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Keywords:

  • methane seep;
  • Timor Sea;
  • pmoA;
  • methanotrophs;
  • real-time PCR;
  • clone libraries

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase (pmoA) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum. Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Geophysical and geochemical evidence suggests that methane seeps are widespread within the tropical Timor Sea (O'Brien et al., 2000), an arm of the Indian Ocean situated between the island of Timor and the north-western coastline of Australia. On the shallow carbonate-rich Yampi Shelf region of the Timor Sea, numerous active seeps have recently been documented and characterized (Rollet et al., 2006). Here, numerous plumes of methane gas associated with petroleum oils of thermogenic origin were observed emanating from the seafloor and into the water column, and at periods of low tide, methane reached the sea surface and escaped into the atmosphere (Rollet et al., 2006). Methane and other hydrocarbons released from seeps are important components of local and global energy and carbon cycles, often sustaining thriving biological communities (Levin, 2005) and contributing large amounts (estimates of up to 48 Tg yr−1) of the potent greenhouse gas into the atmosphere (Judd, 2004). It is therefore important to understand the factors, such as microorganisms, that are responsible for influencing the fate of such large quantities of energy and carbon.

Studies of seep-associated microbial communities and activities have recognized the importance of microbial-driven metabolic processes such as the anaerobic oxidation of methane (AOM) (Hinrichs et al., 2000; Joye et al., 2004), aerobic oxidation of methane (Kruger et al., 2005; Niemann et al., 2006), methanogenesis (Inagaki et al., 2004), sulfate reduction (Joye et al., 2004), sulfide oxidation (Aharon & Fu, 2000) and petroleum hydrocarbon oxidation (Formolo et al., 2004). These processes, especially methane and other hydrocarbon oxidation processes, are especially important in reducing fluxes of methane to the atmosphere (Kruger et al., 2005), transferring hydrocarbon-derived energy and carbon to higher trophic levels via grazing or symbiotic interactions (Bauer et al., 1990), and reducing the accumulation of toxic petroleum hydrocarbon compounds.

Previous microbiological and geochemical studies of marine hydrocarbon seeps have focused primarily on characterizing microbial communities and biogeochemical processes associated with the AOM. The AOM acts as an efficient ‘benthic filter’ in typical diffusion-controlled marine sediments and ‘low- and medium-flux’ advection-controlled seep sediments, often reducing the concentrations of ascending methane to negligible amounts before reaching oxygenated surface sediments (Luff & Wallmann, 2003; Luff et al., 2004). At seep sites where high fluid and gas fluxes reduce, overwhelm and/or bypass AOM activity, aerobic oxidation of methane is an important additional means of regulating methane flux (Niemann et al., 2006; Sommer et al., 2006). Furthermore, it has been shown that shallow anoxic subsurface sediments have the potential for aerobic methane oxidation when oxygen becomes available (Kruger et al., 2005), as may occur in sediments subject to oxygen flushing due to physical processes such as bioturbation, currents or gas ebullition (Zimmermann et al., 1997). Together, these findings suggest that aerobic methane oxidation may be important at some seep sites, especially at sites where methane reaches and passes through oxygenated surface sediments, as was observed over relatively large areas of the current study site.

Few investigations have examined communities of aerobic methanotrophs associated with hydrocarbon seeps, and in general, there is limited information regarding the diversity and community structure of aerobic methanotrophs in marine environments (Inagaki et al., 2004; Elsaied et al., 2005; Yan et al., 2006; Hayashi et al., 2007a, b). In addition, no studies have been conducted on environments of tropical marine methane seeps. Studies of aerobic methanotrophs in sediments associated with seeps off the coast of Japan (Inagaki et al., 2004), in the Gulf of Mexico (Yan et al., 2006) and along the Californian margin (Tavormina et al., 2008) have revealed relatively diverse communities dominated by novel phylotypes of Type I methanotrophs of the Gammaproteobacteria. Collectively, these studies have revealed that marine hydrocarbon seeps can support communities of diverse and novel methanotrophic bacteria, and therefore the study of such environments contributes to our understanding of the diversity and ecology of methanotrophic bacteria and provides an insight into the microorganisms carrying out this important biogeochemical process.

In this study, microbial diversity in surface sediments overlying a carbonate hardground structure associated with active methane and petroleum hydrocarbon seepage in the Timor Sea was investigated. Particular focus was on examining the diversity of aerobic methanotrophic bacteria. The results of this study provide an insight into the composition of microbial life in this unique environment and reveals evidence for the existence of a novel microbial diversity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Study sites, sampling and sample processing

Samples were collected from the Cornea region on the Yampi Shelf of the Timor Sea, Australia, during the RV Southern Surveyor cruise SS05/06 of June 2005. Active seepage was identified using 120 kHz echo-sounders and used to guide sampling using a heavily weighted Smith-MacIntyre (0.25 m2 surface area) grab sampler that provided sediment grabs with undisturbed surface sediments. Grabs were then subsampled with a sterile core device (5 cm inner diameter and 30 cm length) shipboard. Three cores (D, E and H) were collected from the seep-associated carbonate structure. Core H (13:39.27°S 124:42.70°E; 89.6 m water depth; 5 cm length) was obtained from sediments associated with the origin of the most intense gas plume observed. Cores D (13:39.31°S 124:42.69°E; 89.2 m water depth; 20 cm length) and E (13:39.15°S 124:42.69°E; 89.6 m water depth; 10 cm length) were also collected from sediments overlying the carbonate hardground structure where less intense seep plumes were observed. Coral fragments and other carbonate debris restricted sampling of deeper sediments in all cores. Two additional cores G (13:30.62°S 124:42.48°E; 109.6 m water depth; 20 cm length) and F (13:24.71°S 124:41.80°E; 107.2 m water depth; 20 cm length) were collected from sediments where no active seepage was detected. All cores were sectioned into 1-cm intervals and stored at −20 °C shipboard, in liquid nitrogen (−180 °C), during transport (5 days) and at −80 °C in the laboratory before molecular analysis.

DNA extraction

Total DNA was extracted from 0.5 g of sediment (wet weight) by a combined physical and chemical lysis treatment using a FastDNA Spin Kit for Soil (Qbiogene) following the manufacturer's instructions, with the exception of the following modifications. Samples were bead beaten in a MiniBeadBeater (BioSpec Products) for 30 s using the set default speed of c. 2000 r.p.m. Bead beating times were optimized on the basis of DNA yield and integrity as determined by agarose gel electrophoresis of crude DNA extracts. DNA was eluted using the supplied DES (DNA elution solution) water to a final volume of 50 μL according to the manufacturer's instructions and 50 μL of 2 × TE buffer [20 mM Tris-HCl (pH 7.5), 2 mM EDTA (pH 8.0)] was added. DNA concentrations were determined using a Quant-iT PicoGreen dsDNA quantitation kit (Molecular Probes) using fluorimetry. Extracted DNA was of sufficient yield and purity to be PCR amplified, with no further dilution or purification necessary. All DNA extracts were stored at −20 °C.

PCR amplification of 16S rRNA and pmoA genes for cloning

Bacterial 16S rRNA genes were amplified using the bacterial-specific forward primer B27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and universal reverse primer U1492R (5′-GGTTACCTTGTTACGACTT-3′) (Lane, 1991). Archaeal 16S rRNA genes were amplified using the archaeal-specific forward primer A8F (5′-TCCGGTTGATCCTGCC-3′) (Teske et al., 2002) and universal reverse primer U1492R. Type I methanotroph 16S rRNA genes were amplified using Type I targeted primers, forward primer Type IF (5′-ATGCTTAACACATGCAAGTCGAACG-3′) and reverse primer Type IR (5′-CCACTGGTGTTCCTTCMGAT-3′) (Chen et al., 2007). Partial pmoA genes were PCR amplified using the forward primer pmoA189-f (5′-GGNGACTGGGACTTCTGG-3′) and reverse primer mb661-r (5′-CCGGMGCAACGTCYTTACC-3′) (Costello & Lidstrom, 1999). This primer pair was selected because it has proven to reveal extensive pmoA diversity within various environments (Bourne et al., 2001) and is specific to pmoA genes and does not coamplify closely related ammonia monooxygenase amoA genes (Dumont & Murrell, 2005). Cloned PCR products were checked for inserts using a direct PCR method using the vector-specific primers M13F (5′-GTAAAACGACGGCCAG-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′). All PCR reactions (final volume of 25 μL) contained 1 × Qiagen PCR Buffer (Qiagen, Germany), 1 U of HotStarTaq DNA Polymerase (Qiagen), 200 μM of each dNTP, 25 pmol of each primer, 0.5 μL of purified bovine serum albumin (BSA) 10 μg μL−1 (New England Biolabs), 0.5 μL of DNA template (c. 50 ng) and MilliQ water up to 25 μL. Thermocycling conditions for the amplification of bacterial and archaeal 16S rRNA genes consisted of an initial ‘enzyme activation’ at 95 °C for 15 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, followed by a final extension step of 72 °C for 10 min to facilitate ‘A-tailing’ of PCR products for cloning. Thermocycling conditions for the amplification of pmoA genes and clone inserts were the same as for bacterial and archaeal 16S rRNA genes, except that 35 cycles were performed. Thermocycling conditions for the amplification of Type I methanotroph 16S rRNA genes were the same as for pmoA genes, except that the annealing temperature was 60 °C.

Clone library construction and DNA sequencing

PCR products from three replicate reactions for each sample were pooled and subject to agarose gel electrophoresis. Bands of expected size were excised and purified using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. Purified DNA was then cloned using a TOPO-TA Cloning Kit (Invitrogen) according to the manufacturer's instructions. Individual colonies were suspended in 100 μL of sterile MilliQ water using a sterile toothpick, vortexed briefly, allowed to stand for 60 min, vortexed again and this cell suspension used as a template for PCR using the M13 primers to confirm inserts. The PCR products generated were dried and sent to Macrogen Inc. (Seoul, South Korea) for purification and sequencing using an ABI3730 XL automatic DNA sequencer using the M13F vector-specific primer.

Real-time PCR assay for quantification of pmoA genes

Real-time PCR assays were carried out using a Rotor-Gene 3000 real-time DNA amplification system (Corbett Research) using a Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) and the pmoA-specific primers pmoA189-f and mb661-r (Kolb et al., 2003). PCR reactions (final volume of 25 μL) contained 12.5 μL of 2 × Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 4.0 mM MgCl2, 25 pmol of each primer, 0.5 μL of purified BSA 10 μg μL−1 (New England Biolabs), 2.0 μL of DNA template and MilliQ water up to 25 μL. Real-time PCR cycling conditions included an initial ‘UDG incubation’ step at 50 °C for 2 min, an ‘enzyme activation’ step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 20 s, annealing at 65 °C for 20 s and extension at 72 °C for 40 s. Acquisition of the fluorescence signal was performed during the 72 °C extension step of each cycle. Melt-curve analysis was not possible with this primer and amplicon combination as described previously (Kolb et al., 2003), and therefore PCR products from all real-time PCR assays were examined using standard agarose gel electrophoresis. Products of the expected size were detected in all assays without the formation of nonspecific products and meant that the curves generated were most likely produced by pmoA genes only and could therefore be used to determine pmoA gene copy numbers using rotor-gene software as detailed below.

The DNA standards used in the real-time PCR assays consisted of serial dilutions of purified PCR product derived from a cloned pmoA gene. Clone TS-SS PA34 (see Results) was PCR amplified directly from a colony using M13 vector-specific primers, checked using standard agarose gel electrophoresis, gel purified using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions and DNA concentrations were determined using a Quant-iT PicoGreen dsDNA quantitation kit (Molecular Probes) using fluorimetry. The measured concentrations of purified PCR product were then converted to copies per microliter and the concentration was adjusted to 1 × 109 copies μL−1 before performing serial dilutions. A five-point standard curve (2 × 107–2.0 × 103 copies per reaction) was run in triplicate with each run. Environmental samples and negative controls (no template DNA) were also run in triplicate. Data and copy numbers of pmoA targets in environmental samples were analyzed using the rotor-gene software version 6.1.71 (Corbett Research) following the manufacturer's guidelines. The final copy numbers of pmoA genes in environmental samples were calculated after accounting for multiple pmoA gene copies per genome (Stolyar et al., 1999, 2001), dilution of DNA template during DNA extraction and assuming 100% DNA extraction efficiency.

Phylogenetic analysis of 16S rRNA and pmoA genes

Retrieved 16S rRNA and pmoA gene nucleotide sequences were initially checked using chromas lite software version 2.01 (Technelysium, Australia) before being truncated to exclude the primer and the vector sequence. 16S rRNA gene sequences were screened for potential chimeras using bellerophon (Huber et al., 2004) and sequences flagged as potential chimeras were discarded from further analysis. 16S rRNA gene sequences were then imported into the arb software package (http://www.arb-home.de) (Ludwig et al., 2004), aligned against the Greengenes database (http://greengenes.lbl.gov) (DeSantis et al., 2006) that is compatible with arb, followed by a manual correction of the alignment when necessary. Additional reference sequences that were not available in the Greengenes database at the time of analysis were identified from blast (Altschul et al., 1997) searches of sequences retrieved in this study and were imported and aligned using arb. Trees were constructed using the neighbor-joining (Jukes–Cantor correction) (Saitou & Nei, 1987) algorithms implemented in arb. The robustness of the inferred tree topologies was evaluated after 1000 bootstrap replicates of the neighbor-joining data. Partial sequences (c. 600 bp) were inserted into the tree without changing the tree topology using the arb parsimony interactive method. Nucleotide sequences of pmoA genes were translated into protein sequences using the Translate tool in the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (http://us.expasy.org/tools/dna.html). Deduced protein sequences were aligned using clustalx version 1.83 (Thompson et al., 1994). Distance matrices were calculated using the protdist program in phylip (Felsenstein, 2005). Phylogenetic trees were generated from distance matrices using the neighbor-joining method and the Kimura substitution algorithm (Kimura, 1983) using phylip. Bootstrapping with 1000 replicates was performed using SeqBoot as integrated in phylip.

Statistical analysis of pmoA clone library data

Rarefaction analysis (Heck et al., 1975), Chao1 nonparametric richness estimates (Chao, 1984) and Simpson's index of diversity (Magurran, 1988) were generated using dotur software (Schloss & Handelsman, 2005). Partial pmoA nucleotide sequences and a distance threshold of 0.10 (90% sequence similarity) were used for all dotur analyses. This distance threshold was selected with regard to the concept that defines 16S rRNA gene sequences showing >97% sequence similarity as belonging to the same species (Stackebrandt & Goebel, 1994) and the 3.5 times higher nucleotide substitution rate of the pmoA gene (Heyer et al., 2002).

Nucleotide sequence accession numbers

The nucleic acid sequences determined in this study have been deposited in the GenBank/EMBL/DDBJ databases. The accession numbers for sequences of the genes are as follows: bacterial 16S rRNA gene, FJ175453FJ175606; archaeal 16S rRNA gene, FJ175607FJ175656; pmoA gene, EU417453EU417476, EU417478EU417545 and EU417547EU417558.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Study site description and physicochemical characteristics

The study site was at a depth of 84 m and consisted of an oval-shaped carbonate hardground structure (500 × 1400 m) that protruded up to 4 m higher than surrounding sediments. A layer of coarse, carbonate-rich, sandy-mud overlaid the carbonate hardground structure. Numerous plumes consisting of almost pure thermogenic methane (99% methane, δ13C=−41‰) emanated from the carbonate hardground area. Petroleum oil was associated with gas bubbles and small petroleum oil slicks were observed on the sea surface above plumes. Towed video observations revealed points of active methane ebullition from sediments, some over relatively large areas (c. 10 m2) of seafloor at periods of low tide. Various benthic organisms such as solitary corals, bryozoans, gorgonians and ophiuroids were observed, while no evidence of typical conspicuous seep-associated chemosynthetic biota (e.g. microbial mats, tubeworms or mussels) was observed. A thin green algal mat was also observed on the surface of sediments. The ambient seawater temperature was 27–28 °C c. 5 m above the seafloor.

Diversity of Bacteria and Archaea detected using 16S rRNA gene clone libraries

Clone libraries of bacterial and archaeal 16S rRNA genes were generated using general bacterial and archaeal primers, respectively, from both surface (0–1 cm b.s.f.) and shallow subsurface (4–5 cm b.s.f.) sediments of core H, which were associated with the most intense methane plume observed. Clone libraries of Type I and II methanotroph 16S rRNA genes were also generated using Type I and Type II specific primers, respectively, from the surface sediments (0–1 cm b.s.f.) of core H. Because of the high sequence diversity within libraries, clones were randomly selected for sequencing.

Bacterial clone libraries generated with general bacterial primers from surface (101 clones) and shallow subsurface (51 clones) sediments were dominated by sequences affiliated with the Gammaproteobacteria (35% and 18% of surface and subsurface libraries, respectively), Deltaproteobacteria (22% and 30% of surface and subsurface libraries, respectively) and Alphaproteobacteria (13% and 20% of surface and subsurface libraries, respectively) (Fig. 1). Other sequences affiliated with the Acidobacteria, Planctomycetes, Bacteroidetes, Nitrospira, Firmicutes, Lentisphaerae, Gemmatimonadales and candidate divisions TM7, GN06, WS3 and OS-K were also detected (Fig. 1).

image

Figure 1.  Relative abundance of taxonomic groups of bacterial 16S rRNA genes present in clone libraries.

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In general, sequences recovered in this study were most closely related to sequences recovered previously from marine and some methane-rich sediment environments (see phylogenetic affiliations in Supporting Information, Fig. S1). Only three phylotypes (B1-6, B1-45 and B1-98) recovered from the surface sediment clone library generated with general bacterial primers clustered with aerobic methanotrophs of the family Methylococcaceae. Only one phylotype (I-3, represented by 3/20 clones) recovered from the surface sediment clone library generated with primers targeting Type I methanotrophs clustered within the Methylococcaceae. Phylotypes B1-6, B1-46, B1-98 and I-3 affiliated with sequences recovered from marine environments and were distantly related (95%, 95%, 94% and 92%, respectively) to other sequences within public databases related to the genera Methylococcus and Methylocaldum (Fig. 2). No Type II methanotroph-related sequences were detected in libraries generated with general bacterial or Type II specific primers.

image

Figure 2.  Phylogenetic tree showing the affiliations of Methylococcaceae-related 16S rRNA gene sequences derived from Timor Sea seep-associated sediments. Sequences with ‘B1’ followed by a number indicate sequences retrieved from the general bacterial surface sediment library (given in bold), while sequences with ‘I’ followed by a number indicate sequences retrieved from the Type I methanotroph-specific surface sediment library (given in bold). Bootstrap values of ≥50% and ≥90% from 1000 resamplings are presented at nodes as filled (•) and open circles (○), respectively. The scale bar represents 10% sequence divergence.

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Archaeal clone libraries generated using general archaeal primers from both the surface (51 clones) and the shallow subsurface (56 clones) were dominated by sequences affiliated with Marine Group I of the Crenarchaea (Vetriani et al., 1999), while other sequences affiliated with the Miscellaneous Crenarchaeotal Group (Inagaki et al., 2003), Thermoplasmatales, Marine Benthic Group B (Vetriani et al., 1999) and Methanococcoides were also detected (Fig. 3). No sequences related to known anaerobic methanotrophs (ANME) were detected, even when using various primer sets targeting 16S rRNA genes of different ANME groups or primers targeting methyl coenzyme M reductase (mcrA) genes involved in AOM via ‘reverse methanogenesis’ by ANME archaea (Hallam et al., 2003).

image

Figure 3.  Relative abundance of taxonomic groups of archaeal 16S rRNA genes identified in clone libraries.

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Detection and estimation of aerobic methanotroph numbers

The existence of aerobic methanotrophs was further investigated by analysis of functional pmoA genes. A general pmoA targeted real-time PCR assay (Kolb et al., 2003) revealed the highest numbers of methanotrophs in sediments of core H that were associated with the most intense methane plume observed. This assay revealed approximately twice the number of cells in the top 1 cm of core H (1.48 × 105±0.17 cells g–1 sediment) when compared with similar core sections (cores D and E) that were taken from the carbonate hardground area (Table 1). Copy numbers steadily declined to undetectable levels in deeper sediment sections below 10 cm in cores D and E. No pmoA genes could be detected in samples (cores G and F) collected from sediments where no evidence for active seepage was observed.

Table 1.   Estimation of methanotroph numbers using real-time PCR
Depth below seafloor (cm)Number of cells per gram of sediment*
Core HCore ECore D
  • *

    Expressed as 105 cells g−1 sediment dry weight (± SD).

0–11.48 (± 0.17)0.75 (± 0.30)0.72 (± 0.07)
2–30.90 (± 0.23)0.17 (± 0.07)0.27 (± 0.01)
4–50.77 (± 0.03)0.08 (± 0.01)0.44 (± 0.07)
9–10 0.12 (± 0.03)0.22 (± 0.06)

Diversity of aerobic methanotrophs detected using pmoA gene clone libraries

Clone libraries generated with primers targeting pmoA genes from surface (0–1 cm b.s.f.) and shallow subsurface (4–5 cm b.s.f.) sediments of core H demonstrated greater methanotroph pmoA gene diversity in surface sediments in comparison with subsurface sediments when subjected to rarefaction analysis (Fig. 4), Chao1 richness estimates and Simpson's index of diversity (Table 2). dotur analysis of sequences established 18 and 13 phylotypes for surface and subsurface libraries, respectively (Table 2).

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Figure 4.  Rarefaction analysis of pmoA gene sequences obtained from surface (•) and subsurface (○) clone libraries of core H. Phylotypes were defined using a 10% nucleotide sequence similarity cut off.

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Table 2.   Diversity analysis of pmoA gene fragment libraries*
LibraryNo. of clonesNo. of phylotypesChao1Simpson's index
  • *

    Phylotypes were defined using a 10% nucleotide sequence similarity cut off.

Surface5518580.15
Subsurface511316.20.08

Phylogenetic analysis of deduced pmoA amino acid sequences indicated that they were most closely related to sequences previously retrieved from marine environments, methane seeps and hydrothermal vents (Fig. 5). A large proportion (62%) of retrieved sequences were only distantly related (≤91% amino acid and≤86% nucleotide sequence identities) to sequences currently present in public databases. None of the sequences retrieved in this study were closely related to any cultured methanotrophs. All sequences were affiliated with Type I methanotrophs of the Gammaproteobacteria, while no sequences were related to Type II methanotrophs of the Alphaproteobacteria. The large majority (90%) of sequences (designated Marine Cluster A) were of an unclear affiliation and were only distantly related to Type Ib methanotrophs (also known as Type × methanotrophs) of the genera Methylococcus and Methylocaldum, with clone TS-SS PB4 identified using blast searches as the closest relative to any formally described cultured representative with only 83% amino acid and 73% nucleotide sequence similarities to Methylococcus capsulatus Bath. The remaining sequences (designated Marine Cluster B) were also of an unclear affiliation and were only distantly related to Type Ia methanotrophs (e.g. Methylobacter, Methylomicrobium and Methylosoma genera), with clone TS-S P34 identified using blast searches as the closest relative to any formally described cultured representative with only 87% amino acid and 75% nucleotide sequence similarities to Methylomicrobium japanense NI.

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Figure 5.  Phylogenetic tree based on deduced pmoA amino acid sequences derived from Timor Sea seep-associated sediments relative to reference sequences from cultured methanotrophs and environmental sequences retrieved from other studies. Unique phylotypes from each library are presented. Numbers presented in parentheses after each sequence retrieved in this study indicate the number of clones represented by the phylotype presented. Bootstrap values of ≥50% and ≥90% from 1000 resamplings are presented at nodes as filled (•) and open circles (○), respectively. The tree was rooted with an ammonia monooxygenase amino acid sequence from Nitrosococcus oceani. The scale bar represents 10% sequence divergence.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This is the first study to investigate the microbial diversity associated with a shallow hydrocarbon seep in a tropical marine environment. The numerous and sometimes intense methane seeps associated with Cornea hardground area suggested that this environment may be favorable for the establishment of microbial communities comprised largely of methane (and possibly other hydrocarbon)-oxidizing microbial populations, similar to those found in other seep environments (Mills et al., 2003; Inagaki et al., 2004; Knittel et al., 2005; Losekann et al., 2007). However, various observations suggest that chemosynthetic microorganisms sustained by hydrocarbons are not a large component of the overall microbial communities in surface sediments at the Cornea seep area. These observations include the fact that bacterial and archaeal communities included few phylotypes typically associated with hydrocarbon oxidation processes (e.g. aerobic methane oxidation), the apparent absence of sulfur-oxidizing microbial mats or seep-associated chemosynthetic macrofauna that often thrive where methanotrophic microorganisms mediate the transfer of carbon and energy from seeping methane to other organisms (Levin, 2005) and the failure to detect isotopically depleted carbon in sedimentary carbonates (Rollet et al., 2006) that are typically associated with AOM processes (Luff & Wallmann, 2003).

Phylogenetic analysis of 16S rRNA genes derived from clone libraries revealed highly diverse and mostly novel phylotypes, while only a few sequences were related to possible methanotrophic or petroleum oil-oxidizing bacteria. No 16S rRNA gene sequences were related to methanotrophic archaea and no methyl coenzyme M reductase (a functional gene for methanogenic and methanotrophic archaea) sequences related to known methanotrophic archaea were retrieved, suggesting that anaerobic methanotrophic archaea were absent in the surface sediment samples analyzed. Aerobic methanotroph sequences constituted only a small proportion of 16S rRNA gene libraries and even the application of primers that target 16S rRNA genes of aerobic methanotrophs detected few methanotroph-related sequences and mostly detected other closely related gammaproteobacterial sequences. This has been similarly reported in water samples from methane plumes from the Californian continental margin (Tavormina et al., 2008) and is probably a result of the limitation of primer specificity against other closely related sequences that were probably more abundant and therefore take preference during the amplification process over less abundant target sequences. Together, these data suggest that methanotrophic bacteria and archaea were not major dominant members of the microbial communities in surface sediments at the study site.

Despite the fact that aerobic methanotrophs did not appear to dominate the overall bacterial community in surface sediments, these bacteria are still likely to play an important biogeochemical role and are therefore worth studying further. Furthermore, the methanotroph-related 16S rRNA genes that were retrieved were novel and suggested that further analysis of this functional group by targeting functional pmoA genes may reveal a more detailed understanding of aerobic methanotroph diversity. In this study, more than half of the retrieved pmoA amino acid sequences were <91% similar to the sequences present in public databases, suggesting that numerous novel putative methanotroph species and genera exist that have not been detected in any previous culture-dependent or -independent analyses. Studies of hydrocarbon seeps on the continental margin of California (Tavormina et al., 2008), in the Gulf of Mexico (Yan et al., 2006) and off the coast of Japan (Inagaki et al., 2004) have also revealed diverse and novel methanotrophs, suggesting that marine hydrocarbon seep environments harbor extensive undocumented methanotroph diversity. Interestingly, the clone libraries of pmoA genes in this study were dominated by novel phylotypes distantly related to the genera Methylocaldum and Methylococcus of the Type Ib group. This is distinct from pmoA gene sequences recovered from previously studied marine methane seeps, which have been dominated by phylotypes related to genera of the Type Ia group. Although it is difficult to directly compare data obtained from clone libraries created using different molecular procedures due to various inherent biases associated with molecular methods, it is likely that a combination of geographical separation and distinct environmental conditions (e.g. warm shallow waters versus low-temperature deep-waters) found at the different seep sites contributed to differences in the groups of methanotrophs found.

The majority of phylotypes identified in this study (Marine Cluster A, Fig. 5) were only distantly related to the genera Methylocaldum and Methylococcus of the Type Ib group and are thus likely to represent sequences belonging to novel uncultured genera of the family Methylococcaceae. Another smaller group of phylotypes identified (Marine Cluster B, Fig. 5) were only distantly related to genera of the Type Ia group and are also likely to represent sequences belonging to a novel uncultured genera of the family Methylococcaceae. Phylotypes from both Marine Clusters A and B were similar to only a few phylotypes found in public databases, all of which were recovered from marine environments. Examination of the phylogeny of these marine sequences shows that they have clearly diverged from previously described phylotypes retrieved from terrestrial environments and therefore possibly represent divergent lineages of methanotrophs that are likely to be marine specialized. These sequence data set provide evidence for the existence of such divergent groups of methanotrophs that have not previously been apparent based on molecular data (McDonald et al., 2005), probably due to the small number of sequences recovered from marine environments present in public databases. Future studies that provide more sequences for comprehensive phylogenetic analyses, other molecular data sourced from techniques such as metagenomics or successful cultivation of these phylotypes may provide additional evidence that these lineages are indeed bona fide marine specialized groups.

In summary, this study provides evidence for the existence of diverse and novel microbial and aerobic methanotroph diversity. Phylotypes discovered in this study were distinct from those previously reported from other hydrocarbon seep and marine environments. Phylogenetic analysis of these novel pmoA phylotypes in relation to previously identified phylotypes lends support to the existence of so far unrecognized lineages of specialized marine methanotrophs related to Type Ia and Ib methanotrophs. Future studies of methanotrophs in this ecosystem should focus on determining the activity and identity of the active members of the communities to confirm that the methanotrophs detected in this study are indeed responsible for this biogeochemical process in situ. Furthermore, attempts should be made to enrich and isolate the marine methanotrophs in order to confirm the sources of these novel pmoA genes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank the crew and scientific support staff of the RV Southern Surveyor during cruise SS05/06 to the Timor Sea for collection of samples during June of 2005. Special thanks are due to Gregg Brunskill and Irena Zagorskis for help in sampling and geochemical analyses. The authors thank the Australian Biological Resources Study and Commonwealth Scientific and Industrial Research Organization for stipend and financial support.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Neighbor-joining phylogenetic trees showing the affiliations of 16S rRNA gene sequences derived from Timor Sea seep-associated sediments compared with reference sequences. Numbers indicate bootstrap percentages from 1000 bootstrap replicates. Only bootstaps >50% are shown. The scale bar represents 10% sequence divergence. The tree is shown in parts that correspond to different phylogenetic groups: a) α-proteobacteria, b) γ-proteobacteria, c) δ-proteobacteria, d) all other bacteria excluding Proteobacteria, e) Crenarchaea and f) Euryarchaea. Sequences with ‘B1’ or ‘A1’ followed by a number are derived from bacterial and archaeal surface (0-1 cm below sea floor) libraries, respectively. Sequences with ‘B5’ or ‘A5’ followed by a number are derived from bacterial and archaeal sub-surface (4-5 cm below sea floor) libraries, respectively. Sequences with ‘I’ or ‘MI’ followed by a number are derived from Type-I methanotroph specific libraries. Sequences with ‘MII’ followed by a number are derived from Type-II methanotroph specific libraries. Numbers in rounded brackets indicate the number of times each phylotype occurred in the corresponding library and numbers in square brackets indicate the Genbank accession number.

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