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ORIGINAL ARTICLE

Life in the dark: metagenomic evidence that amicrobial slime community is driven by inorganic

nitrogen metabolismSasha G Tetu1, Katy Breakwell1, Liam DH Elbourne1, Andrew J Holmes2,Michael R Gillings3 and Ian T Paulsen11Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales,Australia; 2School of Molecular Bioscience, Sydney University, Sydney, New South Wales, Australia and3Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia

Beneath Australias large, dry Nullarbor Plain lies an extensive underwater cave system, wheredense microbial communities known as slime curtains are found. These communities exist inisolation from photosynthetically derived carbon and are presumed to be chemoautotrophic. Earlier

work found high levels of nitrite and nitrate in the cave waters and a high relative abundance ofNitrospirae in bacterial 16S rRNA clone libraries. This suggested that these communities may besupported by nitrite oxidation, however, details of the inorganic nitrogen cycling in thesecommunities remained unclear. Here we report analysis of 16S rRNA amplicon and metagenomicsequence data from the Weebubbie cave slime curtain community. The microbial community iscomprised of a diverse assortment of bacterial and archaeal genera, including an abundantpopulation of Thaumarchaeota. Sufficient thaumarchaeotal sequence was recovered to enable apartial genome sequence to be assembled, which showed considerable synteny with thecorresponding regions in the genome of the autotrophic ammonia oxidiser Nitrosopumilusmaritimus SCM1. This partial genome sequence, contained regions with high sequence identity tothe ammonia mono-oxygenase operon and carbon fixing 3-hydroxypropionate/4-hydroxybutyratecycle genes of N. maritimus SCM1. Additionally, the community, as a whole, included genesencoding key enzymes for inorganic nitrogen transformations, including nitrification anddenitrification. We propose that the Weebubbie slime curtain community represents a distinctivemicrobial ecosystem, in which primary productivity is due to the combined activity of archaeal

ammonia-oxidisers and bacterial nitrite oxidisers.The ISME Journal (2013) 7, 12271236; doi:10.1038/ismej.2013.14; published online 21 February 2013Subject Category: microbial ecology and functional diversity of natural habitatsKeywords: ammonia oxidising archaea (AOA); chemolithotrophy; metagenomics; microbialcommunity; nitrogen cycling; Nullarbor caves

Introduction

Life in the dark recesses of the planet comes in manystrange forms. Exploration of these environmentsprovides key insights into the metabolic flexibilityof micro-organisms that do not rely on sunlight forenergy. Along the southwestern coast of Australia

lies the Nullarbor Plain, the worlds largest contig-uous karst system, covering more than 200 000 km2,which arose from the sea in the Middle Miocene(B14 MYA) (Webb and James, 2006). Although thesurface environment is semiarid, a number ofaquatic, subterranean caves extend to considerabledistances below the Nullarbor karst, intersecting the

water table at a depth of around 100m below thesurface. Exploration of these underwater passagesby cave divers has revealed the presence of unusualmicrobial biofilms, dense mantles or curtains ofbiological material, known as Nullarbor cave slimes(Holmes et al., 2001). Cave slime communitiesconsist of long, tendril-like structures or amorphous

biofilms attached to cave surfaces. These microbialformations are observed in areas of complete dark-ness, deep within submerged regions of the cavesystems. They comprise a high standing biomassand can be up to 1 m in length (Holmes et al., 2001).No aquatic macrofauna has been recorded in theseregions of the caves (Richards, 1971). The cavewaters are unlikely to receive significant organicinput from the surface, as rainfall in these regionsis typically very low, ranging from 150200 mmannually (Gillieson and Spate, 1992). Indeed, filteredwater samples collected from the vicinity of themicrobial material were previously shown to contain

Correspondence: IT Paulsen, Department of Chemistry & Bio-molecular Sciences, Macquarie University, North Ryde, Sydney2109, New South Wales, Australia.E-mail: [email protected] 19 July 2012; revised 12 December 2012; accepted 2January 2013; published online 21 February 2013

The ISME Journal (2013) 7, 12271236

& 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13

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no detectable organic carbon (James and Rogers,1994). Chemical analysis of the water samples didshow, however, that there are relatively high levels ofnitrite as well as significant sulphate and nitrate inmany of the caves (Holmes et al., 2001). Thecomposition and metabolism of these communitiesis therefore of particular interest, with the micro-

organisms comprising these structures presumablycapable of some form of chemolithotrophy.

Initial work by Holmes et al. (2001) provided thefirst assessment of the microbial communitiesassociated with the Nullarbor cave slimes. In thisstudy, the microbial community from one Nullarborcave, Weebubbie, was sampled and bacterial 16SrRNA gene clone libraries were generated andanalysed via restriction fragment length polymorph-ism mapping and sequencing of selected restrictionfragment length polymorphism pattern representa-tives. The majority of phylotypes were found tobelong to Proteobacteria, with Pseudomonas andPseudoalteromonas related sequences being parti-cularly abundant. A relatively high proportion(B12%) of clones were classified to the phylumNitrospira, of which all characterised members areable to oxidise nitrite to nitrate (Holmes et al., 2001).This suggested that nitrite oxidation could have asignificant role in the trophic structure of the slimecommunities. There were also a large number ofclones which represented novel phylotypes, high-lighting the unusual nature of this microbialecosystem. Examination of the Weebubbie slimecommunity was also carried out using scanningelectron microscopy, revealing a dense matrix ofunbranched filaments interspersed with spherical-,

rod- and spiral-shaped cells and calcite microcrys-tals. The observation of a microscopic structure mostlikely comprising an abundant filamentous organ-ism, whose morphology did not match with theknown cell shape of any abundant clone type, led tothe hypothesis that this environment might alsocontain additional key organisms not effectivelydetected using a PCR-based approach.

In this study, we have significantly expanded ourunderstanding of this community by utilisingpyrosequencing to generate metagenomic and 16SrRNA amplicon data sets for the Weebubbie micro-bial cave slime. We have found evidence for anabundant archaeal population with sequence simi-

larity to Nitrosopumilus maritimus SCM1, anammonia-oxidising archaeon, thus considerablyextending our knowledge of both the phylogeneticcomposition and metabolic capabilities of thisunusual community.

Materials and methods

Site description and sample preparationWeebubbie cave is located in the Nullarbor Plainregion of South Australia in the middle of the GreatAustralian Bight (Latitude 31.654180526, Long-itude 128.774993896). The entrance of the cave isvia a 70m descent from the surface through acollapsed doline. At the base a B200 m talus filledpassage leads to two large lakes, which whentraversed, lead into a number of submerged passageswhere the microbial slime communities are found(Figure 1).

DNA samples used in this work were aliquots ofgenetic material extracted by Holmes et al. (2001)that had been stored at 80 1C. Descriptions ofsample collection, DNA extraction methodology,microscopy and water chemistry analyses are pro-vided in Holmes et al. (2001). Physicochemicalcharacteristics of Weebubbie cave water were mod-erate salinity falling in the brackish range (16.2%),relatively high levels of sulphate (1200 mg l 1),nitrate (98 mg l1) and nitrite (510 mg l1).

As a minimum of 1 mg of DNA was needed formetagenomic pyrosequencing, 33 ng of the remain-ing DNA from Weebubbie samples was amplifiedusing the multiple strand displacement Phi29 DNApolymerase, following the manufacturers instruc-

tions (GenomiPhi V2 DNA Amplification Kit, GEHealthcare, Little Chalfont, UK). Duplicate whole-genome amplifications were performed and pooledto reduce potential amplification bias. Each ampli-fication used 1-ml template and was subsequentlycleaned using ethanol precipitation and resus-pended in a final volume of 75 ml Tris EDTA buffer.DNA concentrations for each amplification reactionwere measured with the Nanodrop 2000 (ThermoScientific, Bremen, Germany) and B600 ng of DNAfrom each reaction was pooled, then sent to theRamaciotti Centre for Gene Function Analysis forsequencing using the GS-FLX pyrosequencing plat-form (Roche, Mannheim, Germany).

Figure 1 Photographs of Weebubbie cave environment and microbial slime communities. (a) Aerial view of Weebubbie cave doline. (b)Submerged chamber of Weebubbie cave. (c) Image of slime tentacles attached to cave walls and roof.

Metagenomic analysis of a cave microbial communitySG Tetu et al

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For 16S rRNA amplicon sequencing, original caveDNA (rather than Phi29-amplified DNA) was usedas a template in PCR with the primer set F515 andR806 of Bates et al. (2011), which have been shownto successfully amplify an B250 bp region of the16S rRNA gene from both bacteria and archaea(Bates et al., 2011). Reactions were performed in

triplicate 25ml reactions using the FastStart HighFidelity PCR System (Roche) according to themanufacturers instructions with 1 ml templateDNA. Reactions were carried out using the followingcycling conditions: 35 cycles (95 1C, 30s; 50 1C, 45 s;72 1C, 60 s) after an initial denaturation 95 1C, 2 min.Pooled triplicate reactions were run out on a 2%agarose gel and B250 bp bands were excised andpurified using the Qiaquick gel cleanup kit (Qiagen,Valencia, CA, USA) before sequencing at theRamaciotti Centre for Gene Function Analysis usingthe GS-FLX pyrosequencing platform (Roche).

Data analysisMetagenome reads were assembled into contigs atthe Ramaciotti Centre for Gene Function Analysisusing Newbler assembly software (Margulies et al.,2005). The unassembled sequence file was pro-cessed to remove potentially low quality sequencesusing the mothur software suite (Schloss et al.,2009). Sequences with an average quality score lessthan 20, below 100 bp in length or containingambiguous nucleotides were removed.

Exploring taxonomic and metabolic composition of

Weebubbie communityProcessed unassembled sequences were uploaded tothe Metagenome Rapid Annotation using SubsystemTechnology (MG-RAST) server, v3.1.2, for annota-tion (Meyer et al., 2008). A taxonomic profile wasgenerated using the phylogenetic identity ofsequence matches to the M5 non-redundant proteindatabase with a maximum e-value cutoff of 1e15. Afunctional comparison was performed using theWeebubbie metagenome, four metagenomes thatwere publicly available on MG-RAST, and onemetagenome that was obtained and uploaded on toMG-RAST as a private metagenome (SupplementaryTable 1). Metagenomes were searched against the

Subsystem database with a maximum e-value of1e5, using a minimum identity cutoff of 75%.Metabolic profiles were determined from normal-ised abundances of SEED subsystems, and a heat-map was produced using complete clustering withthe Bray-Curtis metric.

For 16S rRNA, amplicon sequencing data readswere processed with both MG-RAST and QIIMEsoftware (Caporaso et al., 2010), which binned readsinto phylotypes (97% similarity) and assigned anidentity based on comparison to sequences in theRibosomal Database Project (Cole et al., 2005).Taxonomic analysis of the metagenomic data set

for comparison with the 16S rRNA data employedPhymmBL (Brady and Salzberg, 2009) and MG-RASTto identify the most probable taxonomic origin ofeach sequence read. The metagenomic sequencereads and contigs, as well as the 16S rRNA ampliconsequences are publically accessible through theMG-RAST server with the accession numbers

4448052.3, 4447951.3, and 4467995.3 respectively.

Examination of Weebubbie ThaumarchaeotapopulationThe programme PhymmBL (Brady and Salzberg,2009) was used to identify the most probabletaxonomic origin of each contig sequence. Contigsbinning to the phylum Thaumarchaeota werealigned against the Ca. Cenarchaeum symbiosum(Hallam et al., 2006) genome draft and N. maritimusSCM1 (Walker et al., 2010) genome using Progressi-veMauve (Darling et al., 2010). Contigs binning tothe phylum Thaumarchaeota were also searched

against the Ribosomal Database Project database andtwo putative thaumarchaeotal 16S rRNA fragmentswere identified, one of which was of sufficientlength for use in phylogenetic analysis (the second,containing 301 bp of 16S rRNA gene sequence at theother end of the gene, spanned insufficient variableregions to allow meaningful phylogenetic analysis).The first contig of 941 bp was aligned with 26known archaea within the phyla Crenarchaeota,Euryarchaeota and Thaumarchaeota, as well as fiveuncultured marine Thaumarchaeota. A bacterialrepresentative, Thermotoga maritima MSB-8, wasincluded in the alignment as an out-group (acces-sion numbers of all included gene sequences areprovided in Supplementary Table 2). Sequenceswere aligned using ClustalX2 and trimmed to a totalof 695 nucleotide positions. Phylogenetic analyseswere performed using PhyML 3.0 (Guindon et al.,2010), with the GTR model of nucleotide substitu-tion, utilising a BioNJ tree as a starting tree, and theSPR heuristic implemented for tree topologysearches. Confidence values at each node werecalculated using the non-parametric bootstrap ana-lysis over 1000 replicates. jModelTest (Darriba et al.,2012) was utilised to assess the effect of variation inthe substitution model and other parameters, thetopology model-averaged phylogeny resulting from

the 280 models in the 100% confidence range wasidentical to the tree shown in Figure 4.

Exploration of nitrogen cycling pathways in theWeebubbie communityGene fragments of key enzymes involved in nitrogenmetabolism were identified and combined withtaxonomic assignations of the sequence reads toproduce a metabolic pathway showing the relativeabundances of bacteria and archaea contributing toeach reaction. PhymmBL was used to identify themost probable taxonomic origin of each readsequence. A local database of representative protein


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sequences was generated for key enzymes andprocessed metagenomic reads were searched againstthis database using BlastX with an e-value cutoff of1e15. The BlastX results and PhymmBL taxonomicassignations for each enzyme were used to deter-mine the relative taxonomic abundances calculatedfor each enzyme.

Results

Taxonomic composition of Weebubbie cave microbialslime communityA total of 548 372 raw reads with an average lengthof 581 bp were generated from metagenomic sequen-cing of Weebubbie microbial slime communityDNA. Following removal of low quality reads andthose of less than 100 bp, a total of 475 608 highquality processed read sequences remained in thedata set and were included in analyses. To extendthe taxonomic analysis of the metagenomic data weadditionally generated a 16S rRNA amplicon library;a total of 48 851 fragments spanning the V4 variableregion were analysed. In both the metagenome and

the 16S amplicon data sets the Weebubbie slimecommunities were principally comprised of a mixof bacterial and archaeal members (Figure 2;Supplementary Figure 1). Both analyses showedsimilar sets of dominant taxa, although there wereminor differences in the rank abundance order. Themain difference between the 16S rRNA data and the

binned metagenomic data was the relative abun-dances of Archaea compared with Bacteria, with the16S rRNA data set showing a higher proportion ofArchaea. Amongst the Bacteria, the phylum Proteo-bacteria had the highest relative abundance. Withinthe Proteobacterial assigned sequences, more thanhalf belonged to gammaproteobacteria with almost allthe remainder fairly evenly distributed between alpha,beta and delta subdivisions. Looking further into thegammaproteobacteria at a genus level, Pseudoaltero-monas was the most commonly detected.

Interestingly, in both the metagenomic data andthe 16S rRNA data, there is evidence of an abundantpopulation of Thaumarchaeota related to the ammo-nia oxidising N. maritimus SCM1 and CandidatusCenarchaeum symbiosum.

Figure 2 The inferred taxonomic composition of the Weebubbie cave community. Taxonomic designations were assigned at phylumlevel based on the (a) complete metagenomic data set, and (b) 16S rRNA amplicon data set; species level for the phylum Thaumarchaeotabased on the (c) complete metagenomic data set, and (d) 16S rRNA amplicon data set; and species level for the phylum Proteobacteriabased on (e) the complete metagenomic data set, and (f) the 16S rRNA amplicon data set. The taxonomic analyses based on themetagenome data used MG-RAST, and sequences were compared with the M5 non-redundant protein database with a maximum e-valuecutoff of 1e15. Unclassified sequences accounted for 7.31% of metagenome reads, and were not included in this figure. The taxonomicanalyses based on the 16S rRNA data used MG-RAST, and sequences were compared with the M5 rRNA non-redundant rRNA databasewith a maximum e-value cutoff of 1e15. Unclassified sequences accounted for 26.35% of 16S rRNA amplicon reads. Full colourbreakdown of the organisms represented in the pie charts in (a) and (b) can be found in Supplementary Figure 1.


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Comparison of the Weebubbie thaumarchaeotalpopulation with other studied ThaumarchaeotaAs a high proportion of reads were taxonomicallyassigned to the phylum Thaumarchaeota, and thesequenced genome of N. maritimus SCM1 and draftgenome of Ca. C. symbiosum both recruited a highnumber of reads, alignments were produced to

compare the thaumarchaeotal component of themetagenome with these genomes. From assembly ofmetagenomic data, a set of 20 233 contigs with aminimum of 4 coverage, ranging in size from 100 bpto 67 Kbp (1697 contigs 41 Kbp, 84 contigs 45 Kbp),was generated. The subset of contigs that binned toThaumarchaeota was used to generate alignments(which included almost all of the largest contigs,82/84 of those greater than 45 Kbp). The globalalignment showing the greatest degree of syntenywith the Weebubbie thaumarchaeotal contigs wasobserved with N. maritimus SCM1 (Figure 3). Align-ment against Ca. C. symbiosum genome showedsubstantially less synteny (Supplementary Figure 2).

It is evident that there are numerous syntenousregions in the Weebubbie Thaumarchaeota contigscompared with N. maritimus SCM1 genome, how-ever the similarity profiles (indicated by vertical lineheight along alignment) indicate nucleic acidsequence similarities were relatively low in manyregions (Figure 3a). There are also a number of

instances where genes with sequence similarity tothose of N. maritimus SCM1 were present in theWeebubbie metagenome but showed a differentglobal arrangement (illustrated in Figure 3a by linesconnecting regions of each alignment which haveshifted in their relative location). Also evident inthis alignment are a large number of gaps, represent-ing both sequence present in the N. maritimusSCM1 genome and absent in the Weebubbiemetagenome data and regions of Weebubbie thau-marchaeotal contigs, which contain genes not foundin the N. maritimus SCM1 genome (Figure 3b).

Perhaps one of the most interesting regions ofthe N. maritimus SCM1 genome is the ammonia

Figure 3 MAUVE alignment of the Weebubbie contigs and N. maritimus SCM1 genome. Coloured, boxed sections show regions ofsynteny, with the connecting lines between the two alignments linking matching regions in the N. maritimus SCM1 genome andWeebubbie contigs. The height of the coloured lines within these regions indicates how similar the gene sequences of the matching genesare, with identical gene sequences having a bar that extends to the top border of the region. Gaps indicate regions of sequence present inone genome, but absent in the other. Panel (a) shows the complete alignment over the entire N. maritimus SCM1 genome while (b) showsa subsection, where a region of aligned Weebubbie contigs contains sequence not observed in N. maritimus (in this panel extended redvertical lines indicate contig boundaries, with one large contig spanning most of the length of this region).


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mono-oxygenase operon, which indicates the poten-tial for these organisms to have a role in nitrogencycling. In the Weebubbie metagenome contigs wererecovered which span large regions of this operon,including a single large contig which covers both theamoB and amoC genes, and a second contig whichcontains part of the amoA gene. Alignments of each

of these translated genes from N. maritimus SCM1and Ca. C. symbiosum genome A with the relevantregion of Weebubbie contigs showed proteinsequence identities of 85% for amoB and 95% foramoC, while the fragment of amoA showed proteinidentities of 92% and 93% (over 131 of 216 aminoacids) with N. maritimus SCM1 and Ca. C. symbio-sum, respectively.

Two contigs containing putative thaumarchaeotal16S rRNA gene fragments were identified in theWeebubbie metagenome data set, one of whichcontained a sufficiently long region of 16S rRNAgene identity for phylogenetic analyses. An align-ment of this sequence with archaeal representativesfrom all three lineages Crenarcheaota, Euryarchaeotaand Thaumarchaeota was used to generate a neigh-bour-joining phylogenetic tree (Figure 4). On thebasis of this analysis there is good support forplacing the Weebubbie 16S rRNA sequence within

the Thaumarchaeota. This sequence groups closelywith Ca. C. symbiosum and an uncultured archaealrepresentative recovered from a metagenomic fos-mid library of hydrothermal vent water (0.2mmfiltrate, 1000 m depth, Adriatic sea, accession num-ber EU686633.2 (Martin-Cuadrado et al., 2008))(Figure 4). Also within this group are other well-

studied group 1.1a Thaumarchaota, includingN. maritimus SCM1 and Ca. Nitrosoarchaeumlimnia SFB1, recently identified in a low-salinityenvironment in San Francisco Bay (Blainey et al.,2011), while representatives of group 1.1 b, Ca.Nitrososphaera spp. EN76 and Ca. Nitrososphaeragargensis, formed a separate group within thethaumarchaotal branch.

Comparison of metabolic profiles from other habitatsThe Weebubbie cave environment has salinity levelsin the brackish range, high levels of sulphate, nitrateand nitrite, no detectable organic carbon and acomplete absence of sunlight (Holmes et al., 2001).The metabolic profile of this community wascompared with that of other aquatic metagenomes,including representatives from a freshwater aquiferlocated in South Australia, deep-sea and other

Figure 4 Phylogenetic tree showing the relationship of recovered archaeal Weebubbie cave 16S rRNA gene to that of other archaealrepresentatives. The maximum likelihood tree was constructed from an alignment of 695 nucleotide positions in the 16S rRNA gene. Thetree was inferred with the GTR genetic distance model, using Thermotoga maritima MSB-8 as an outgroup. Bootstraps were calculatedbased on a total of 1000 replications, with bootstrap values displayed at the nodes.


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marine environments (Figure 5). The Weebubbiecave metagenome grouped most closely with thedeep Marmara Sea metagenome and together withthe South Australia Groundwater Aquifer metagenomeform a separate branch from the other examinedmarine metagenomes, which represent photic zone

habitats (Figure 5).The grouping of the Weebubbie metagenomemetabolic profile with that of the Marmara deep-sea environment reflects similar abundances formany SEED categories. The iron acquisition andmetabolism group in particular stands out asrelatively abundant in these two environmentscompared with the others analysed (Figure 5).Indeed the relative abundance of iron acquisitiongene fragments was even higher in Weebubbie thanthe deep Marmara Sea, suggesting an environmentlow in biologically available iron. Examination ofthe predicted gene products within this subsystemindicated that it includes numerous iron transporter

components, receptors and siderophores.

Nitrogen and Carbon Cycling in the Weebubbie CaveCommunityPrevious examination of the Weebubbie cave envir-onment and slime community indicated that theymay represent chemolithotrophic communities reli-ant on nitrite oxidation (Holmes et al., 2001). Theapparent abundance of an archaeal population inour Weebubbie metagenome related to ammonia-oxidising N. maritimus SCM1 indicated that ammo-nia oxidation might also be an important source of

energy in this environment. To gain a more completeappreciation of the nitrogen cycling processescarried out by the Weebubbie slime community themetagenome reads were analysed to determine theabundance and likely taxonomic affiliation of keynitrogen metabolism enzymes (Figure 6). This

analysis indicated that genes encoding enzymes foreach step of the nitrogen cycle, except nitrogenfixation, were present in this community. Of thereads matching ammonia mono-oxygenase subunit-encoding genes, 82% were most likely of archaealorigin (best match to N. maritimus SCM1 amogenes), while the remainder were most likelyencoded by Nitrosomonadales representatives. Asmall number of reads matching hydroxylamineoxidase, which carries out the next step in nitrifica-tion in bacteria were also observed in the metagen-ome, further evidence that bacteria were responsiblefor only a small proportion of ammonia oxidation inthis environment. As archaeal ammonia-oxidisers

(AOA) are thought to utilise a divergent pathway forthis second step, for which there is little informationat present (Walker et al., 2010), no candidate gene(s)were available to search for evidence of this part ofthe process in AOA. There is also evidence for thesecond stage of nitrification, oxidation of nitrite tonitrate, in a number of sequence hits to nitrateoxidoreductase, with both bacterial and archaealforms observed in the metagenome. A relativelylarge number of putative nitrate reductase encodinggenes were also observed in the Weebubbie meta-genome sequence, the majority of which were likelybacterial in origin.

Figure 5 Functional comparison of the Weebubbie cave metagenome with other selected aquatic metagenomes. A complete clusteranalysis with Bray-Curtis distances was performed using normalised abundances of SEED Subsystem categories, comparing theWeebubbie cave metagenome (~) with freshwater (K) and marine () metagenomes (scale bar indicates log transformed, normalised

abundance values). SEED categories from left to right: (1) Fatty Acids, Lipids, and Isoprenoids; (2) Phages, Prophages, TransposableElements, Plasmids; (3) Cofactors, Vitamins, Prosthetic Groups, Pigments; (4) Virulence, Disease and Defence; (5) Carbohydrates; (6)Protein Metabolism; (7) Clustering-based Subsystems; (8) Respiration; (9) Stress Response; (10) DNA Metabolism; (11) Amino Acids andDerivatives; (12) Miscellaneous; (13) RNA Metabolism; (14) Nitrogen Metabolism; (15) Cell Wall and Capsule; (16) Metabolism ofAromatic Compounds; (17) Membrane Transport; (18) Nucleosides and Nucleotides; (19) Cell Division and Cell Cycle; (20) SulphurMetabolism; (21) Motility and Chemotaxis; (22) Regulation and Cell Signalling; (23) Phosphorus Metabolism; (24) Potassium Metabolism;(25) Secondary Metabolism; (26) Dormancy and Sporulation; (27) Photosynthesis; (28) Iron Acquisition and Metabolism.


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Carbon fixation in N. maritimus SCM1 has beenhypothesised to be carried out using the 3-hydroxy-propionate/4-hydrobutyrate pathway, with homologuesof all the genes implicated in this pathway observedwithin this organisms genome (Walker et al., 2010).Reads recruiting to all of these genes (biotin dependent

acetyl-CoA/propionyl-CoA carboxylase, methylmalo-nyl-CoA epimerase and mutase and 4-hydroxybutyratedehydratase) were observed in the Weebubbie meta-genome. Evidence for ribulose-1,5-bisphosphatecarboxylase-oxygenase was also recovered from thebacterial binned component of metagenome reads.

Discussion

Weebubbie cave slime comprises a taxonomicallydiverse microbial communityThe Nullarbor cave slime communities provide afascinating ecosystem, in which microbes dominate

a habitat which appears to be completely isolatedfrom sunlight or photosynthetically derived carbon.Our examination of the phylogenetic composition ofthe Weebubbie microbial communities based ontaxonomic binning of metagenome reads and 16SrRNA amplicon sequencing reveals a diverse com-munity comprising both bacterial and a relativelyhigh proportion of archaeal representatives span-ning a range of described phyla, as well as aproportion of sequences which at this time areunable to be characterised. Both the Thaumarch-aeota and Nitrospirae have lower representation inthe metagenomic binning compared with the 16S

rRNA amplicon phylogenetic analysis, this probablyreflects the relative paucity of both taxa in genomicdatabases, leading to an under identification ofsequences from these phyla. As the 16S rRNA genecan be present in different copy numbers in differentgenomes this potentially affects the estimation of the

relative abundances of phylum. However, as thesequenced Thaumarchaeota generally have a singlecopy of the rRNA gene it seems unlikely that the 16SrRNA amplicon sequence data is overestimating therelative abundance of this phylum. Conversely, theproteobacteria and firmicutes have higher represen-tation in the metagenomic data compared with the16S rRNA amplicon analysis, probably becausethese phyla are the two most heavily sampled bygenome sequencing.

Discovery of a new, abundant Thaumarchaeote in theWebbubbie cave communityThe most striking taxonomic observation from this

work is the finding that Thaumarchaeota comprise asizable proportion of assigned sequences, from1845% (Figure 2) based on metagenomic and 16SrRNA analyses, respectively. On the basis of theobservation of an abundant filamentous organism intheir s.e.m. examination of the microbial slimeswhose morphology did not resemble that of theprevalent clone types, (Holmes et al., 2001) hypothe-sised the potential for a missing link, an abundantorganism not effectively amplified with their primerset. The observations made here confirm that anabundant phylum, namely Thaumarchaeota, wasindeed missed in the cloning based approach, and

Figure 6 Bacterial and archaeal abundances for key nitrogen metabolism enzymes in the Weebubbie cave metagenome. The totalabundance of gene fragments for each enzyme is shown in parentheses next to the enzyme name. The grey and white shading with theboxes indicates the relative proportions of bacterial and archaeal reads matching each enzyme. Solid arrows represent reactionsperformed by a known enzyme that was found in the Weebubbie cave metagenome. Dotted lines represent known nitrogentransformations for which no evidence was obtained from the Weebubbie cave metagenome.


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potentially comprise this missing filamentous mor-photype. Examples of filamentous Thaumarchaeoteshave been reported (Muller et al., 2010) indicating thepotential for the microbial slime filaments to similarlyrepresent examples of novel thaumarchaeotal celltypes.

A partial genome assembly of the Thaumarch-

aeota in the Weebubbie cave environment showedsignificant synteny, albeit with a number of largerearrangements, compared with the sequencedN. maritimus SCM1 genome. There are clearlyclusters of genes within aligned Weebubbie contigsthat are not observed in N. maritimus SCM1indicating that cave environment likely containsnovel sets of thaumarchaeotal genes. It is also likelythat there are genomic regions in N. maritimusSCM1 which are not present in the cave environ-ment, as indicated by a number of sizableN. maritimus SCM1 genome sections which haveno matches at all in the metagenome reads.A recent analysis of metagenome reads most likelyderived from environmental planktonic Thau-marchaeota in the Gulf of Maine, which whencompared with N. maritimus also appeared to lacka number of genomic regions (Tully and Nelson,2012). Combination of these three data sets puta-tively represents a core genome conserved amongstthe group 1.1a Thaumarchaeota.

Phylogenetic analysis of a partial 16S rRNA genesequence recovered from the Weebubbie metagenomeprovides further confirmation of the presence of atleast one thaumarchaeotal representative in thisenvironment. While two contigs containing partialthaumarchaeotal 16S rRNA genes were recovered, one

was of insufficient length to carry out meaningfulphylogenetic analysis and did not overlap with theother, making it difficult to say whether multiplelineages of Thaumarchaeota are present within thecave community. Analysis of the longer sequenceindicates this organism grouped most closely with themarine sponge symbiont Ca. C. symbiosum (Prestonet al., 1996) and a presumably planktonic metagenomerepresentative found in deep-sea hydrothermal ventwater (Martin-Cuadrado et al., 2008). It is of interestthat the Weebubbie sequence groups together withrepresentatives from marine environments, group1.1a, rather than 1.1b, the group predominantlydetected in terrestrial environments. Another recent

metagenomic analysis of AOA in a radioactive thermalcave in the Austrian Alps found that five of six 16SrRNA gene fragments recovered clustered with the1.1b group (Bartossek et al., 2012). The Weebubbiethaumarchaeotal representative may indeed have hada marine origin, as periodic inundations of theNullarbor caves by the sea are thought to haveoccurred a number of times in the geological past,during periods of high sea levels, however, it isthought that the final retreat of the sea occurred ca. 14Ma after which the region experienced uplifting andhas remained relatively geologically stable (Webb andJames, 2006).

An unusual chemolithotrophic community utilisingmultiple nitrogen forms?It has been hypothesised previously that theNullarbor cave slime represents a chemolithotrophiccommunity supported by nitrite oxidation, withwater analysis indicating very high nitrite (510 p.p.m.) and relatively high nitrate levels

(98 p.p.m.) around Weebubbie communities(Holmes et al., 2001). Metagenomic sequencing hasallowed us to look directly for genes relating tonitrogen cycling in this community. This analysisindicated that both stages of nitrification are likelyto be key to energy generation in this community.Examination of the detected ammonia mono-oxygenase (amo) gene copies indicates that thethaumarchaeotal AOA dominate in the Weebubbiecommunity, as they have been shown to do in themajority of soil, estuarine and hot spring sediments,coastal and marine environments for which relativeabundances have been investigated (reviewed byErguder et al. (2009)). Ratios of archaeal to bacterialamoA have been shown to be quite variable,determined to be around 10100 in coastal waters,101000 in the open ocean (Wuchter et al., 2006)and as much as 80 times greater in estuarineenvironments (Caffrey et al., 2007). In the Weebubbiemetagenome reads containing archaeal amo genefragments were roughly five times more abundantthan bacterial copies, suggesting that while AOA arepredominant, AOB activity may still contribute tothe first stage of nitrification in this community.

The second stage of nitrification, oxidation ofnitrite to nitrate also appears to rely on both archaealand bacterial activity, however in this instance it

appears the nitrite oxidising bacteria may bepredominantly responsible. Our metagenome ana-lyses found no evidence of nitrogen fixing pathwaysin this environment.

Conclusion

In this work metagenomic and 16S rRNA ampliconsequencing has provided a more complete view ofthe taxonomic composition of the Weebubbie micro-bial community, revealing that archaeal, as well asbacterial representatives live together within theseunusual slime curtain structures. In 2001, Holmes

and colleagues applied up-to-date molecularmethods to examine the phylotypic structure of thiscommunity, providing the first glimpse into theecology of this rare system (Holmes et al., 2001). Alittle more than 10 years later microbiologists canmake use of a range of new technologies, includingnext-generation sequencing of environmental DNAfor metagenomics, to take an increasingly in-depthlook at the composition, both taxonomic andmetabolic, of such communities. Our analyses havesignificantly broadened our understanding of whatorganisms make up the Weebubbie cave slimecommunity and what they may be doing to survive


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in such conditions. On the basis of metabolicprofiling we hypothesise that the Weebubbie slimecurtains represent novel chemolithotrophic com-munities driven by nitrification.

Acknowledgements

ITP received support from a Life Science Research Award,provided by the New South Wales Office of Science andMedical Research. We would also like to acknowledge thecave divers responsible for sample collection and photo-graphy: Peter Rogers, Cheryl Bass, Bob Davis, Phil Prustand Steve Trewavas.

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