APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2007, p. 5642–5647 Vol. 73, No. 17 0099-2240/07/$08.00ϩ0 doi:10.1128/AEM.00461-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Distribution and Diversity of Archaeal Ammonia Monooxygenase Genes Associated with Coralsᰔ† J. Michael Beman,1‡ Kathryn J. Roberts,1§ Linda Wegley,2 Forest Rohwer,2 and Christopher A. Francis1* Department of Geological and Environmental Sciences, Stanford University, Stanford, California 94305,1 and Department of Biology, San Diego State University, San Diego, California 921822 Received 28 February 2007/Accepted 18 June 2007 Corals are known to harbor diverse microbial communities of Bacteria and Archaea, yet the ecological role of these microorganisms remains largely unknown. Here we report putative ammonia monooxygenase subunit A(amoA) genes of archaeal origin associated with corals. Multiple DNA samples drawn from nine coral species and four different reef locations were PCR screened for archaeal and bacterial amoA genes, and archaeal amoA gene sequences were obtained from five different species of coral collected in Bocas del Toro, Panama. The 210 coral-associated archaeal amoA sequences recovered in this study were broadly distributed phylogenetically, with most only distantly related to previously reported sequences from coastal/estuarine sediments and oceanic water columns. In contrast, the bacterial amoA gene could not be amplified from any of these samples. These results offer further evidence for the widespread presence of the archaeal amoA gene in marine ecosystems, including coral reefs. The exceptional biological diversity of coral reef ecosystems available. However, it has become increasingly clear that many is relatively well characterized for many eukaryotic species mesophilic Crenarchaeota may be capable of ammonia oxida- (see, e.g., reference 32). However, diverse assemblages of mi- tion (9), based on the cultivation of an ammonia-oxidizing croorganisms from the domains Bacteria and Archaea are also archaeon (AOA) (21) and accumulating (meta)genomic (12, found in association with corals (2, 8, 11, 15, 20, 22, 30, 33, 34, 13, 21, 45, 46), molecular (1, 6, 10, 24, 25, 29, 31, 50), and 43, 49), yet very little is known about the ecological function of geochemical (19, 23) evidence. More specifically, amoA genes the microorganisms that comprise these communities (34, 35). putatively encoding the ␣-subunit of the ammonia monooxy- Among other important roles, it has been hypothesized that genase enzyme appear to be present in many mesophilic Cre- coral-associated microorganisms are critical for nutrient cy- narchaeota (10, 12, 13, 21, 25, 50), and Lam et al. (24) recently cling (34), and in fact corals have been shown to harbor sym- demonstrated that this gene is actively expressed by some biotic nitrogen (N)-fixing cyanobacteria, which may represent Crenarchaeota in the ocean. an appreciable source of this essential nutrient (26). In addi- Ammonia oxidation is the first and rate-limiting step of tion, specific bacterial ribotypes appear to be associated with chemoautotrophic nitrification—the overall oxidation of am- Ϫ Ϫ particular coral species (33, 34), with many of these types being monia (NH3) to nitrite (NO2 ) to nitrate (NO3 )—and there most closely related to known nitrogen fixers and antibiotic is clear evidence for nitrification in association with individual producers (34). corals (47), within coral reefs (48), and within reef cavities While these studies have provided some insight into the (37). Given the association between Crenarchaeota and corals ecological function of coral-associated Bacteria, members of (20, 49), we investigated the distribution and diversity of ar- the domain Archaea are also known to be associated with chaeal amoA gene fragments in DNA extracts recovered from corals: both mesophilic Euryarchaeota and Crenarchaeota are coral colonies in Panama, Bermuda, Hawaii, and Puerto Rico. widely distributed across different reef locations and coral spe- Our results indicate that archaeal amoA genes are associated cies (20, 49), and Archaea appear to be abundant on some coral with a variety of coral species. 8 Ϫ2 colonies, numbering Ͼ10 cells cm (49). Until recently, few Sample collection, DNA preparation, and amplification and insights into the ecology and physiology of these coral-associ- sequencing of archaeal amoA genes. Samples were collected ated Archaea—and the mesophilic Archaea in general—were from coral colonies at Whale Bone Bay, Bermuda (August 1999), Bocas del Toro, Panama (April 1999, June 2000, Feb- ruary 2005), La Parguerra, Puerto Rico (January 2002), and * Corresponding author. Mailing address: Department of Geologi- Kane’ohe Bay, HI (June 2003) by use of previously described cal and Environmental Sciences, Building 320 – Room 118, Stanford methods (49). Briefly, a punch and hammer or bone clippers University, Stanford, CA 94305-2115. Phone: (650) 724-0301. Fax: (650) 725-2199. E-mail: [email protected]. were used to collect a single sample from each healthy coral † Supplemental material for this article may be found at http://aem colony; samples were then placed in Ziploc bags underwater, .asm.org/. washed with 0.2 ␮M filtered and autoclaved seawater at the ‡ Present address: Department of Biological Sciences, University of surface, placed on ice, returned to the lab, and stored at Ϫ80°C Southern California, Los Angeles, CA 90089. Diploria strigosa Montastraea franksi § Present address: Ocean Sciences Department, University of Cali- until DNA extraction. , , fornia Santa Cruz, Santa Cruz, CA 95064. and Porites astreoides were sampled in both Bermuda and Pan- ᰔ Published ahead of print on 22 June 2007. ama, Colpophyllia natans and Porites furcata were sampled only 5642 VOL. 73, 2007 ARCHAEAL amoA GENES ASSOCIATED WITH CORALS 5643 in Panama, Acropora cervicornis and Acropora prolifera samples TABLE 1. Richness of archaeal amoA libraries from coralsa were collected in Puerto Rico, and samples were collected No. of No. of OTUs: from Porites compressa in Hawaii (see Table S1 in the supple- Coral HЈ clones Estimated Estimated mental material). For DNA extraction, each frozen coral sam- library Observed value sequenced by Chao1 by ACE ple was airbrushed (Ͻ2.7 bar) with 10ϫ TE (100 mM Tris [pH 8.0]-10 mM EDTA) to remove the tissue and associated mi- CN8C 23 9 31 27 1.9 crobes. Two milliliters of the coral tissue-TE slurry was pel- DS2 32 12 15 16 2.3 ϫ DS4 31 9 10 12 1.9 leted for 30 min at 10,000 g at 4°C. Total DNA was extracted MA7 33 7 17 31 1.1 from the pellet by use of the Ultra Clean soil DNA kit (Mo- PF1 32 3 3 3 0.93 Bio). PA6 29 7 7 7 1.8 Archaeal amoA gene fragments were amplified using the PA22 30 2 2 2 0.69 PCR primers Arch-amoAF (5Ј-STAATGGTCTGGCTTAGA a All OTUs were defined as having Յ5% difference in nucleic acid sequence CG-3Ј) and Arch-amoAR (5Ј-GCGGCCATCCATCTGTATG alignment, and all indices were calculated using DOTUR. T-3Ј) and conditions as previously described (10). Triplicate PCRs were pooled, gel purified, and cloned using the TOPO-TA cloning kit (Invitrogen). White transformants were observed coverage of our libraries. Significant P values were transferred to 96-well plates containing LB broth (with 50 evaluated after correcting for multiple pairwise comparisons by ␮ ϭ Ϫ g/ml kanamycin), grown overnight at 37°C, and PCR use of the Dunn-Sida´k method (42), where Pcorrected 1 Ϫ 1/k screened for the presence of inserts by use of T7 and M13R (1 Puncorrected) , where k is the total number of compari- vector primers. Sequencing of T7/M13 PCR products was per- sons (for seven archaeal amoA libraries, each compared with formed using vector primers on ABI 3730xl capillary sequenc- the corresponding six other libraries, k ϭ 7 ϫ 6 ϭ 42). ers (PE Applied Biosystems). Coral DNA extracts were also Distribution and richness of archaeal amoA genes associ- PCR screened for the presence of betaproteobacterial amoA ated with corals. DNA was extracted from coral colonies in genes by use of primers (AmoA-1F* and AmoA-2R) and con- Bocas del Toro, Panama, Whale Bone Bay, Bermuda, ditions described previously (36, 44). Kane’ohe Bay, HI, and La Parguerra, Puerto Rico and Richness, phylogenetic, and statistical analyses. Nucleotide screened for the archaeal amoA gene. These DNA samples sequences were assembled and edited using Sequencher v.4.2 were drawn from nine different species of coral: Acropora cer- (GeneCodes, Ann Arbor, MI), and nucleotide and amino acid vicornis, Acropora prolifera, Colpophyllia natans, Diploria stri- alignments were generated using MacClade (http://macclade gosa, Montastraea annularis, Montastraea franksi, Porites as- .org). Operational taxonomic units (OTUs) were defined as treoides, Porites compressa, and Porites furcata. From 40 sequence groups in which sequences differed by Յ5%, and all samples that were screened, the archaeal amoA gene was am- analyses of richness—including rarefaction analysis (14) and plified from a total of 12 samples (see Table S1 in the supple- both ACE (5) and Chao1 (3) nonparametric richness estima- mental material). In contrast, in no case could the betapro- tions—were performed using DOTUR (38). teobacterial amoA gene be PCR amplified. Neighbor-joining phylogenetic trees (based on Jukes-Can- Of the 12 samples from which the archaeal amoA gene was tor-corrected distances) and parsimony trees were constructed amplified, 10 were previously screened for archaeal 16S rRNA based on alignments of DNA sequences by use of ARB (http: genes, and all contained 16S rRNA genes from Crenarchaeota //www.arb-home.de) (27). Nucleic acid sequences (rather than (49). In addition, Wegley et al. (49) quantified Crenarchaeota predicted amino acid sequences) were analyzed in order to and Euryarchaeota for 3 of the 40 coral colonies included in this highlight the genetic (rather than protein-level) heterogeneity study via fluorescent in situ hybridization, and Archaea num- among communities.