RESEARCH ARTICLE
Intracellular Oceanospirillales bacteria inhabit gills of Acesta bivalves ˚ Birkeland1,3 & Martin Hovland3,4 ´ Sigmund Jensen1, Sebastien Duperron2, Nils-Kare 1
´ Department of Biology, University of Bergen, Bergen, Norway; 2UMR7138 Systematique Adaptation Evolution, Universite´ Pierre et Marie Curie, Paris, France; 3Centre for Geobiology, University of Bergen, Bergen, Norway; and 4Statoil, Stavanger, Norway
Correspondence: Sigmund Jensen, Department of Biology, University of Bergen, PO Box 7800, N-5020 Bergen, Norway. Tel.: 147 5558 4400; fax: 147 5558 4450; e-mail:
[email protected] Received 4 November 2009; revised 16 September 2010; accepted 22 September 2010. Final version published online November 2010. DOI:10.1111/j.1574-6941.2010.00981.x
MICROBIOLOGY ECOLOGY
Editor: Michael Wagner Keywords Acesta excavata; intracellular; Oceanospirillales; rRNA.
Abstract A novel bacterium was discovered in the gills of the large bivalve Acesta excavata (Limidae) from coral reefs on the northeast Atlantic margin near the shelf break of the fishing ground Haltenbanken of Norway, and confirmed present in A. excavata from a rock-wall in the Trondheimsfjord. Purified gill DNA contained one dominant bacterial rRNA operon as indicated from analysis of broad range bacterial PCR amplicons in denaturant gradient gels, in clone libraries and by direct sequencing. The sequences originated from an unknown member of the order Oceanospirillales and its 16S rRNA gene fell within a clade of strictly marine invertebrate-associated Gammaproteobacteria. Visual inspection by fluorescent in situ hybridization and transmission electron microscopy indicated a pleomorphic bacterium with no visible cell wall, located in aggregates inside vacuoles scattered within the gill cells cytoplasm. Intracellular Oceanospirillales exist in bathymodiolin mussels (parasites), Osedax worms and whiteflies (symbionts). This bacterium apparently lives in a specific association with the Acesta.
Introduction The large bivalve Acesta excavata is part of the dominant fauna of many coral reefs and rock-walls along the continental margin of the northeast Atlantic Ocean (Buhl´ Mortensen & Mortensen, 2004; Lopez Correra et al., 2005; Hovland, 2008; Roberts et al., 2009). Reefs near the shelf break of the fishing ground Haltenbanken of Norway were surveyed in September 2004 with the remotely operated vehicle (ROV) Hirov6, during the development of the Kristin hydrocarbon field. At least 120 reefs were seen, growing outside, inside and on the rim of small craters (pockmarks) in cool 6 1C water and at depths of 310–385 m. The largest reef measured 90 m in length and rose 3.5 m above the seafloor (Hovland, 2005, 2008). Major reef animals were in order of apparent abundance the hard coral Lophelia pertusa, sponges including Geodia, the soft corals Paragorgia arborea and Primnoa resedaeformis, and in clusters attached to hard surfaces, the A. excavata. Acesta excavata also occur at the Mid-Atlantic Ridge (Mortensen et al., 2008), off Newfoundland (Haedrich & Gagnon, 1991; Gagnon & Haedrich, 2003), at the Azores, the Canary FEMS Microbiol Ecol 74 (2010) 523–533
´ Islands (Lopez Correra et al., 2005) and in the Mediterranean Sea (Ghisotti, 1979). This bivalve belongs to the family Limidae and has been found at depths of 33–3200 m, first described from Norwegian fjords by Fabricus in 1779 (Vokes, 1963). The characteristic features of A. excavata are its large size (up to 20 cm), orange- to red-coloured body, very large gill area and a high filtration rate ( 4 100 L h1) ´ (Lopez Correra et al., 2005; J¨arnegren & Altin, 2006). Acesta ´ excavata feeds on micrometer-sized plankton (Lopez Correra et al., 2005; J¨arnegren & Altin, 2006), but has an unusual relative, Acesta oophaga in the Gulf of Mexico (J¨arnegren et al., 2007), which feeds on eggs of a vestimentiferan tubeworm at cold seeps (Kohl & Vokes, 1994; J¨arnegren et al., 2005). No microbiological study has, to our knowledge, been performed on the Acesta bivalves. Deep-water bivalves are best known from reduced environments such as vents, seeps, sediments and wood and whale falls, where they benefit from a nutritional relationship with chemoautotrophic and methanotrophic bacteria. Representatives of the families Solemyidae, Thyasiridae, Lucinidae, Vesicomyidae and Mytilidae harbour specialized bacteria-filled cell types in their gills (bacteriocytes) from 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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which the bacteria provide them with otherwise inaccessible sources of carbon, nitrogen and chemical energy (Southward et al., 2001; Stewart et al., 2005; Dubilier et al., 2008). The bacteria live in the cytoplasm and utilize sulphide (thiotrophs) or methane (methanotrophs). Additionally, there are intracellular bacteria that excrete enzymes to free carbon and energy from wood to Teredinidae bivalves (Distel et al., 1991; Sipe et al., 2000). Apart from these gammaproteobacterial endosymbionts, a related, but parasitic ‘Candidatus Endonucleobacter bathymodioli’ has been discovered recently inside the nuclei of gill cells of bathymodiolin mussels (Mytilidae) from hydrothermal vents in the Pacific–Antarctic Ridge and in the Mid-Atlantic Ridge, and from cold seeps in the Gulf of Mexico (Zielinski et al., 2009). Acesta excavata represent animals that thrive in permanently cold and dark marine environments, despite the lack of the light that sustains shallow-water fauna and tropical coral reefs, and the sulphide and methane that sustains vent and seep fauna. The existence of corals in the deep was known by fishermen for centuries until technology made a visual mapping of their reefs possible (Hovland, 2008; Roberts et al., 2009). Little is known about reef ’s nutrition, although the influx of plankton (Thiem et al., 2006), coral organic matter (Wild et al., 2008) and possibly pockmarkreleased sulphides and hydrocarbons (Hovland & Risk, 2003) stimulate reef life. As part of a study of the role played by bacteria, we collected A. excavata from coral reefs on the northeast Atlantic margin vest of Trondheim (Norway), and for comparison, from a fjord rock-wall, and examined the bivalves for gill-associated bacteria using a combination of molecular, microbiological and microscopic methods.
AeJ1,J2 (from rock), appeared as healthy adults of about 11 8 cm in size and with no obvious damage. Onboard the ROV support vessels, animals were kept in seawater, live and below 10 1C (AeJ1,J2), cooled to 4 1C (Ae2,7,8) or frozen to 18 1C (Ae1, Ae3-6). Reef animals were transported to the laboratory in a cooler bag with ice. Within 24 h, gill tissue of Ae2 was dissected and homogenized in 3% NaCl. The homogenate was plated on marine agar plates (DIFCO 2216) incubated at 6 1C. Gill tissue of Ae2 was stored in 96% ethanol at 4 1C, while within 48 h, gill tissue of Ae7,8 was fixed directly. The animals were deepfrozen to 80 1C. Rock animals were transferred within 5 h to a holding tank with flow-through water from 130 m depth and 8 1C (J¨arnegren & Altin, 2006) and no feeding. After being held for 1 year in the tank, gill tissue from AeJ1,J2 was dissected and posted to the laboratory in plastic tubes filled with 96% ethanol (August 2005).
Materials and methods Sampling and preparations Acesta excavata were collected using ROVs at Haltenbanken reefs in the Kristin hydrocarbon field (reef KA2 and reef Geosund; 64159 0 4000 N, 06133 0 1500 E) and in the adjacent hydrocarbon field Morvin (reference reef MRR08; 65108 0 1000 N, 06128 0 0200 E). Additionally, A. excavata were collected from a geographically separated rock-wall in the Trondheimsfjord (63128 0 2800 N, 09154 0 3000 E) (Table 1, Fig. 1). The 10 bivalves, prefixed Ae1-8 (from reefs) and
Fig. 1. Map indicating the geographical position of coral reefs (64159 0 4000 N, 06133 0 1500 E; KA2) and rock-wall (63128 0 2800 N, 09154 0 3000 E; 300 m depth) sampled for Acesta excavata bivalves. The reefs are located at fishing ground Haltenbanken, reef KA2 at 325 m depth and 5 m down an 8 m deep pockmark in the Kristin hydrocarbon field. Reefs Geosund and MRR08 are at a distance of c. 100 m (at 310 m depth) and c. 20 km (at 365 m depth), respectively.
Table 1. Collected Acesta excavata individuals, main analysis and findings as indicated by the bullets Individual
Collected
Site
DGGE
Library
FISH
TEM
afs
ivc
Ae1,3 Ae2 Ae4,5,6 Ae7,8 AeJ1,J2
June 2003 September 2004 June 2007 October 2009 August 2004
Reef KA2 Reef KA2 Reef Geos. Reef MRR08 Rock Sbn.
– –
– – –
– –
– –
Geos, Geosund; Sbn, Stokkbergneset; afs, autofluorescent structure; ivc, invertebrate clade.
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PCR, denaturing gradient gel electrophoresis (DGGE) and DNA sequencing DNA was extracted from 4–11 mg wet weight gill tissue, using a DNeasy Tissue Kit (Qiagen). Ethanol-preserved tissue was rinsed once in 1 mL sterile 3% NaCl before DNA extraction. Bacterial 16S rRNA gene fragments (200 bp), genes (1500 bp), 16S–23S intergenic spacers (150 bp) and 23S gene fragments (up to 1400 bp) were PCR amplified using the primers 338fGC/518r (Muyzer et al., 1993), 27f/1492r (Lane, 1991) and 27f/457r, 1608r (Lane, 1991; Hunt et al., 2006), respectively. Archaeal 16S rRNA genes (660 bp) and Eukaryal 18S rRNA genes (1800 bp) were PCR amplified using the primers A751f/UA1406r (Baker et al., 2003) and EukF/EukR (DeLong, 1992). Additionally, we tested DNA from the gill tissues for genes encoding enzymes involved in the oxidation of methane (pmoA), methanol (mxaF), in sulphur metabolism (aprA) and in autotrophy (cbbL), using the primers A189/A682 (Holmes et al., 1995), 1003f/1555r (McDonald & Murrell, 1997; Neufeld et al., 2007), aprA1FW/ aprA5RW (Meyer & Kuever, 2007) and IAB595f/IAB1385r (Elsaied et al., 2007), respectively. The reaction mixture contained 1 U of the DNA polymerase Dynazyme II in the supplied buffer (Finnzymes), 1.5 mM MgCl2, 125 mM each dNTP, 0.5 mM each primer and bovine serum albumin (BSA) to a final concentration of 2 mg mL1. Aliquots were transferred to 0.2 mL eppendorf tubes and template DNA (0.2 ng mL1) was added. Positive controls were DNA from Escherichia coli, Archaeoglobus fulgidus and Methylococcus capsulatus. PCR was performed in a thermal cycler (PTC-100 or a Perkin Elmer) with initial denaturation at 92–95 1C for 2–5 min, followed by cycles of denaturation at 92 1C for 1 min; annealing at 55 1C for 30 s (338fGC/518r), 55 1C for 1 min (A751f/UA1406r), 60 1C for 1 min (27f/1492r, 27f/457r, 1608r, M13f/M13r); and elongation at 72 1C for 1 min, before a final extension at 72 1C for 5–6 min. A modification to enhance sensitivity was performed for the amplification of the pmoA, mxaF, aprA and cbbL genes and also applied for the amplification of the 18S rRNA gene. The modification involved the use of the Phusion DNA polymerase in the GC buffer supplied (Finnzymes), 250 mM of each dNTP and a BSA concentration of 20 mg mL1. The corresponding PCR was 1 min of denaturation at 98 1C, followed by 35 cycles of 10 s at 98 1C, 30 s at 53 1C and 40 s at 72 1C, before a final extension at 72 1C for 5 min. All other PCR reactions were run for 30 cycles, but for amplicons to be cloned, precautions were taken to obtain representative 16S rRNA gene libraries that reflected the diversity of bacterial populations associated with the gills. The accumulation of miss-incorporated nucleotides, chimeras and heteroduplexes (Qiu et al., 2001) was minimized by reducing the cycling number to 18 (Ae1), 20 (Ae2) and 25 (Ae3) cycles, and by mending putative heteroduplexes in a FEMS Microbiol Ecol 74 (2010) 523–533
postamplification reconditioning step of three cycles (Acinas et al., 2004). Reconditioning was run with a 10-fold dilution using 5 mL amplicons in a 45 mL fresh reaction mixture. PCR products were checked for size and purity in 2% agarose gels following standard methods (Sambrook et al., 1989). DGGE was performed with a V20-HCDC system of SciePlas Limited (Southam), using a previous method (Jensen et al., 2004). Briefly, gels (0.75 mm) were prepared with 8% (w/v) 37.5 : 1 bisacrylamide in 0.5 TAE [20 mM Tris acetate (pH 7.4), 10 mM acetate, 0.5 mM Na2EDTA], yielding a linear gradient typically ranging from 30% to 65% denaturant [100% denaturant corresponds to 7 M urea and 40% (v/v) formamide deionized with AG501-X8 mixed bed resin (Bio-Rad)]. Gels were run for 20 h at 70 V and 60 1C in 0.5 TAE, stained with SYBR Gold (Molecular Probes) and photographed using a UV transillumination table (GeneGenius Syngene). Bands of interest were excised from the gels, eluted in water overnight, reamplified, checked for the correct positioning in a new DGGE, cleaned (USB Amersham) and sequenced using primers 338f (without GC clamp) and 518r. Sequencing was performed using an ABI 3700 PE sequencer (Applied Biosystems), following the protocol of the BigDye 3.1 kit (Perkin Elmer).
Clone libraries, isolates and phylogeny Pooled 27f/1492r reactions (3–19 parallels) were concentrated by sodium acetate ethanol precipitation (Ae1,2) or by elution in a smaller volume after passage through a Strataprep presequencing column (Ae3) and ligated into the PCR4 vector and transformed into E. coli, according to the manufacturer’s instructions (Invitrogen). Clones were checked for inserts by PCR using vector primers (M13f/ M13r) and fragments from inserts amplified with the primers 338fGC/518r. Amplicons were categorized in the DGGE by their position to the nearest defined reference position (Fig. 2). Plasmids were purified (Stratagene or ENZA kit) and inserts were sequenced as described above, from representative clones using the primers 27f, 530f, 518r, 926f, 1091r and 1492r. Additionally, direct sequencing was performed on 27f/1492r and 27f/457r, 1608r amplicons without a prior cloning of the genes. 16S rRNA gene fragments from bacteria readily cultivated on the marine agar plates were sequenced using a minimum of one reaction (primer 27f), on 27f/1492r products PCR amplified from whole cells. The 18S rRNA gene fragment of bivalves was sequenced using primers EukF, EukR and 530f on a EukF/EukR PCR product amplified from muscle tissue DNA. The sequences were inspected for chimeras using the RDP program CHIMERA CHECK (http://rdp8.cme.msu.edu) and no potential chimeras were detected. Related 16S rRNA gene 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Microscopy, fluorescent in situ hybridisation (FISH) and transmission electron microscopy (TEM)
Fig. 2. DGGE profiles of ribosomal gene fragments PCR amplified from Acesta excavata gills. Lanes 1–3, reef individuals Ae1,2,3; lanes 4–5, rock-wall individuals AeJ1,J2. M is marker with the corresponding gene fragments from Escherichia coli. Triangles and their numbers indicate the denaturing positions of reference for the categorization of 16S rRNA genes captured in clone libraries (OTUs; D is dominant).
sequences were identified using BLASTN (Altschul et al., 1997), SEQMATCH and CLASSIFIER (Cole et al., 2009), and NEXT RELATIVES within ARB (Ludwig et al., 2004). Phylogenetic analyses were performed in ARB. Imported sequences were automatically aligned to a prealigned data set (SILVA; Pruesse et al., 2007) using the FAST ALIGNER assisted with manual corrections according to the secondary structure of the 16S rRNA molecule. A filter was generated that omitted alignment at positions of sequence ambiguity and where sequence data were not available for all near-full-length sequences. Phylogeny was reconstructed with various data sets using Distance, Maximum-Parsimony and MaximumLikelihood methods, and topologies were compared visually, and evaluated by bootstrapping. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Crushed tissues of fresh and frozen gills were examined directly using a phase-contrast microscope (Nikon Eclipse E400). FISH and TEM were performed on five bivalves (Table 1). The ethanol-preserved gill tissue (Ae2,J1,J2) was rehydrated by transfer through 96% and 70% ethanol (3 15 min each) rinsed twice in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 130 mM NaCl, pH 7.3) and fixed overnight in 4% paraformaldehyde in PBS. Fresh gill tissue (Ae7,8) was fixed for 2 h, either in paraformaldehyde (for FISH) or in 3% glutaraldehyde and 1% osmium tetraoxide in 0.4 M NaCl buffered with 0.1 M cacodylate, pH 7.4 (for TEM), solutions changed twice. Rinses (2–3 ) in PBS or cacodylate buffer followed before being stored frozen in PBS and ethanol (50 : 50) or refrigerated in 70% ethanol, respectively. In preparation for FISH, the tissue was subsequently rinsed three times in PBS, dehydrated through 70%, 96% and 100% ethanol (3 minimum 15 min each), soaked in xylene and embedded in paraffin. Tissue was cut to 4 mm using a microtome (Leica RM2155) and the sections were collected on SuperFrostPlus slides (Menzel-Gl¨aser). The further FISH protocol was carried out essentially as described by Duperron et al. (2005). In brief, the paraffin was removed from sections by xylene (three 10-min treatments) and sections were rehydrated in decreasing series of 95%, 80% and 70% ethanol (10 min each), permeabilized in 0.2 M HCl (12 min), rinsed in 20 mM Tris-HCl pH 8.0 (10 min), permeabilized in the Tris buffer supplemented with 0.5 mg proteinase K mL1 (5 min at 37 1C), rinsed in the Tris buffer (10 min) and air-dried. Circled sections were covered with a mixture containing 100 ng probe in a hybridization buffer of 30 mL of 0.9 M NaCl, 0.02 M Tris-HCl, 0.01% SDS and formamide, and hybridized for 3 h at 46 1C. The probes applied were Eub338, NON338 (Amann et al., 1995), Euk1209 (Giovannoni et al., 1988) and Ae128 (5 0 CCTCTACCGGGCAAATTC-3 0 ). Ae128 (E. coli numbering) was designed specifically for the Acesta bacterium using clone Ae3p8 that was scanned by eye for a unique region. Promising 15–20 nt stretches in regions with good accessibility to the ribosomal structure (Behrens et al., 2003) were checked for matches in public databanks using BLASTN (Altschul et al., 1997) and PROBEMATCH (Cole et al., 2009). Ae128 has at least 1 nt mismatch with all entered GenBank sequences, including relevant Vibrio species (a single mismatch) and intracellular bivalve bacteria (two mismatches), as of September 2010). The Ae128 signal breaks down at 40% formamide concentration. Texas Red and Cy5 fluorochrome-conjugated probes were ordered from Thermo Electron Corporation (Ulm). After hybridization, slides were washed for 15 min at 48 1C in 0.1 M NaCl, 0.02 M Tris-HCl, 5 mM EDTA and 0.01% SDS, rinsed by dipping in FEMS Microbiol Ecol 74 (2010) 523–533
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MilliQ water (48 1C) and air-dried. The sections were covered with 1 mg mL1 4 0 6-diamidino-2-phenylindole (DAPI; Fluka) for 3 min, washed in water and 96% ethanol, air-dried and mounted in the antifade VectaShield (Vector) under a cover slip. The sections were examined using an epifluorescence microscope (Zeiss Axioplan) and a confocal laser scanning microscope (Leica TCS SP2 AOBS). For TEM, the dehydrated tissue was soaked in propylene oxide and embedded in Agar 100 resin. Tissue was cut to 1 mm (for light microscopy to aid TEM studies) and to 60 nm (for TEM) using a microtome (Reichert Ultracut S). Semi-thin sections were collected on SuperFrostPlus slides and ultrathin sections were collected on 100-mesh copper grids. Semi-thin sections were stained with 2% toluidine blue for 2–3 min at 60 1C, rinsed in tap water, air-dried and mounted in DPX. Ultrathin sections were stained with 1% uranyl acetate for 20 min at room temperature, rinsed in distilled water and stained with Reynolds lead citrate for 10 min before being air-dried and examined using a JEOL JEM-1230 transmission electron microscope.
Sequence accession numbers Sequences of 16S rRNA genes from Ae1, EF508132, HQ412804; Ae2, GQ240891, HQ412803, HQ412805; Ae3, GQ240892, HQ412802; Moritella sp., GQ240894; Colwellia sp., GQ240895; isolates, GQ240896, GQ240897, GQ240898, GQ240899; and of the 16S–23S rRNA intergenic spacer and 23S rRNA gene fragment of Ae7, HM216958; and the 18S rRNA gene of Ae1, GQ240893 are available in GenBank under the corresponding accession numbers.
Results and discussion Gill-associated microorganisms Bacterial, but not archaeal, 16S rRNA genes were successfully PCR amplified from gill DNA purified from A. excavata bivalves, first collected at coral reef KA2 and rock-wall Stokkbergneset (Fig. 1, Table 1). DGGE profiling of the amplicons revealed two distinct bands (Fig. 2). Sequence analysis of DNA from these bands identified two different rRNA genes, also from individual AeJ1 with a faint band. The upper band (Fig. 2) yielded a DNA sequence similar to a fragment of the 18S rRNA gene of another Limidae. At the relatively low PCR stringency-used primer 338fGC probably mismatched (Jensen et al., 2004). Comparison of the sequence with an extended sequence from a EukF/EukR amplification of the A. excavata 18S rRNA gene confirmed the mismatch and revealed high similarity to Lima lima. From the lower DGGE band, we obtained a single 197 bp 16S rRNA gene sequence from a novel Gammaproteobacterium. PCR-DGGE screening of DNA from A. excavata muscle and mantle yielded FEMS Microbiol Ecol 74 (2010) 523–533
only visible bands in the upper position, representing the 18S rRNA gene. The sequence was not found among 40 clones in a library prepared from a 200 mL water sample of the KA2 reef bacterioplankton (Jensen et al., 2008). Cultivated gill associates revealed 16S rRNA gene sequences 97% or more identical to Vibrio splendidus (e.g. from Crassostrea gigas), Vibrio spp. (from seawater), Shewanella kaireitica (from deep-sea sediment) and clone ctg NISA248 (Sulfitobacter-like) from Alaskan octocoral bacterioplankton (Penn et al., 2006). Because of their affiliations, and for being undetected in the DNA extracts, these sequences most likely represent free-living bacteria, or in the case of Vibrio, possible pathogens.
Prevalence and phylogeny The 16S rRNA genes from the KA2 reef bivalves (Table 1) were PCR amplified in almost full length and 181 random clones were screened for DGGE bands with migration properties similar to the gammaproteobacterial DNA fragment (Fig. 2). Matching bands appeared at the following frequencies: 52/57 (Ae1 library), 68/115 (Ae2 library) and 8/9 (Ae3 library). Matches represented 70% of the clones and these sequences (1505 bp) shared 99.7% identity among the three libraries, variable sites being scattered throughout the genes. Sequencing of clones falling into the four other migration categories detected revealed o 2% differences to the dominant sequence type (36 clones) and to sequences from Colwellia (16 clones) and Moritella (1 clone). Clones very similar to the dominant sequence type may represent a second rRNA operon in the same bacterium (Cilia et al., 1996) or a second bacterium. Clones very similar to Colwellia and Moritella probably fall into the category of the cultivated gill bacteria and likely represent free-living bacteria of this cool deep-water environment. The dominant sequence type was also obtained by direct sequencing of the PCR products, as has been found with monospecific symbionts (e.g. Polz & Cavanaugh, 1995). Further direct sequencing of the rock-wall individuals, and individuals later collected from reefs adjacent to KA2 (Table 1, Fig. 1), yielded clean sequence chromatograms indicative of a sequence type exactly matching clone Ae2p1d1 across the 1450 examined nucleotide positions. All 10 individuals revealed a single dominant 16S rRNA gene sequence, suggesting bacterial host specificity (Distel et al., 1988). The finding of a single 16S–23S rRNA gene spacer and 23S rRNA gene fragment, following direct sequencing, supported the putative homogeneity (sequences relate to Arctic EU837026 and Hawaiian EU795190 seawater bacteria, respectively). Phylogenetic analysis of the dominant sequences confirmed the gammaproteobacterial affiliation and revealed a 16S rRNA clade of associates of marine invertebrates (Fig. 3). The clade contains no sequence from recognized bivalve symbionts, their relatives or other methanotrophs or 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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clone CN34 Chondrilla nucula (AM259915) Spongiobacter nickelotolerans (AB205011) clone EC79 Erythropodium caribaeorum (DQ889911) clone ME19 Muricea elongata (DQ917863) clone PDA-OTU3 Pocillopora damicornis (AY700601) clone Gven_B17 Gorgonia ventalina (GU118479) clone C19 Cystodytes dellechiajei (DQ884169) Endozoicomonas elysicola (AB196667) clone Bathymodiolus childressi parasite (FM244838) clone Bathymodiolus puteoserpentis parasite (FM162189) clone JI4_12 Phacoides pectinatus (EU487853) clone JI4_17 Phacoides pectinatus (EU487858)
ASSOCIATES OF MARINE INVERTEBRATES
clone HOC2 Halichondria okadai (AB054136) clone OTU7 Alcyonium antarcticum (DQ312240) isolate MOLA 531 Petrosia ficiformis (AM990755) clone Dstr_I24 Diploria strigosa (GU118168) clone Mfav_K11 Montastraea faveolata (GU118649)
Isolate KMD001 Kistimonas asteriae (EU599216) clone Past_C21 Porites astreoides (GU118970) clone BY ocean water Bohai Bay (FJ154998) clone Ae2p1d1 Acesta excavata (OTU D, 68 clones), AeJ1,J2,4-8 clone Ae1pa1 Acesta excavata (OTU D, 52 clones) clone Ae3p8 Acesta excavata (OTU D, 8 clones) clone Ae3p7 Acesta excavata (OTU 3, 1 clone) clone Ae2p1c2 Acesta excavata (OTU 4, 6 clones) clone Ae1p1e12 Acesta excavata (OTU 4, 5 clones) clone Ae2p1c4 Acesta excavata (OTU 3, 24 clones) clone HOC25 Halichondria okadai (AB054156) clone HOC25 Halichondria okadai (AB054159) clone LQ-2001 seawater (AJ315452) Zooshikella ganghwensis (AY130994) Bankia setacea symbiont (AF102866) WOOD Teredinibacter turnerae (EU604078) clone T742_10_B2 Osedax frankpressi (DQ911529) BONE Oceanospirillum beijerinckii (AB006760) Marinobacter hydrocarbonoclasticus (AB021372) Pseudomonas fluorescens (D86003) clone Bathymodiolus platifrons (AB036710) clone M3.33 Idas sp. (AM402955) METHANE Methylomicrobium japanese (D89279) clone BC1007 Mytilidae sp. (AM503921) Calyptogena magnifica (AF035721) Codakia orbicularis (X84979) Thyasira flexuosa (L01575) SULFIDE Solemya reidi (L25709) Moritella sp. ODA02 (AB011353) clone Ae2p1f2 Acesta excavata (OTU 5, 1 clone) Vibrio splendidus (AJ874363) Shewanella kaireitica (AB201780) Colwellia sp. IE7-5 (AY829231) clone Ae2p2h1 Acesta excavata (OTU 1-2, 16 clones) clone EV818EB5CPSAJJ40 Kalahari Shield (DQ337043) C. ’Portiera aleyrodidarum’ (AY268081) Zymobacter palmae (AF211871) Hahella chejuensis (AF195410) Agrobacterium tumefaciens (D14500) 0.1
Fig. 3. Phylogenetic relationship between the intracellular Oceanospirillales bacterium and its closest relatives, including representatives of the parasitic ‘Candidatus Endonucleobacter bathymodioli’ (FM162189, FM244838) and nutritional symbionts from the families Solemyidae, Thyasiridae, Lucinidae, Vesicomyidae, Mytilidae and Teredinidae (sulphide, methane and wood utilizers). The tree was constructed in ARB using maximum likelihood analysis (PHYML, HKY model) of 16S rRNA genes filtered to 1201 aligned nucleotide positions, excluding ambiguities, missing data and positions where the frequency of a nucleotide occurring was o 50%. Circles indicates branch points supported by both maximum likelihood and maximum parsimony bootstraps ( 4 75 open, 4 95 filled; of 100 iterations performed in PHYLIP) (Felsenstein, 2004). The outgroup was the Alphaproteobacterium Agrobacterium tumefaciens. Scale bar represents 0.1 changes per nucleotide.
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thiotrophs, but contains sequences from corals, sponges, a sea slug, an ascidian, a sea star, seawater and gills of other bivalves. The bivalve sequences originate from intranuclear parasites found in Bathymodiolus mytilids from hydrothermal vents and cold seeps (Zielinski et al., 2009), from Siliqua cultellids from Washington State beaches (Elston, 1986; Kerk et al., 1992) and from uncharacterized bacteria in the lucinid Phacoides pectinatus from Florida seagrass beds (A.M. Green-Garcia, M. Thiessen & A.S. Engel, unpublished data). The Siliqua sequence is divided into three separate fragments (M94380–82; sum up to 695 nt) and is difficult to recognize in homology searches. It represents the first 16S rRNA gene sequence from an intranuclear Gammaproteobacterium (Kerk et al., 1992) and shares 93% identity with the Acesta, Bathymodiolus and Phacoides bacteria, all belonging to the Oceanospirillales family Hahellaceae (RDP classifier). These bacteria are evolutionarily related, but may be differently adapted in hosts at beaches, seagrass beds, reefs and vents. Two members of the invertebrate clade are cultured and characterized. Both are aerobic pigmented and rod-shaped heterotrophs. They are the Endozoicomonas elysicola from the gut of the Japanese sea slug Elysia ornata (Kurahashi & Yokota, 2007) and the Kistimonas asteriae from the skin of the Korean starfish Asterias amurensis (Choi et al., 2009). The clade also includes the isolate MOLA 531 from the Mediterranean sponge Petrosia ficiformis (M. Bourrain, R. Belbes, L. Intertaglia & P. Lebaron unpublished data). Spongiobacter spp. members from the clade were recently shown to degrade dimethylsulphoniopropionate (DMSP) (Raina et al., 2009). The clade’s closest characterized relative (Fig. 3) is the free-living curved rod Zooshikella ganghwensis from tidal flat sediment in Korea (Yi et al., 2003).
and always with little to no DAPI signal (Fig. 4a). Members of the family Hahellaceae produce pigments. Zooshikella excretes a yellowish-red to red pigment (Yi et al., 2003) and Hahella excretes an algicidal and pinkish-red pigment
Visualization A characteristic zone of bacteriocytes, as seen in the gills of methane seep Bathymodiolus mussels (Duperron et al., 2005) and wood-boring Teredinidae bivalves (Sipe et al., 2000), was not seen in the gills of the A. excavata. Bacterialike morphotypes were, however, observed. Scattered throughout the gill filaments, there were aggregates of bacteria-like spheres with intense autofluorescence following ethanol fixation, packed in round to oval or short tubelike 5–10 mm vacuoles. Specific Ae128 probing yielded above background Cy5 signals (Fig. 4a), indicating that the structures are the actual Oceanospirillales bacterium. From probing using Eub338, signals were seen from the same aggregates and no other signals were observed (Fig. 4b). This indicates few other bacteria within the gill tissue, bearing in mind that Eub338 is not targeting all bacteria (Daims et al., 1999). The size of the aggregates varied and the spheres were sometimes seen individually, but they were always seen in the lateral zone and not in the filament tip, FEMS Microbiol Ecol 74 (2010) 523–533
Fig. 4. Fluorescent in situ hybridization of gill tissue from the Acesta excavata using bacteria-specific probes and confocal microscopy. Hybridizations were performed on the same section with 30% formamid as follows: Cy5-labelled (green) Acesta bacterium-specific probe Ae128 (a) and texas red-labelled (red) broad-range bacterial domain-specific probe Eub338 (b). The nucleic acid stain DAPI was included to reveal all nuclei and bacteria (blue). For localization, insets show the bacteria without the Ae128 probe overlay (a) and the bacteria with the overlay of Ae128 and Eub338 (b). A different section of the same experiment was hybridized with the Cy5labelled negative control probe NON338 (not shown). Scale bars = 10 mm (individual Ae8).
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Fig. 5. Transmission electron micrographs of intracellular bacteria within gill tissue of the Acesta excavata. In the longitudinal section of the gill filament (a), four pleomorphic cells are seen aggregated in a vacuole (arrow) to the right of host nuclei (N). The inset shows details and the cell membrane (arrowhead), but no visible cell wall. Pleomorphic bacteria-like morphotypes in a vacuole adjacent to a nucleus (b). Deeper within gill tissue (c), several pleomorphic bacteria can be seen. Scale bars = 2.0 mm except for inset and (c) where 1.0 mm (individuals Ae7 and Ae8).
(Jeong et al., 2005). Fluorescent yellow-green pigments are known for the Oceanospirillales (Garrity et al., 2005) and the autofluorescence may originate from a pigment produced by the bacterium. TEM inspection revealed a pleomorphic morphotype with no visible cell wall (Fig. 5), supposedly corresponding to the FISH-positive structures as they showed the same size, shape and distribution in the gill tissue (Fig. 4). A comparable ultrastructure was noted for the primary symbiont of whiteflies, the cell wall-lacking and pleomorphic Oceanospirillales bacterium ‘Candidatus Portiera aleyrodidarum’ (Costa et al., 1995; Baumann, 2005). No clearly distinguishable bacterium inside nuclei (Zielinski et al., 2009) could be recognized (Fig. 5). Based on the images and on the superposition of images, the probe signals were concluded to be localized outside of nuclei. The probe signals do not superimpose with nuclei, but instead superimpose with weaker DAPI signals that most likely correspond to the nucleic acid content of bacteria. The large electron dense structures (Elston, 1986) occasionally seen inside nuclei could not be identified as bacteria and it remains unknown whether this bacterium enters the nuclei.
Ecology of the A. excavata association Oceanospirillales are rod-shaped, curved and helical marine bacteria with pigments and a distinct resting stage (coccoid body); they are widespread and perform aerobic degradation of complex organic compounds by the excretion of hydrolytic enzymes and emulsifying agents, and they utilize organic acids, amino acids, ammonium, linear alkanes, polycyclic aromatics (Garrity et al., 2005) and DMSP (Raina et al., 2009). Some are closely associated with invertebrates and also insects, such as the parasites, which probably utilize chromatin and threaten Siliqua (Elston, 1986) and Bathymodiolus (Zielinski et al., 2009), and the symbionts, which make Osedax utilize whalebones (Goffredi et al., 2005) and 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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whiteflies live on plant sap (Baumann, 2005). Oceanospirillales are heterotrophs and genes encoding Rubisco or ATP citrate lyase, necessary for autotrophic CO2 fixation, were not detected in the Osedax symbiosis (Goffredi et al., 2005). Also using the PCR, we were unable to detect genes encoding pmoA and mxaF involved in the utilization of methane in methanotrophs (McDonald et al., 2008) and aprA and cbbL involved in the utilization of sulphur and carbon fixation in thiotrophs (Elsaied et al., 2007; Meyer & Kuever, 2007). We found few clues on the intracellular Acesta bacterium’s lifestyle and significance. The rRNA gene sequences investigated differ from other sequences by minimum 7%, indicating novelty. However, although sequences from characterized bacteria are distant and the nearest sequences belong to uncharacterized bacteria, the 16S rRNA gene fall in a clade of strictly marine invertebrate-associate bacteria (except for ocean water FJ154998) that share a common ancestor. This unique relationship indicates an adaptation for the invertebrate cell environment. In the gills of other investigated bivalves, Oceanospirillales exist together with methane- and sulphide-utilizing symbionts. Details from the Bathymodiolus implied a separate localization because the parasite was absent in the bacteriocytes and was therefore considered to be harmless to Bathymodiolus (Zielinski et al., 2009). The Siliqua have no reported symbionts and its associated bacterium was suggested to cause mass mortalities (Elston, 1986). The A. excavata analysed revealed no previously known symbionts and the ROV surveyed individuals appeared to be healthy. Currently, there are no characterized symbionts from the invertebrate clade (Fig. 3), and Zielinski et al. (2009) suggest that clade members may instead have a parasitic lifestyle. The relatively low bacterial numbers in the gill tissue resemble the parasites, but the visualization indicated a localization to the cytoplasm and the low numbers can also be attributed to slow FEMS Microbiol Ecol 74 (2010) 523–533
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growth with a corresponding low energy demand as suggested from cold seep tubeworm symbionts (L¨osekann et al., 2008). Oceanospirillales have several properties, which include direct organisms impacts by parasitism (Zielinski et al., 2009), symbiosis (Baumann, 2005; Goffredi et al., 2005) and algicidal activity (Jeong et al., 2005). The resemblance of the bacterium to the Portiera may suggest that it is a heterotroph, which supplements the diet of A. excavata with an essential missing nutrient.
Conclusions Only marine Proteobacteria were found to be associated with the gills of the large deep-water bivalve A. excavata in this first study with animals from Norwegian coral reefs and a rock-wall. The association was dominated by the Acesta bacterium, a novel intracellular representative from a clade of strictly marine invertebrate-associated bacteria from the ubiquitous and potentially ecologically relevant order Oceanospirillales. For its preliminary characterization, we propose the name ‘Candidatus Acestibacter aggregatus’ (A.cest.i.bac 0 ter ag.gre.ga 0 tus; Acesta a bivalve genus; L. masc. n. bacter a staff, a short rod; L. masc. adj. aggregatus add to, joined together; rod-shaped and rounded bacteria joined together in bivalves of the genus Acesta).
Acknowledgements We thank Statoil, especially the Kristin and the Morvin development projects, and the crew on Edda Fonn and Normand Tonjer for sampling coral reefs and on Vita for sampling rock-wall. Johanna J¨arnegren provided the rockwall individuals and helped with constructive comments on the manuscript. Nicole Dubilier and Silke Wetzel kindly hosted studies using CARD-FISH. Tissue preparation, sectioning, confocal microscopy and TEM were supported by Anne Nyhaug, Helen Olsen, Endy Spriet and Trygve Knag, Molecular Imaging Center, FUGE, Norwegian Research Council, University of Bergen. This work was funded by the Norwegian Academy of Science and Statoil (VISTA project 6146).
References Acinas SG, Klepac-Ceraj V, Hunt DE, Pharino C, Ceraj I, Distel DL & Polz MF (2004) Fine-scale phylogenetic architecture of a complex bacterial community. Nature 430: 551–554. Altschul SF, Madden TL, Sch¨affer AA, Zhang J, Zhang Z, Miller W & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic identification and in-situ detection of individual microbialcells without cultivation. Microbiol Rev 59: 143–169.
FEMS Microbiol Ecol 74 (2010) 523–533
Baker GC, Smith JJ & Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbial Meth 55: 541–555. Baumann P (2005) Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annu Rev Microbiol 59: 155–189. Behrens S, R¨uhland C, In´acio J, Huber H, Fonseca A´, SpencerMartins I, Fuchs BM & Amann R (2003) In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl Environ Microb 69: 1748–1758. Buhl-Mortensen L & Mortensen PB (2004) Symbiosis in deepwater corals. Symbiosis 37: 33–61. Choi EJ, Kwon HC, Sohn YC & Yang HO (2009) Kistimonas asteriae gen. nov., sp. nov., a gammaproteobacterium isolated from Asterias amurensis. Int J Syst Evol Micr 60: 938–943. Cilia V, Lafay B & Christen R (1996) Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level. Mol Biol Evol 13: 451–461. Cole JR, Wang Q, Cardenas E et al. (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37: D141–D145. Costa HS, Westcot DM, Ullman DE, Rosell R, Brown JK & Johnson MW (1995) Morphological variation in Bemisia endosymbionts. Protoplasma 189: 194–202. Daims H, Br¨uhl A, Amann R, Schleifer K-H & Wagner M (1999) The domain-specific probe Eub338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434–444. Delong EF (1992) Archaea in coastal marine environments. P Natl Acad Sci USA 89: 5685–5689. Distel DL, Lane DJ, Olsen GJ, Giovannoni SJ, Pace B, Pace NR, Stahl DA & Felbeck H (1988) Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences. J Bacteriol 170: 2506–2510. Distel DL, DeLong EF & Waterbury JB (1991) Phylogenetic characterization and in situ localization of the bacterial symbiont of shipworms (Teredinidae: Bivalvia) by using 16S rRNA sequence analysis and oligodeoxynucleotide probe hybridization. Appl Environ Microb 57: 2376–2382. Dubilier N, Bergin C & Lott C (2008) Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat Rev Microbiol 6: 725–740. Duperron S, Nadaling T, Caprais JC, Sibuet M, Fiala-M´edioni A, Amann R & Dubilier N (2005) Dual symbiosis in a Bathymodiolus sp. mussel from a methane seep on the Gabon continental margin (southeast Atlantic): 16S rRNA phylogeny and distribution of the symbionts in gills. Appl Environ Microb 71: 1694–1700. Elsaied HE, Kimura H & Naganuma T (2007) Composition of archaeal, bacterial, and eukaryal RuBisCO genotypes in three Western Pacific arc hydrothermal vent systems. Extremophiles 11: 191–202.
2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
532
Elston RA (1986) An intranuclear pathogen [nuclear inclusion X (NIX)] associated with massive mortalities of the Pacific razor clam, Siliqua patula. J Invertebr Pathol 47: 93–104. Felsenstein J (2004) PHYLIP (Phylogeny Inference Package) version 3.6. Department of Genome Sciences, University of Washington, Seattle. Gagnon JM & Haedrich RL (2003) First record of the European giant file clam, Acesta excavata (Bivalvia: Pectinoidea: Limidae), in the northwest Atlantic. Can Field Nat 117: 440–447. Garrity GM, Bell JA & Lilburn T (2005) Order VIII. Oceanospirillales ord. nov. Bergey’s Manual of Systematic Bacteriology, Vol. 2 (Brenner DJ, Krieg NR, Staley JT & Garrity GM, eds), pp 270–292. Springer, New York. Ghisotti F (1979) Ritrivamente di Acesta (Acesta) excavata (Fabricius, 1779) vivente in Mediterraneo. Boll Malacol 15: 57–66. Giovannoni SJ, DeLong EF, Olsen GJ & Pace NR (1988) Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol 170: 720–726. Goffredi SK, Orphan VJ, Rouse GW, Jahnke L, Embaye T, Turk K, Lee R & Vrijenhoek RC (2005) Evolutionary innovation: a bone-eating marine symbiosis. Environ Microbiol 7: 1369–1378. Haedrich RL & Gagnon JM (1991) Rock wall fauna in a deep Newfoundland fjord. Cont Shelf Res 11: 1199–1207. Holmes AJ, Costello A, Lidstrom ME & Murrell JC (1995) Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionary related. FEMS Microbiol Lett 132: 203–208. Hovland M (2005) Pockmark-associated coral reefs at the Kristin field off Mid-Norway. Cold-Water Corals and Ecosystems (Freiwald A & Roberts JM, eds), pp 623–632. Springer-Verlag, Berlin. Hovland M (2008) Deep-Water Coral Reefs, Unique Biodiversity Hot-Spots. Praxis Publishing Ltd, Chichester, UK. Hovland M & Risk M (2003) Do Norwegian deep-water coral reefs rely on seeping fluids? Mar Geol 198: 83–96. Hunt DE, Klepac-Ceraj V, Acinas SG, Gautier C, Bertilsson S & Polz MF (2006) Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of bacterial diversity. Appl Environ Microb 72: 2221–2225. J¨arnegren J & Altin D (2006) Filtration and respiration of the deep living bivalve Acesta excavata (J.C. Fabricius, 1779) (Bivalvia; Limidae). J Exp Mar Biol Ecol 334: 122–129. J¨arnegren J, Tobias CR, Macko SA & Young CM (2005) Egg predation fuels unique species association at deep-sea hydrocarbon seeps. Biol Bull 209: 87–93. J¨arnegren J, Schander C, Sneli JA, Ronningen V & Young CM (2007) Four genes, morphology and ecology: distinguishing a new species of Acesta (Mollusca; Bivalvia) from the Gulf of Mexico. Mar Biol 152: 43–55. Jensen S, Øvre˚as L, Bergh Ø & Torsvik V (2004) Phylogenetic analysis of bacterial communities associated with larvae of the
2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
c
S. Jensen et al.
Atlantic halibut propose succession form a uniform normal flora. Syst Appl Microbiol 27: 728–736. Jensen S, Neufeld JD, Birkeland NK, Hovland M & Murrell JC (2008) Insight into the microbial community structure of a Norwegian deep-water coral reef environment. Deep-Sea Res PT I 55: 1554–1563. Jeong H, Yim JH, Lee C et al. (2005) Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent. Nucleic Acids Res 33: 7066–7073. Kerk D, Gee A, Dewhirst FE, Drum AS & Elston RA (1992) Phylogenetic placement of ‘‘nuclear inclusion X (NIX)’’ into the gamma subclass of Proteobacteria on the basis of 16S ribosomal RNA sequence comparisons. Syst Appl Microbiol 15: 191–196. Kohl B & Vokes HE (1994) On the living habits of Acesta bullisi (Vokes) in chemosynthetic bottom communities, Gulf of Mexico. Nautilus 108: 9–14. Kurahashi M & Yokota A (2007) Endozoicomonas elysicola gen nov., sp. nov., a g-proteobacterium isolated from the sea slug Elysia ornata. Syst Appl Microbiol 30: 202–206. Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt E & Goodfellow M, eds), pp. 115–175. John Wiley & Sons, New York. ´ Lopez Correra M, Freiwald A, Hall-Spencer J & Taviani M (2005) Distribution and habitats of Acesta excavata (Bivalvia: Limidae) with new data on its shell ultrastructure. Cold-Water Corals and Ecosystems (Freiwald A & Roberts JM, eds), pp. 173–205. Springer-Verlag, Berlin. L¨osekann T, Robador A, Niemann H, Knittel K, Boetius A & Dubilier N (2008) Endosymbioses between bacteria and deepsea siboglinid tubeworms from an arctic cold seep (Haakon Mosby mud volcano, Barents Sea). Environ Microbiol 10: 3237–3254. Ludwig W, Strunk O, Westram R et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32: 1363–1371. McDonald IR & Murrell JC (1997) The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs. Appl Environ Microb 63: 3218–3224. McDonald IR, Bodrossy L, Chen Y & Murrell JC (2008) Molecular ecology techniques for the study of aerobic methanotrophs. Appl Environ Microb 74: 1305–1315. Meyer B & Kuever J (2007) Phylogeny of the alpha and beta subunits of the dissimilatory adenosine-5 0 -phosphosulfate (APS) reductase from sulfate-reducing prokaryotes – origin and evolution of the dissimilatory sulfate-reduction pathway. Microbiology 153: 2026–2044. Mortensen PB, Buhl-Mortensen L, Gebruk AV & Krylova EM (2008) Occurrence of deep-water corals on the Mid-Atlantic Ridge based on MAR-ECO data. Deep-Sea Res PT II 55: 142–152. Muyzer G, DeWaal EC & Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microb 59: 695–700. Neufeld JD, Sch¨afer H, Cox MJ, Boden R, McDonald IR & Murrell JC (2007) Stable-isotope probing implicates
FEMS Microbiol Ecol 74 (2010) 523–533
533
Acesta excavata bivalve bacterium
Methylophaga spp. and novel Gammaproteobacteria in marine methanol and methylamine metabolism. ISME J 0: 1–12. Penn K, Wu D, Eisen JA & Ward N (2006) Characterization of bacterial communities associated with deep-sea corals on Gulf of Alaska seamounts. Appl Environ Microb 72: 1680–1683. Polz MF & Cavanaugh CM (1995) Dominance of one bacterial phylotype at a Mid-Atlantic ridge hydrothermal vent site. P Natl Acad Sci USA 92: 7232–7236. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J & Gl¨ockner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acid Res 35: 7188–7196. Qiu XY, Wu LY, Huang HS, McDonel PE, Palumbo AV, Tiedje JM & Zhou JZ (2001) Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl Environ Microb 67: 880–887. Raina JB, Tapiolas D, Willis BL & Bourne DG (2009) Coralassociated bacteria and their role in the biogeochemical cycling of sulfur. Appl Environ Microb 75: 3492–3501. Roberts JM, Wheeler A, Freiwald A & Cairns S (2009) Cold-Water Corals – The Biology and Geology of Deep-Sea Coral Habitats. Cambridege University Press, New York. Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York.
FEMS Microbiol Ecol 74 (2010) 523–533
Sipe AR, Wilbur AE & Cary SC (2000) Bacterial symbiont transmission in the wood-boring shipworm Bankia setacea (Bivalvia: Teredinidae). Appl Environ Microb 66: 1685–1691. Southward EC, Gebruk A, Kennedy H, Southward AJ & Chevaldonn´e P (2001) Different energy sources for three symbiont-dependent bivalve molluscs at the Logatchev hydrothermal site (Mid-Atlantic Ridge). J Mar Biol Assoc 81: 655–661. Stewart FJ, Newton ILG & Cavanaugh CM (2005) Chemosynthetic endosymbioses: adaptations to oxic–anoxic interfaces. Trends Microbiol 13: 439–448. Thiem Ø, Ravagnan E, Foss˚a JH & Berntsen J (2006) Food supply mechanisms for cold-water corals along a continental shelf edge. J Marine Syst 60: 207–219. Vokes HE (1963) Studies on tertiary and recent giant Limidae. Tulane Stud Geol 2: 75–92. Wild C, Mayr C, Wehrmann L, Sch¨ottner S, Naumann M, Hoffmann F & Rapp HT (2008) Organic matter release by cold water corals and its implication for fauna-microbe interaction. Mar Ecol Prog Ser 372: 67–75. Yi H, Chang YH, Oh HW, Bae KS & Chun J (2003) Zooshikella ganghwensis gen. nov., sp. nov., isolated from tidal flat sediments. Int J Syst Evol Micr 53: 1013–1018. Zielinski FU, Pernthaler A, Duperron S, Raggi L, Giere O, Borowski C & Dubilier N (2009) Widespread occurrence of an intranuclear bacterial parasite in vent and seep bathymodiolin mussels. Environ Microbiol 11: 1150–1167.
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