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Deep-Sea Research II 54 (2007) 1682–1690

www.elsevier.com/locate/dsr2

The archaebacterial communities inAntarctic bathypelagic sediments

David C. Gillana,�, Bruno Danisb

aMarine Biology Laboratory, CP160/15, Universite Libre de Bruxelles, 50 av. Roosevelt, 1050 Brussels, BelgiumbRoyal Belgian Institute of Natural Sciences, 29 Rue Vautier, 1000 Brussels, Belgium

Accepted 4 July 2007

Available online 1 August 2007

Abstract

16S ribosomal DNA clone library analysis was performed to assess archaeal diversity within three surficial sediment

samples obtained from the bathypelagic zone (depth: 2165–3406m) of the Weddell Sea, Antarctica. The nearly complete

16S rDNA gene (1440 bp) was obtained for 146 clones and 46 phylotypes were defined. The majority of the sequences

(499%) formed three clusters within the Marine Group I Crenarchaeota. The most important cluster, with 78.8% of the

clones, included Candidatus Nitrosopumilus maritimus, a mesophilic archaeon able to oxidize ammonia. The most

important subgroup in that cluster was the APA4-0cm subgroup (with 62.3% of the clones). This subgroup might

represent important Crenarchaeota in the functioning of the bathypelagic sedimentary ecosystems of the Weddell Sea

because it dominated the clone libraries in all sampling stations, and was found in sediments separated by very large

geographic distances. Only one clone grouped within the Euryarchaeota. This euryarchaeal clone could not be affiliated

with any of the previously defined clusters and might represent a novel euryarchaeal lineage.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Archaebacteria; Crenarchaeota; Sediments; Antarctic; 16S rDNA; Marine Group I

1. Introduction

Phylogenetic analyses based on ribosomal DNAhave shown that the archaeal domain of life ismainly composed of two kingdoms, the Euryarch-aeota and the Crenarchaeota (Woese et al., 1990).Other deeply branching archaeal clades such as theKorarchaeota and the Nanoarchaeota also havebeen found (Huber et al., 2002; Auchtung et al.,2006). Within the last several years, the two main

e front matter r 2007 Elsevier Ltd. All rights reserved

r2.2007.07.002

ng author. Tel.: +32 2 6502970;

796.

ss: dgillan@ulb.ac.be (D.C. Gillan).

lineages have been recovered from many realmssuch as coastal seawater (e.g., DeLong, 1992), deep-sea plankton (e.g., Karner et al., 2001), marinesediments (e.g., Vetriani et al., 1999), freshwaterlakes (e.g., Jurgens et al., 2000), terrestrial soils (e.g.,Leininger et al., 2006) and macrobiota (e.g., van derMaarel et al., 1998), and it is becoming evident thatEuryarchaeota and Crenarchaeota are importantmembers of marine and terrestrial ecosystems(Herndl et al., 2005; Konneke et al., 2005; Leiningeret al., 2006). Despite the high abundance andrichness of archaea in the deep-sea plankton (Karneret al., 2001; Bano et al., 2004) and in the SouthernOcean (Massana et al., 1998; Murray et al., 1999;

.

ARTICLE IN PRESSD.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–1690 1683

Church et al., 2003; Bano et al., 2004), little workhas been carried out on the diversity of the archaealcommunities in deep-sea Antarctic sediments. Onlytwo studies addressing mesopelagic sediments(150–1000m) have been published so far and werecarried out in the Eastern sector of the SouthernOcean (Bowman and McCuaig, 2003; Bowmanet al., 2003). The other reports dealing with archaealdiversity in Antarctic sediments have been restrictedto shallow coastal environments (Bowman et al.,2000; Powell et al., 2003; Purdy et al., 2003).

In the study of Bowman and McCuaig (2003),conducted in the continental shelf area (depth,761m), the Marine Group I Crenarchaeota pre-dominated in the sediment 16S ribosomal DNAlibraries, particularly in the surface sediments(0–2 cm), where the group represented 88.4% (76sequences) of the archaeal 16S rDNA clones. Therest of the archaeal clones in the surface sedimentsgrouped within the crenarchaeal APA3-11 group(5.8%; five sequences) and the Euryarchaeota(5.8%; five sequences) (Bowman and McCuaig,2003). In the latter group, only one clone in the

Fig. 1. Position of the sampling stations.

Table 1

Characteristics of stations sampled during ANDEEP3 for Archeal com

Station names Official names Date (2005) Latitud

PS2 PS67-78 21.02.05 7119.49

PS7 PS67-121 14.03.05 63141.7

PS8 PS67-142 18.03.05 62111.6

surface sediments grouped with the previouslydescribed Marine Benthic Group D, the otherclones being affiliated with the pBRC84, the ACE-6or the PENDANT-33 groups (Bowman andMcCuaig, 2003). In the second study, focused onthe mesopelagic zone, sediments were sampled at adepth of 709–940m (Bowman et al., 2003) and therelative abundance of archaebacteria was assessedby using the group-specific ARC915 oligonucleotideprobe, which targets the 16S rRNA molecule.The results indicated that archaeal 16S rRNArepresented only 1–6% of the total 16S rRNA poolin the surface sediments. Similar quantities ofarchaeal 16S rRNA have been observed at otherdepths in other sediments such as Arctic sediments(Ravenschlag et al., 2001) and Atlantic sediments(Vetriani et al., 1999).

The aim of the present work was to investigatethe archaeal diversity in three bathypelagic(1000–3500m) stations of the Weddell Sea (Antarc-tica) using a 16S rDNA cloning and sequencingstrategy (Gillan et al., 2005). To the best of ourknowledge, sediments from the bathypelagic zonehave never been investigated in the Southern Oceanusing this approach.

2. Methods

2.1. Sampling

Archaeal communities were analyzed in sedi-ments in three bathypelagic stations (2200–3400m)sampled during the ANTXXII-3 cruise of R.V.Polarstern (ANDEEP3) (Fig. 1). Characteristics ofthe sampling stations are described in Table 1.Sediments were sampled using a box-corer. Thesediments were immediately and aseptically sampledusing 3ml polyethylene (PET) cryovials (top 2 cm ofthe sediments). The cryovials were immediatelyfrozen in liquid nitrogen and the samples were thenstored at �20 1C. Six replicate samples of the samecore were collected at each site.

munities analysis

e (S) Longitude (W) Depth (m) Seafloor

0 13159.920 2164 Mud, rocks

40 50142.990 2621 Mud

10 49129.450 3406 Mud

ARTICLE IN PRESSD.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–16901684

2.2. DNA extraction

For each station, 2 g of sediment—wet weight—(0.33 g of each replicate were combined) was placedin 6ml of TES buffer pH 8.0 (Tris-HCl 50mM,EDTA 40mM, sucrose 0.75M) and incubated at37 1C for 20min with vigorous shaking (400 rpm).Then, 60 ml of lysozyme (100mg/ml), 48 ml ofpronase E (20mg/ml) and 120 ml of mutanolysin(1040U/ml) were added to the sample, and themixture was incubated at 37 1C for 1 h with gentleshaking (150 rpm) (Vetriani et al., 1999). Lysozymewas dissolved in MilliQ water, pronase E was dis-solved in Na acetate 0.01M (pH 7.5) and mutano-lysin was dissolved in a K2HPO4 buffer (0.1M, pH6.2). Next, 60 ml of proteinase K (20mg/ml) wereadded to the mix followed by incubation at 37 1Cfor 30min (at 150 rpm) and at 55 1C for 30min(tubes were agitated two times manually). Then,900 ml of 20% SDS and 600 ml of 20% N-laurylsar-cosine were added and the tubes were incubated at65 1C for 1 h (tubes were agitated four timesmanually). The samples were extracted once withan equal volume of water-saturated phenol (pH8.0), once with 0.5 volume of phenol–chloroform–isoamyl alcohol (25:24:1), and once with an equalvolume of chloroform–isoamyl alcohol (24:1). Theaqueous phase was precipitated with 0.6 volumes ofisopropanol (overnight precipitation at �20 1C).The DNA was collected by centrifugation (20minat 14,000 rpm, 4 1C), washed once in cold 70%ethanol, dried in the dark for 3 h at ambienttemperature and resuspended in 50 ml of sterileUV-treated MilliQ water. The DNA was thenfurther purified using QIAquick columns (Qiagen).Relative amounts of DNA were estimated bydensitometry after 1.5% agarose gel electrophoresisand ethidium bromide staining, by comparison witha Gene Ruler molecular mass ladder (Fermentas).At this stage, DNA yields were estimated as 0.53 mg(PS2), 0.34 mg (PS7) and 0.09 mg DNA per gram ofwet sediments. As PCR reactions were unsuccessful,DNA was further purified using 1.5% agarose gelelectrophoresis and the QIAquick gel extraction kit(Qiagen). DNA was then eluted in 30 ml of sterileMilliQ water.

2.3. Cloning and sequencing

The almost complete 16 rDNA archaeal gene wasamplified using primers Arch21F (50-TTCCGGTT-GATCCYGCCGGA-30) (DeLong, 1992) and U1492R

(50-GGTTACCTTGTTACGACT-30) (Orphan et al.,2001). Eight independent PCR reactions (finalvolume: 25 ml) were performed for each station.Half of these PCR reactions were performed using2 ml of purified DNA and an annealing temperatureof 55 1C, the other half being performed using 5 mlof DNA and an annealing temperature of 54 1C.The PCR amplification procedure was performedwith an Eppendorf Mastercycler using the PCR kitRed’y’StarMix (Eurogentec) as described elsewhere(Gillan et al., 2005). The final concentration ofprimers was 1 mM. Tubes were first incubated10min at 95 1C to activate the HotGoldStar DNApolymerase. The PCR was performed by using 30cycles consisting of denaturation at 94 1C for 1min,annealing at 54 1C (or 55 1C) for 1min and primerextension at 72 1C for 1.5min. For the last extensionstep, tubes were incubated for 10min at 72 1C. Asingle discrete band was obtained at ca. 1500 bpafter agarose gel electrophoresis and ethidiumbromide staining (one band corresponded to1.7–9.4 ng of DNA in 4 ml of PCR products). TheDNA from the eight independent PCR reactionswas then combined (160 ml per station, correspond-ing to 141–297 ng of DNA) and purified using theQIAquick PCR purification kit (Qiagen). DNA wasthen eluted in 30 ml of sterile MilliQ water and usedimmediately for the cloning reaction.

The PCR products were cloned into TOP10chemically competent Escherichia coli cells using theTOPO TA Cloning Kit featuring the pCR 2.1-TOPOvector (invitrogen). Four microliters of DNA wasused for the ligation. Clones containing the complete16S rDNA gene, as revealed by PCR with vectorprimers M13F (50-GTAAAACGACGGCCAG-30)and M13R (50-CAGGAAACAGCTATGAC-30),were selected for plasmid isolation with the QIAprepspin miniprep kit (Qiagen). Plasmids were sequencedcompletely on an ABI Prism 3100 genetic analyzerusing vector primers M13F and M13R as wellas primer GM1F-ARC (50-TCAGCCGCCGCGG-TAA-30). This primer corresponded to a conservedarea of the 16S rDNA located between nucleotide 451and 466 (Methanococcus vannielli numbering) andpresent in all the sequences of this study as indicatedby M13F and M13R sequencing.

2.4. Phylogenetic analysis

PCR-generated chimeric sequences were firstdetected and eliminated using three different soft-wares: CHIMERA_CHECK version 2.7 of the

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Fig. 2. Species richness curves of the three clone libraries (PS2,

PS7 and PS8). The phylotypes were determined with a 99%

similarity cutoff value over 1440 nucleotides. Nb, number.

D.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–1690 1685

Ribosomal Database Project II (Cole et al., 2003),Bellerophon (Huber et al., 2004) and Pintail version0.33 (Ashelford et al., 2005). Sequences were thensubmitted to BLAST version 2.2.12 to identify theclosest relatives and download their 16S rDNAsequence (Madden et al., 1996). All sequences weremanually aligned and analyzed using Se–Al version2.0a11 (Rambaut, 1996). Alignments and similaritycalculations also were performed using theEMBOSS-Align tool of the EMBL-EBI (http://www.ebi.ac.uk). Distance and maximum-likelihoodtrees were generated with the Phylip programpackage, version 3.6 (Felsenstein, 2002). Distancetrees were generated with ‘‘Dnadist’’ using Jukes-Cantor distances and Neighbor-Joining. Maxi-mum-likelihood trees were generated with ‘‘Dnaml’’(Ti/Tv ¼ 2.0; empirical base frequencies, one cate-gory of sites with a constant rate of variation). Thestatistical significance of the phylogenetic groupswithin the trees was tested by using bootstrapanalysis with the Phylip programs ‘‘Seqboot’’ and‘‘Consense’’ (100 bootstrap replicates, using usingJukes-Cantor distances and Neighbor-Joining).Trees were created using the program Treeview(version 1.6.6.).

2.5. Coverage of the clone libraries

The coverage (C) was derived from the equationC ¼ 1� n=N

� �, where n is the number of clones

that occurred only once, and N is the total numberof clones examined. For this calculation, we haveassumed that clones that are more than 99% similarare identical (Mullins et al., 1995).

2.6. Nucleotide sequence accession numbers

Fifty-three sequences obtained in this study (atleast one for each of the 47 phylotypes) have beenassigned in the GenBank database under accessionno. EF069336–EF069388.

3. Results and discussion

Clone library coverages were estimated at 76%for site PS2, 90% for site PS7 and 71% for site PS8.The number of sequenced clones in relation tothe number of phylotypes obtained is presented inFig. 2. From a total of 146 clones sequencedcompletely, 47 phylotypes were defined: 24 for sitePS2, 9 for site PS7 and 14 for site PS8 (a phylotypefeatured sequences that were more than 99%

similar) (Figs. 3 and 4). Four phylotypes werecommon to the PS7 and the PS8 sampling stations(for instance, the PS8ARC40-PS7bARC15 phylo-type). This may be the result of the close proximityof these two stations. All of the 146 clones with theexception of clone PS2ARC29 (which was placed inthe Euryarchaeota) grouped in the Marine Group ICrenarchaeota.

The sequences that grouped within the MarineGroup I Crenarchaeota formed three clusters,which were supported by high bootstrap values:the Nitrosopumilus group (78.8% of the clones), thePS2ARC15 group (7.5% of the clones) and theSeAq RB09 group (13.0% of the clones) (Fig. 3).Each of these clusters featured sequences from allthe three sampling sites, except the PS2ARC15group, which does not contain any sequence fromthe PS7 site. Most of the new Antarctic sequences inthe Nitrosopumilus cluster were grouped within theAPA4-0cm subgroup. This important subgroup wasalmost exclusively composed of the sequencesobtained in this study (91 sequences and sixphylotypes representing 62.3% of the clones). Theclosest relative of the subgroup was clone APA4-0cm isolated from deep-sea sediments (depth:4500m) in the NW Atlantic Ocean abyssal plain(Vetriani et al., 1999). In each of the threestations analyzed in this study the majority of theclones were members of the APA4-0cm subgroup.By comparison, in the study of Bowman andMcCuaig (2003) only one sequence grouped withclone APA4-0cm, and the majority of the sequencesgrouped close to clone pMC1A103 (data notshown). The APA4-0cm subgroup might represent

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Fig. 3. Maximum-likelihood tree showing the relationship among the 16S rDNA clones of the Antarctic sediments (in boldface) that

belong to the Marine Group I Crenarchaeota. Clone SCA1151 (Soil Crenarchaeota) served as the outgroup. A matrix of 1307 nucleotides

was used. GenBank accession numbers are listed for the close relatives of the clones. Bootstrap values of 450% (obtained with 100

resamplings) are shown. The bar represents 100 expected substitutions for the 16S rDNA region analyzed (1307bp).

D.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–16901686

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Fig. 4. Maximum-likelihood tree showing the relationship among the 16S rDNA clones of the Antarctic sediments (in boldface) that

belong to the Euryarchaeota. Clone pOWA19 (Korarchaeota) served as the outgroup. A matrix of 1124 nucleotides was used. Variable

regions V1–V3, V7 and V9 (Neefs et al., 1990) were removed for the analysis. The scale bar represents 100 expected substitutions for the

16S rDNA matrix analyzed (1124bp). See the legend to Fig. 3 for other information.

D.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–1690 1687

important Crenarchaeota in the functioning of thebathypelagic sedimentary ecosystems of the WeddellSea because the subgroup dominated the clonelibraries, in each sampling station, and over verylarge geographic distances. Future studies shouldfocus on the biomass of this group using in situhybridization. Besides the APA4-0cm subgroup, therest of the Nitrosopumilus group featured 24 newsequences (12 phylotypes). The Nitrosopumilus

group features Candidatus Nitrosopumilus mariti-

mus, the first mesophilic Crenarchaeote isolated sofar in pure culture, and able to grow chemolithoau-totrophically by aerobically oxidizing ammonia tonitrite (Konneke et al., 2005). The closest relative ofthis organism in the present study was clonePS2ARC05 (similarity: 97.7%). However, it shouldbe remembered that nearly identical 16S rDNAsequences are sometimes found in bacteria with highlydivergent genomes and ecophysiologies (Jaspers andOvermann, 2004). Most of the sequences in the

ARTICLE IN PRESSD.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–16901688

Nitrosopumilus group were obtained from deep-seasediments or water. This suggests that the deep-seaarchaeal communities, in the Weddel Sea andelsewhere, are not a simple accumulation ofplanktonic Archaea from the shallow zones of thewater column.

The PS2ARC15 group was exclusively composedof new Antarctic sequences (10 sequences, 8 phylo-types). The closest relatives of that group were twosponge symbionts in the marine environment: Cen-

archaeum symbiosum (host: Axinella mexicana) andclone A7G11 (host: Axinella polycapella) (Hallamet al., 2006). Environmental genomic analyses ofthese symbiotic Crenarchaeota have suggested theirability to use reduced nitrogen compounds (ammoniaoxidation) as energy sources to fuel an autotrophicmetabolism (Hallam et al., 2006).

The SeAq RB09 group featured 19 Antarcticsequences (eight phylotypes). The closest relatives ofthese sequences were clones retrieved in the recentlydiscovered Lost City Hydrothermal Field near theMid-Atlantic ridge (clones LC1231) (Schrenk et al.,2004) as well as clone SeAq RB09 retrieved from atropical seawater tank substratum (Konneke et al.,2005) and clone APA3-0cm from the NW AtlanticOcean abyssal plain (Vetriani et al. 1999).

The only sequence that grouped within theEuryarchaeota was clone PS2ARC29 (Fig. 4).Although the sequence possessed all the signaturenucleotides of the Euryarchaeota (Winker andWoese, 1991; Vetriani et al., 1999) it was notincluded in any of the previously defined euryarch-aeal clusters. Consequently, clone PS2ARC29 mightrepresent a novel lineage within the Euryarchaeotawhose closest relatives are the Marine Group II(DeLong, 1992), the Marine Group III (Fuhrmanand Davis, 1997) and the Marine Benthic Group D(Vetriani et al., 1999). This was supported by thebootstrap analysis in Fig. 4. Marine Benthic GroupD and Marine Group III feature sequences isolatedfrom marine deep-sea water or sediments (Vetrianiet al., 1999). Marine Group II is almost entirelycomposed of planktonic organisms (DeLong, 1992).The most closely related sequence to clonePS2ARC29 was clone C1_R046 from the MarineBenthic Group D (1186 identities over 1447nucleotides; similarity: 82.0%). As in the presentstudy, very low numbers of Euryarchaeota werefound in surficial sediments of the eastern sector ofthe Southern Ocean: only five clones, from a total of86, were found in the 0–2 cm sediment layer(Bowman and McCuaig, 2003). Although the in

situ abundance of the Euryarchaeota remains to bedetermined in all the Antarctic sediments investi-gated so far, the 16S rDNA cloning approachsuggests that Euryarchaeota are not abundant indeep Antarctic sediments.

Although entirely based on a 16S rDNA sequen-cing approach, the results obtained in this studysuggest that the Marine Group I Crenarchaeota isan important component of the microbial commu-nities in deep-sea Antarctic sediments. This result isin accordance with previous research conductedwithin East Antarctica deep-sea sediments (Bow-man and McCuaig, 2003) as well as with previousstudies conducted in deep Pacific waters. Forinstance, in the North Pacific the abundance ofthe pelagic Marine Group I Crenarchaeota in-creased below 100m and the abundance of pelagicEuryarchaeota remained low over the entire watercolumn up to 5000m deep (Karner et al., 2001).Similarly, in the marine waters near the AntarcticPeninsula, the abundance of the Marine Group ICrenarchaeota increased significantly with depthand comprised 9–39% of the total picoplanktonabundance in the meso- (150–1000m) and bath-ypelagic (1000–3500m) circumpolar deep water(Massana et al., 1998; Murray et al., 1999; Churchet al., 2003). In total, it seems that the MarineGroup I Crenarchaeota is an important componentof the deep-sea Antarctic ecosystem. Similar con-clusions were drawn for the deep Atlantic Ocean(Herndl et al., 2005).

In conclusion, our data suggest high abundanceof Marine Group I Crenarchaeota in the archaealcommunities in Antarctic bathypelagic sediments,and phylogenetic inference indicates that thesecommunities might be able to oxidize ammonia.This conclusion is supported by a recent reportconducted in the soil environment that concludedthat Crenarchaeota may be the most abundantammonia-oxidizing organisms in soil ecosystems onEarth (Leininger et al., 2006). The 16S rDNAsequences generated in this study can now be usedto design specific oligonucleotide probes to measurethe in situ abundance of the phylotypes detected inthe sediments. It also remains to be shown whetherthese organisms dominate with respect to theirnitrification activities.

Acknowledgments

This paper is a contribution to the ANDEEPProject (publication # 87) and contribution #3 to

ARTICLE IN PRESSD.C. Gillan, B. Danis / Deep-Sea Research II 54 (2007) 1682–1690 1689

the Census of Antarctic Marine Life (CAML). Weare extremely grateful to Profs. Angelika Brandt(Zoological Museum, University of Hamburg),Brigitte Hilbig (Senckenberg Museum, Frankfurt)and Eberhard Fahrbach (Alfred Wegener Institute,Bremerhaven) for their support and for giving usthe opportunity to join the Polarstern ANTXXII-3(ANDEEP3) cruise. We would also like to thank thePolarstern crew and all other ANDEEP3 partici-pants for their help and advice during the wholeexpedition. Many thanks to the reviewers whoclearly improved the manuscript. D.C.G. is a SeniorResearch Assistant of the Belgian National Fundfor Scientific Research (NSFR). The research wassupported by a Belgian federal research program(SSTC, contract EV/11/23A) and by contributionsfrom the Centre Interuniversitaire de BiologieMarine (CIBIM) and from the National Bank ofBelgium (BNB).

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