PCR primers and procedures used in this study

Triplicate PCR products were pooled and clone libraries constructed following the manufacturer's instructions (Takara Inc., Dalian, China). PCR clones from the libraries were randomly selected for sequencing (Sangon Inc., China). Phylogenetic trees were generated using the PHYLIP package (4) and the maximum-likelihood, neighbor-joining, and maximum-parsimony methods. Bootstrap analysis was used to estimate the reliability of phylogenetic tree constructions (200 replicates). Trees were created using the program Treeview (version 1.6.6).

Positive and specific PCR bands were obtained for the archaeal amoA genes from all the three samples, while no PCR band was obtained for the bacterial amoA gene (for the primers and procedures used, see Table Table1).1). In addition, sample 4136-1 was found by Q-PCR analysis to contain the highest number of archaeal amoA genes (with 7.36 ± 0.37 × 104 copies per g of chimney), followed by samples 4143-1A (with 1.88 ± 0.08 × 104 copies per g of chimney) and 4148-1A (with 1.37 ± 0.07 × 102 copies per g of chimney).

Clone libraries of archaeal amoA from the three samples were constructed. A total of 93 clones (33 from sample 4136-1, 30 from sample 4143-1A, and 30 from sample 4148-1A) were sequenced and divided into 33 operational taxonomic units (OTUs) based on 99% nucleotide identity. The majority (81.7%) of the retrieved archaeal amoA OTU sequences exhibited relatively low identity (≤94.56%) to other archaeal amoA sequences deposited in GenBank. The phylogenetic relationships among the retrieved amoA and some published amoA sequences are shown in Fig. Fig.1.1. The chimney archaeal amoA sequences fell into five clusters (chimney group I, chimney group II, sediment A-1, and water column A and B clusters), except the sequence of clone 4143-1A-10, which did not fall into any cluster and exhibited the highest identity (90%) to the sequence of clone HB_B_0805A06, which was derived from coastal sediment (18). Chimney group I contained 52 sequences (30 from sample 4148-1A, 11 from sample 4143-1A, and 11 from sample 4136-1); chimney group II contained 23 sequences (20 from sample 4136-1 and 3 from sample 4143-1A). Fourteen sequences from sample 4143-1A grouped into water column A and B clusters (5); and one sequence from sample 4143-1A grouped into the sediment A-1 cluster (13). The sequences from chimney group I exhibited the highest identity (94%) to clone CR-G3N006, derived from a cold seep of the Japan Sea (13). Sequences in chimney group II exhibited the highest identity to clone OA-MA-122 from a water column of a coastal aquarium biofilter, with 84% nucleotide identity (21). The sequences of chimney group II did not cluster with any other sequences. Although showing low bootstrap values (<50%), the chimney group II sequences always clustered into a separate group (Fig. (Fig.1)1) according to different calculation methods, including the maximum-likelihood, neighbor-joining, and maximum-parsimony methods.

FIG. 1.
Phylogenetic tree showing the affiliations of archaeal amoA gene sequences from chimneys (in bold), sediments, soil, water, and the isolated AOA. Bootstrap values were calculated from 200 replications with 585 characters. Maximum-likelihood (left), distance ...

Sample 4136-1 contained the highest number of archaeal amoA gene copies. Q-PCR using primers 344F and 518R (15) showed that sample 4136-1 contained 1.10 ± 0.05 × 106 copies of archaeal 16S rRNA genes per g of chimney. Assuming that each crenarchaeal cell possessed only one copy of the amoA gene (8), the AOA constituted at least 6.1% of the archaeal community in sample 4136-1. To explore the potential sources of these amoA sequences in sample 4136-1, an archaeal 16S rRNA clone library was constructed and a total of 82 clones were sequenced. These sequences divided into 20 OTUs based on 98% nucleotide identity. Fifteen OTUs (accounting 76.8% of the total sequences) belonged to hyperthermophilic Desulfurococcales species, and two OTUs (accounting for 15.9% of the total number of sequences) belonged to hyperthermophilic Thermoproteales species of the Crenarchaeota phylum, whereas three OTUs (accounting 7.32% of the total number of sequences) belonged to Thermococcales species of the Euryarchaeota kingdom (Fig. (Fig.2).2). Members of the crenarchaeal MGI, which was thought to be the source of nonthermophilic AOA (6, 8), were not detected in this library. Therefore, PCR using MGI-specific primers was performed to further detect MGI species (for PCR primers and conditions, see Table Table11 and reference 14). MGI species were easily detected in sample 4143-1A, but not in samples 4136-1 and 4148-1A, by direct PCR amplification. A nested PCR method employing generic archaeal 16S rRNA gene primers was then performed for the first round of PCR followed by MGI-selective PCR primers for the second round of PCR. This procedure created a PCR band of the correct size for MGI species from sample 4136-1; that band was later shown by cloning and sequencing to represent an MGI 16S rRNA gene fragment (see Fig. S1 in the supplemental material). The data implied that some of the amoA genes detected in the chimney samples may have come from MGI species; however, to determine the origin of the amoA genes, especially those in the chimney groups I and II, isolation or enrichment of the organisms would be necessary.

FIG. 2.
Phylogenetic tree showing the affiliations of 16S rRNA gene sequences retrieved from hydrothermal vent chimney 4136-1 (in boldface) with selected reference sequences of the Archaea domain. Bootstrap values were calculated from 200 replications with 790 ...

Supplementary Material

[Supplemental material]


We thank the crews of the research vessels Atlantis and Alvin for their help in collecting the chimney samples. We also thank Chuanlun Zhang from University of Georgia for his help in editing the paper.

This work was supported by the Natural Science Foundation of China (NSFC grants 40532011 and 40625016), COMRA (grant DYLY0202-01), and the National High-Tech Program (grant 2007AA091904).


[down-pointing small open triangle]Published ahead of print on 24 April 2009.

Supplemental material for this article may be found at http://aem.asm.org/.


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