• microbial mats;
  • Lucky Strike vent field;
  • metabolic diversity;
  • Bathymodiolus azoricus symbionts;
  • sulfur oxidation;
  • methane oxidation


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

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge) were investigated using molecular approaches. DNA and RNA were extracted from mat samples overlaying hydrothermal deposits and Bathymodiolus azoricus mussel assemblages. We constructed and analyzed libraries of 16S rRNA gene sequences and sequences of functional genes involved in autotrophic carbon fixation [forms I and II RuBisCO (cbbL/M), ATP-citrate lyase B (aclB)]; methane oxidation [particulate methane monooxygenase (pmoA)] and sulfur oxidation [adenosine-5′-phosphosulfate reductase (aprA) and soxB]. To gain new insights into the relationships between mats and mussels, we also used new domain-specific 16S rRNA gene primers targeting Bathymodiolus sp. symbionts. All identified archaeal sequences were affiliated with a single group: the marine group 1 Thaumarchaeota. In contrast, analyses of bacterial sequences revealed much higher diversity, although two phyla Proteobacteria and Bacteroidetes were largely dominant. The 16S rRNA gene sequence library revealed that species affiliated to Beggiatoa Gammaproteobacteria were the dominant active population. Analyses of DNA and RNA functional gene libraries revealed a diverse and active chemolithoautotrophic population. Most of these sequences were affiliated with Gammaproteobacteria, including hydrothermal fauna symbionts, Thiotrichales and Methylococcales. PCR and reverse transcription-PCR using 16S rRNA gene primers targeted to Bathymodiolus sp. symbionts revealed sequences affiliated with both methanotrophic and thiotrophic endosymbionts.


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

Microbial mats – layered biofilms containing different types of cells – are complex systems in which representatives of various groups of organisms co-occur in various types of ecosystems. These mats are good models to study biogeochemical processes, such as chemical element cycles, in which a variety of microorganisms cooperate and interact in complex ways (Seckbach & Oren, 2010).

Recent studies on microbial mat communities from chemosynthesis-based ecosystems such as cold-seep areas (Arakawa et al., 2006; Gilhooly et al., 2007), mud volcanoes (Heijs et al., 2005; Omoregie et al., 2008) or sulfide-rich caves (Engel et al., 2004; Dattagupta et al., 2009) have shown they may be host to a huge diversity of bacterial species, dominated by Proteobacteria, including filamentous species belonging to the order Thiotrichales.

White filamentous mats have also been observed in various oceanic hydrothermal vent fields; large bacteria, morphologically affiliated with the genera Beggiatoa and Thiothrix, are the dominant morphotypes (Jannasch & Wirsen, 1981; Jacq et al., 1989; Nelson et al., 1989; Kalanetra et al., 2004). These organisms, members of the order Thiotrichales (Gammaproteobacteria), are known for their ability to form biofilms on oxic/anoxic interfaces, using dissolved free oxygen to oxidize reduced sulfur compounds (Teske & Nelson, 2006).

Despite this fact, only a few studies have attempted to characterize microbial communities of deep-sea hydrothermal vent mats, i.e. along the Mariana Arc (Davis & Moyer, 2008), at the Loihi Seamount (Hawaii) (Moyer et al., 1995), at the 17°S East Pacific Ridge (EPR) hydrothermal vent field (Longnecker & Reysenbach, 2001), the Lost City vent fields [Mid-Atlantic Ridge (MAR)] (Gerasimchuk et al., 2010) and at the Fryer and Kaiko hydrothermal areas in the southern segment of the Mariana Trench (Kato et al., 2009). In the present study, microbial mats were sampled from the Tour Eiffel edifice located on the Lucky Strike (LS) hydrothermal vent field on the MAR (37.29°N; 32.28°W) (Sarradin et al., 1999). The LS vent field spreads around a central lava lake and where both high-temperature, active black smokers (324 °C) and lower-temperature diffuse flow areas (170 °C) are observed. The Eiffel Tower is a 12-m-high sulfide edifice located at 1650–1700 m depth. At this edifice, the vast majority of fluid-exposed hydrothermal deposits and Bathymodiolus azoricus mussels are covered by white filamentous mats whose role is still not understood. Bathymodiolus azoricus mussels dominate the megafauna and form large assemblages in low-temperature flow areas around the central edifice (De Busserolles et al., 2009). Although the environment near mussel communities undergoes a temporal variation, it is characterized by a pH of 5.9–7.3 and sulfide and methane concentrations that allow chemosynthetic activity (De Busserolles et al., 2009; Cuvelier et al., 2011).

Bathymodiolus azoricus mussels harbor both thiotrophic and methanotrophic symbionts within their gills (Duperron et al., 2006). Two species, B. azoricus and Bathymodiolus puteoserpentis, found in the northern MAR (Menez Gwen, LS, Rainbow and Logatchev vent fields), share the same two dominant bacterial symbiont phylotypes that belong to the Gammaproteobacteria (Duperron et al., 2006). These chemosynthetic microorganisms enable the host to colonize sulfide- and/or methane-rich environments. The acquisition pathways of symbionts are not yet fully understood, although some studies provide support for the environmental transmission of thiotrophic symbionts (Won et al., 2003). To date, no free-living forms of the symbionts have ever been described in this environment.

To better understand the function of microbial mats in the LS ecosystem and their possible relationships with other biological communities, we characterized microbial mats using molecular approaches. To do so, both DNA and RNA were extracted from mat samples overlaying hydrothermal deposits or B. azoricus assemblages. 16S rRNA gene-based phylogeny coupled with functional gene analyses can provide a better understanding of the microbial contribution to chemosynthetic ecosystems (Elsaied & Naganuma, 2001; Elsaied et al., 2007). We therefore used universal primers to amplify the 16S rRNA gene from both DNA and RNA extracts, as well as the functional genes implicated in sulfur (aprA, soxB), methane (pmoA) and carbon (cbbM/L, aclB) metabolism. Moreover, primers specific to Bathymodiolus sp. symbionts were used to screen for free-living symbiont-like phylotypes (Duperron et al., 2006).

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

Sampling site and procedure

The LS vent field (1700 m depth) is located at 31°17′N, 32°16′W on the MAR. Microbial mat filaments covering B. azoricus assemblages and hydrothermal deposits were sampled on the eastern and southern sides of the Tour Eiffel edifice during the French oceanographic cruises EXOMAR (2005), MoMARETO (2006) and MoMAR-08 (2008). Mat samples were retrieved from mussel assemblages 1 and 2b and substratum 1b, as defined by Cuvelier (Supporting Information, Table S1) (Cuvelier et al., 2009). Using a water-pumping device on the ROV Victor 6000, samples were collected in 5-L pouches or by scraping mussel shells brought to the surface in previously decontaminated boxes. Before the dive, the tubes of the water-pumping device were washed twice with Desibac HPS (7 d'Armor), rinsed with sterile water, then with alcohol and finally filled with sterile seawater. Once on board (2–5 h after sampling), the contents of the pouches were filtered through 0.2 μm pore-size filters. Both types of samples (filters and scrapings) were transferred into sterile 2 mL Nunc Cryotubes filled with sterile seawater or RNAlater and immediately frozen at −80 °C.

DNA extraction, primer selection and PCR amplification

Total DNA was extracted using the FastDNA® Spin kit for soil (Qbiogene Inc., CA) protocol as modified by Webster and Roussel (Webster et al., 2003; Roussel et al., 2009).

For phylogenetic analyses, amplifications of 16S rRNA genes were performed using universal primers for Bacteria and Archaea: U1492R as reverse primer and E8F for Bacteria or A8F for Archaea as a forward primer (all the primers used in this study are listed in Table 1). Given the chemical composition of the fluids in LS, we focused our study on the main chemoautotrophic metabolisms that may occur within microbial mat communities. The presence of sulfur oxidizers was investigated using two pairs of primers (APS1F/APS4R and Sox432F/Sox1446R) specific to the adenosine-5′-phosphosulfate (APS) reductase α-subunit gene (aprA) and to the soxB component of the multienzyme thiosulfate-oxidizing complex gene (soxB), respectively. soxB and aprA genes constitute efficient functional markers for the two major biochemical pathways of bacterial sulfur oxidation (Meyer & Kuever, 2007a). To study methane oxidation, the primer pair A189F/MB661R was used; it is specific to the particulate methane monooxygenase gene (pmoA), which has been reported in all methanotrophs, except the genus Methylocella (Dedysh et al., 2000). pmoA gene phylogeny is congruent with 16S rRNA gene phylogeny (Bourne et al., 2001); thus, pmoA is the preferred molecular marker to study the diversity of methane oxidizers (Fuse et al., 1998; Shigematsu et al., 1999). For autotrophic carbon fixation, we amplified forms I and II of RuBisCO (d-ribulose-1,5-bisphosphate carboxylase-oxygenase) using, respectively, cbbLF/cbbLR and cbbMF/cbbMR/cbbMR primer combinations, plus the ATP-citrate lyase β-subunit gene with aclB892F/aclBR primers. The Calvin–Benson–Basham (CBB) and the reductive tricarboxylic acid (rTCA) cycles are the two main pathways for carbon dioxide assimilation. CbbL/M are the usual markers for forms I and II RuBisCO (Badger & Bek, 2008); aclB is a gene encoding a key enzyme in the rTCA cycle, which appears to be used by a variety of deep-sea Epsilonproteobacteria (Campbell & Cary, 2004; Takai et al., 2005).

Table 1.   Primers used in the study
DesignationSpecificityPrimer sequence (5′–3′)References
U1492RUniversal 16S rRNA geneGTT-ACC-TTG-TTA-CGA-CTTLane (1991)
A8FArchaeal 16S rRNA geneCGG-TTG-ATC-CTG-CCG-GAKolganova et al. (2002)
907RUniversal 16S rRNA geneCCG-TCA-ATT-CMT-TTG-AGT-TTLane et al. (1985)
Meth138F16S rRNA gene methanotrophic Bathymodiolus sp. symbiontTCT-GCC-TAT-TAG-TGG-GGG-ACA-ACA-TGG-TDuperron et al. (2006)
Sulfo193F16S rRNA gene thiotrophic Bathymodiolus sp. symbiontCTC-TAT-GGA-GTA-AAG-TGG-AGG-ACC-TTC-G 
cbbL_1b FRuBisCO form ICAC-CTG-GAC-CAC-VGT-BTG-GBlazejak et al. (2006)
aclB892FATP-citrate lyase β-subunitTGG-ACM-ATG-GTD-GCY-GGK-GGTCampbell et al. (2003)
aclBRATP-citrate lyase β-subunitATA-GTT-KGG-SCC-ACC-TCT-TC 
APS1FAdenosine-5′-phosphosulfate reductase α-subunitTGG-CAG-ATC-ATG-ATY-MAY-GGMeyer & Kuever (2007b)
soxB432FsoxB component of the periplasmic thiosulfate-oxidizing Sox enzyme complexGAY-GGN-GGN-GAY-CAN-TGGPetri et al. (2001)
A189FParticular pMMoGGN-GAC-TGG-GAC-TTC-TGGHolmes et al. (1995)

Bulk DNA was amplified in a 25 μL reaction mix containing 5 μL of 5 × GoTaq® Flexi DNA polymerase buffer (Promega), 1.5 μL of 25 mM MgCl2 solution, 0.2 μL of 10 mM dNTP solution, 0.1 μL of each primer at 100 pM and 0.12 μL of 5 U μL−1 GoTaq® Flexi DNA polymerase (Promega). Domain-specific 16S rRNA gene primers targeting Bathymodiolus sp. symbionts, which are based on specific FISH probes (Duperron et al., 2007a, b), were tested. For methanotrophic symbionts, Meth138F and Meth845R (5′-GCT-CCG-CCA-CTA-AGC-CTA-TAA-ATA-GAC-C-3′) primers were used. For thiotrophic symbionts, Sulfo193F and Sulfo642R were used.

PCR conditions were the same as those described in the references cited for each primer pair.

RNA extraction and reverse transcription (RT)-PCR amplification

Total RNA was extracted using the FastRNA® Pro Soil Direct kit (Qbiogene Inc.) according to the manufacturer's instructions. To digest trace amounts of DNA, the extraction products were incubated for 1 h at 37 °C with 1 × TURBO DNase® buffer and 18 U of TURBO DNase® (Ambion). The digestion was stopped by adding EDTA to a final concentration of 15 mM and heating for 10 min at 65 °C before a purification step using the RNeasy minikit (Qiagen) following the manufacturer's instructions. The absence of DNA was tested by direct PCR; all were negative.

RNA reverse transcriptions, followed by DNA amplification (RT-PCR) were performed using the OneStep RT-PCR kit (Qiagen). Amplifications were performed using the same primer sets as for PCR amplifications, except to amplify the 16S rRNA gene, where the primers 907R and E8F were used for Bacteria and 915R and A8F were used for Archaea (Table 1).

The PCR conditions were the same as those indicated in the references for each primer pair with an additional reverse-transcription step (30 min at 50 °C) and an initial PCR activation step at 95 °C for 15 min before the standard cycles.

Clone library construction and sequencing

Before cloning, all PCR products were purified using the Qiaquick® Gel Extraction kit (Qiagen) according to the manufacturer's instructions. Clone libraries were constructed by transforming Escherichia coli TOP10F' using the TOPO XL Cloning kit (Invitrogen) according to the manufacturer's instructions. Plasmid extraction, purification and sequencing were carried out by the OUEST-Genopole® sequencing facilities at the Roscoff Biological Station (France). For each gene library, mat samples from different assemblages (1 and 2b) and from substratum 1b were used. As we evidenced no difference between the obtained libraries, they were pooled for phylogenetic analysis.

Phylogenetic analysis

Sequence alignments, edition and analysis were performed using both bioedit 7.0.9 ( and muscle (Edgar, 2004) software. Phylogenetic analyses of the 16S rRNA gene sequences were performed using phylo_win (Galtier et al., 1996) to construct trees based on the neighbor-joining algorithm and Kimura 2-parameter correction. Phylogenetic analyses of the other gene sequences were performed on amino acid-deduced sequences using the same software and algorithm, but with PAM correction. Robustness was tested by bootstrap resampling (1000 bootstrap samples). Sequences displaying more than 97% similarity were clustered within the same operational taxonomic unit (OTU). Rarefaction curves at 97% sequence similarity levels were performed using the dotur program (Schloss & Handelsman, 2005).

Nucleotide sequence accession numbers

For each OTU, one sequence was deposited in the GenBank database under accession numbers FR670348FR670522 for 16S rRNA gene sequences and FR670535FR670580 for functional gene sequences.


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

Macroscopic and microscopic description

At the LS vent field, the microbial mats appeared as attached white filaments covering a large part of the mussel assemblages and fluid-exposed hydrothermal deposits (Fig. 1). Light microscopy observations revealed a wide diversity of morphology, both in size and shape, among microbial populations. We observed various kinds of filaments that resembled filamentous bacterial populations reported at White Point, CA vent fields (Jacq et al., 1989; Kalanetra et al., 2004), in addition to numerous coccoid- and rod-shaped cells. Large, vacuolated filaments (14–65 μm in diameter), containing inclusions that could be sulfur granules, were the dominant morphotype in the mat communities, but thinner filaments (2–6 μm in diameter), probably nonvacuolate, and rosettes or tangles of smaller filaments, were also observed (Fig. 1). Most of the filament morphotypes (including the large dominant ones) hybridize with the GAM42a TRITC-labeled fluorescent probe, specific to the Gammaproteobacteria.


Figure 1.  White filamentous mats on Bathymodiolus azoricus mussels from the LS vent field (top left and right panels). Scale bar=10 cm [IFREMER©, EXOMAR (2005) and Bathyluck (2009) cruises]. Light and differential interference contrast micrographs of large mat filaments found on the LS vent field (middle left and right panels, respectively). Scale bar=50 μm. Whole-cell hybridization of various bacteria with a TRITC-labeled fluorescent probe (GAM42a) Gammaproteobacteria (in green) (bottom left and right panels, respectively). Scale bar=50 μm (same for both pictures).

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Archaeal and bacterial diversity in LS hydrothermal microbial mats

We identified 61 archaea-related sequences from the 16S rRNA gene library. They were distributed in only four OTUs defined at a similarity threshold of 97%. The archaeal 16S RNA transcript library was even less diversified, with only one OTU clustering the 22 sequences obtained.

From the bacterial 16S rRNA gene library, we retrieved 323 bacterial-related sequences. They were distributed in 163 OTUs defined at a similarity threshold of 97% (Table 2). The bacterial RNA transcript library was less diversified, with 76 sequences distributed in only three OTUs.

Table 2.   Analysis of bacterial and archaeal diversity in LS microbial mats
LibraryNumber of clonesNumber of OTUs (with ≥97% similarity)Good's coverage (%)1−HSimpson*
  • *

    Simpson index values are presented as 1−D for better readability, diversity ranges from 0 (one species) to 1 (maximal diversity).

Archaeal transcripts2211000
Bacterial transcripts76397.40.152

The diversity and representativeness of the microbial communities were estimated using rarefaction curves and diversity and coverage indices (Table 2 and Fig. S1). Coverage rates were high and curves reached saturation for the archaeal 16S rRNA gene and transcript libraries and also for the bacterial RNA transcript library. In contrast, for the bacterial 16S rRNA gene library, the coverage rate was only 55% and curves did not reach saturation, indicating that the molecular analysis only retrieved the dominant phyla. Simpson indices confirmed that the bacterial diversity was high, even if the active members of this community seemed limited to a few species.

Archaeal community structure

Archaeal diversity, according to the retrieved 16S rRNA gene sequences, was restricted to the marine archaeal group 1 Thaumarchaeota lineage. Almost all sequences were closely related (99% similarity) to the Nitrosopumilus maritimus 16S rRNA gene sequence and also, but more distantly (97% similarity), to species Candidatus Giganthauma karukerense and Candidatus Giganthauma insulaporcus (Fig. 2). The same N. maritimus-related sequence was also the only OTU identified in the 16S RNA transcript library.


Figure 2.  Neighbor-joining phylogenetic tree showing the relationship of 16S rRNA genes sequences obtained with universal Archaea 16S rRNA gene primers. Bootstrap values >60% (from 1000 bootstrap samples) are indicated near their corresponding nodes. Representative sequences obtained in this study are in bold and all RNA transcript sequences are also underlined. Numbers in parentheses indicate the number of identical clones.

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Bacterial community structure

Analysis of the 323 bacterial 16S rRNA gene sequences indicated that mats were made up of a phylogenetically diverse bacterial population (Table 3 and Figs S2 and S3). Retrieved bacterial sequences were affiliated with seven phyla, but among them, the phyla Proteobacteria and Bacteroidetes were clearly dominant. Within Proteobacteria, various OTUs were related to genera implied in sulfur oxidation such as Roseobacter, Sulfitobacter and Thalassobacter for the Alphaproteobacteria, Beggiatoa and Leucothrix for the Gammaproteobacteria and Sulfurimonas and Sulfovorum for the Epsilonproteobacteria. A few Gammaproteobacteria sequences related to methane oxidizers were also retrieved with affiliations with the genera Methylobacter and Methylomonas (Costello & Lidstrom, 1999; Hirayama et al., 2007); moreover, two sequences were closely affiliated (99.7% similarity) with the B. azoricus methanotrophic symbiont (Duperron et al., 2006). Most of the OTUs observed were affiliated with clones from various chemosynthetic ecosystems including hydrothermal-fauna-associated clones and microbial mats from the Milano mud volcano (Heijs et al., 2005), cold seeps in the Sea of Japan (Arakawa et al., 2006), shallow White Point (CA) hydrothermal vents (Kalanetra et al., 2004) and Okinawa (East China Sea) and Lost City (North Atlantic Ocean) vent fields (Hirayama et al., 2007; Brazelton et al., 2010).

Table 3.   Phylogenetic positioning and abundance of bacterial OTUs in LS microbial mats based on the 16S gene
Phylogenetic groupNumber of related clonesNumber of OTUs (≥97% similarity)% of OTUs
 Other α clones2  
 B. azoricus methanotrophic symbiont2  
 Other γ clones51  
 Other δ clones5  
 Group F65  
Total323 (100%)163 (100%)100

Analysis of the 16S RNA transcript library indicated that the active bacterial community was restricted to three OTUs. Almost all sequences were closely affiliated (99% similarity) with the 16S rRNA gene sequence of a filamentous Beggiatoa from White Point, also retrieved by direct PCR (Table 4 and Fig. S2). This vacuolated, filamentous, sulfur-oxidizing bacterium has been described as attached to diverse biotic and abiotic substrates in the shallow White Point hydrothermal vents (Kalanetra et al., 2004). The other two sequences were affiliated, respectively, with an uncultivated Gammaproteobacteria Methylococcales from the Lost City vent field (Brazelton et al., 2006) and with an Alphaproteobacteria from the EPR (Santelli et al., 2008).

Table 4.   Phylogenetic positioning and abundance of bacterial clones sequenced from the 16S RNA transcript library
Clone designationNumber of clonesPhylogenetic groupNearest relative (NCBI GenBank)Similarity (%)
  • *

    Partial sequence (200 bp).

LSmat_RNA.B0174Gammaproteobacteria ThiotrichalesFilamentous bacterium from White Point (AY496953)99
 Gammaproteobacteria ThiotrichalesFilamentous bacterium from Escanaba Trough (AY883934)99
LSmat_RNA.B021Gammaproteobacteria (Methylococcales)Kazan mud volcano clone (FJ712402)97
  Methylomonas sp. (AB01603)93
LSmat_RNA.B03*1Alphaproteobacteria (Rhizobiales)Bacterium clone EPR3968-O8a-Bc54 (EU491721)95

Presence of Bathymodiolus symbiont 16S DNA and RNA

Two sequences from our 16S DNA library were closely affiliated (>99% similarity) with the monophyletic methanotrophic B. azoricus and B. puteoserpentis symbiont (Gammaproteobacteria) from the MAR, but none were affiliated to the thiotrophic symbiont. To confirm this result and explore the possibility of a free-living mussel symbiont, we used new 16S rRNA gene primers specific to Bathymodiolus sp. methanotrophic or thiotrophic symbionts (Duperron et al., 2006).

Eleven 16S DNA clone sequences and 10 RNA clone sequences were retrieved with the Meth138F/845R primer pair targeted to methanotrophic symbionts. All sequences were identical to each other and closely matched (>99% similarity) the monophyletic methanotrophic B. azoricus and B. puteoserpentis symbiont sequence from the MAR (Duperron et al., 2006) (Fig. 3a).


Figure 3.  Neighbor-joining phylogenetic tree showing the relationship of 16S rRNA gene sequences obtained with domain-specific primers targeted to Bathymodiolus sp. symbionts in PCR and RT-PCR (a). Neighbor-joining phylogenetic tree constructed using PAM correction of the amino acid sequences deduced from a fragment of the gene encoding for the particulate methane monooxygenase gene (LSmat.pmoA#) and for the APS reductase α-subunit gene (LSmat.aprA#) from LS mats (b and c, respectively). Bootstrap values >60% (from 1000 bootstrap samples) are indicated near their corresponding nodes. Representative sequences obtained in this study are shown in bold and all RNA transcript sequences are also underlined. Numbers in parentheses indicate the number of identical clones.

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Twelve 16S DNA clone sequences and 10 RNA clone sequences were retrieved with the Sulfo193F/642R primer pair targeted to thiotrophic symbionts. Three OTUs were detected in both gene DNA and RNA transcript sequences; two of them were related (93.7% and 100% similarity) to B. azoricus thioautotrophic symbionts from LS (Duperron et al., 2006; Won et al., 2008) (Fig. 3a).

Characterization of the chemolithoautotrophic populations

Six DNA and four RNA transcript clone libraries targeting functional genes were constructed. Although we failed to amplify transcripts using Sox432F/Sox1446R and aclB892F/aclBR primer pairs in RT-PCR, successful PCR amplifications indicated at least the presence of microorganisms having soxB and aclB genes in the community.

Sulfur oxidation

Forty clones from the DNA library and 16 clones from the RNA library were retrieved using APS (aprA) primers (Fig. 4c). Clone sequences were distributed between sulfur-oxidizing bacteria lineages I and II, and were all affiliated with sequences related to Gammaproteobacteria. Most transcript and gene sequences were related to a Siboglinidae tubeworm symbiont from eastern Mediterranean cold seeps. One transcript sequence related to a bacterial endosymbiont of the bivalve Idas sp. (Duperron et al., 2008) was also identified. Among the gene sequences, some were related to clones associated with symbionts of tubeworms such as Sclerolinum contortum and Oligobrachia haakonmosbiensis from Håkon Mosby mud volcano (Lösekann et al., 2008) and others with symbionts of species such as Asterechinus elegans, an echinoderm associated with wood falls (Becker et al., 2009), or Kiwa hirsuta, a galatheid collected from hydrothermal vents in the Pacific-Antarctic Ridge (Goffredi et al., 2008).


Figure 4.  Neighbor-joining phylogenetic tree constructed using PAM correction of amino acid sequences deduced from a fragment of the gene encoding for an element of the periplasmic thiosulfate-oxidizing Sox enzyme complex (LSmat.soxB#), the form I and II RuBisCO (LSmat.cbbL/M#) and the β-subunit of ATP-citrate lyase (LSmat.aclB#) (a,b, c and d, respectively). Bootstrap values >60% (from 1000 bootstrap samples) are indicated near their corresponding nodes. Representative sequences obtained in this study are in bold and all RNA transcript sequences are also underlined. Numbers in parentheses indicate the number of identical clones.

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We retrieved 31 gene clones using soxB primers, but no transcripts were amplified (Fig. 4a). All retrieved sequences were related to Alphaproteobacteria and Gammaproteobacteria sulfur oxidizers (Mukhopadhyaya et al., 2000; Meyer et al., 2007).

Methane oxidation

We obtained 33 clones of particulate methane monooxygenase (pmoA) genes and 15 transcript sequences using pmoA primers (Fig. 3b). This transcript library confirmed the presence of active methane oxidizers, all affiliated with Gammaproteobacteria in the microbial mat community. They were related to uncultured bacteria from Pacific Northwest marine sediments (Nold et al., 2000) or the Rainbow vent field (MAR) (Nercessian et al., 2005) and to Bathymodiolus childressi symbionts (Duperron et al., 2007a, b). In addition, sequences related to B. azoricus symbionts, Methylococcales Methylohalobius crimeensis (Heyer et al., 2005), Idas sp. symbionts (Duperron et al., 2008) and uncultured bacteria from methane seep habitats at the Kuroshima Knoll in the southern Ryukyu Arc (Inagaki et al., 2004b) were identified in the pmoA gene library.

Carbon fixation

We retrieved 54 form I RuBisCO (cbbL) genes and 12 transcript sequences, all related to Gammaproteobacteria (Fig. 4b). Several clones from both libraries were affiliated with a Lucinoma aff. kazani endosymbiont (Duperron et al., 2007a, b) and Solemya velum gill sulfur-oxidizing symbionts (Schwedock et al., 2004). In the cbbL gene library, sequences distantly affiliated with Chromatiales Alkalilimnicola ehrlichii MLHE-1 and Halothiobacillus neapolitanus (Baker et al., 1998) were also identified.

We retrieved 38 form II RuBisCO (cbbM) genes and 12 transcript sequences (Fig. 4c). Most were related to Gammaproteobacteria. Sequences related to a Thiobacillus sp. Lamellibrachia symbiont (Elsaied & Naganuma, 2001) and to a vestimentiferan tubeworm symbiont (Vrijenhoek et al., 2007) were identified both in gene and in transcript libraries. In the gene library, sequences related to tubeworm symbionts of Lamellibrachia from the Håkon Mosby mud volcano (Lösekann et al., 2008) were also retrieved. Additionally, sequences related to the nitrogen-fixing Alphaproteobacteria Rhodobacter capsulatus (Larimer et al., 1995) were identified, as well as sequences related to organisms from diverse marine environments.

Using aclB primers, 28 ATP-citrate lyase B gene sequences were obtained, but we failed to construct a transcript library (Fig. 4d). All aclB gene sequences were affiliated with Epsilonproteobacteria, including relatives of the genera Sulfurimonas and Arcobacter (Campbell et al., 2003; Takai et al., 2005) and hydrothermal invertebrate epibionts associated with K. hirsuta (Goffredi et al., 2008) and Alvinella pompejana (Campbell et al., 2003).


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

Limits of the molecular approaches

Phylogenetic analysis of 16S rRNA gene sequences from the LS microbial mats revealed the presence of a highly diversified bacterial community, but a relatively low diversity of archaeal taxa. Because of low Good's coverage values (55.1%, Table 2) for the bacterial 16S rRNA gene sequence library, and due to biases associated with PCR, DNA/RNA extraction and cloning (Wintzingerode et al., 1997), the abundance of a particular OTU in a library does not necessarily reflect its real abundance in the original sample (Teske & Sorensen, 2008). Furthermore, free-living microorganisms from the surrounding environment not directly involved in mat structure and activity may also have been trapped in the mat and thereby contribute to an inaccurate picture of microbial diversity.

In addition, our results underline the need for specific primers to amplify 16S rRNA genes of poorly represented species or specific groups, which cannot be retrieved with universal primers. For example, we noted that although we identified some Planctomycetes-related sequences, none of them were affiliated with anaerobic ammonium oxidation (anammox) clones already retrieved from the same mat samples analyzed in a previous study (Byrne et al., 2008). In this study, the use of domain-specific primers revealed 16S thiotrophic symbiont signatures, although universal 16S rRNA gene primers failed to detect them.

mRNA has a short intracellular lifetime and therefore directly represents active metabolic processes. mRNA molecules are therefore more suitable as microbial activity indicators than 16S rRNA gene molecules, which can persist for up to a few days in the environment (Chin et al., 2008; Lloyd et al., 2010). In this study, both approaches were combined. In our RNA studies, we attempted to minimize as much as possible the lapse of time between sampling and on-board treatment (2–5 h) to avoid the rapid degradation of mRNA.

Finally, it should be noted that phylogenetic assignment and the metabolic role of bacterial communities were difficult to determine due to the presence of numerous bacterial 16S rRNA gene sequences that had sequence similarities of <94% with their closest cultured relatives. Many sequences retrieved in this study may therefore represent new, undiscovered genera or families. Conferring a possible phenotype to the organisms detected in this study must therefore be carried out with caution. This is particularly crucial for the phylogenetic groups that contain phenotypically diverse organisms, such as the Proteobacteria. The analysis of functional gene libraries is even more complicated due to the lack of data and the over-representation of symbiont-related sequences in gene banks.

Prevalence of sulfur oxidizers among microbial populations

Low, but constant levels of sulfides surrounding the mat samples (0.8–2 μM) and the presence of oxygen (230–240 μM) (Sarradin et al., 2009; Cuvelier et al., 2011) make the mats a good habitat for sulfur-oxidizing populations (Fig. 5). The highest number of clone sequences retrieved in our 16S rRNA gene bacterial library (Table 3) was affiliated with the Gammaproteobacteria. Most of them were closely related to environmental sequences identified from deep-sea sediments and some clustered within groups containing sulfur-/methane-oxidizing isolates and gill symbionts. Functional gene libraries were also dominated by clone sequences affiliated with similar populations: the nine OTUs detected using primers based on the aprA gene all belonged to the Gammaproteobacteria symbiont cluster (Meyer & Kuever, 2007a, b) (Fig. 3c). Likewise, among the eight OTUs detected with the soxB gene, three were related to Gammaproteobacteria sulfur oxidizers (Fig. 4a). In addition, form I and II RuBisCO gene sequences confirmed the presence of chemoautotrophs belonging to this group (Fig. 4b and c).


Figure 5.  Detected metabolisms occurring in LS microbial mats and hypothetical relationships with mussels Bathymodiolus azoricus. Main orders or genera for each metabolism are indicated in parentheses. Groups detected as active in RT-PCR are underlined. Data on Planctomycetes anammox are from a previous study (Byrne et al., 2008).

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However, despite this high taxonomic diversity, the 16S rRNA gene transcript library was dominated by sequences affiliated with filamentous Beggiatoa species. This may indicate that this genus dominates active microbial communities. However, because it was not possible to construct a soxB transcript library, we cannot conclude as to Beggiatoa sulfur-oxidizing activity. Furthermore, this pathway may be present, but not active at the time of sampling or its mRNA may be more sensitive than others, degrading more rapidly when the samples were recovered. On the contrary, in the analysis of the transcript library, the use of aprA gene primers detected three OTUs belonging to the Gammaproteobacteria symbiont cluster that includes active sulfur oxidizers (Fig. 3c). Form I and II RuBisCO gene transcript libraries also indicated active carbon fixation for three OTUs related to potential Gammaproteobacteria symbionts (Fig. 4b and c). No transcripts belonging to B. azoricus thiotrophic symbionts were found in the aprA gene library despite the symbionts' presence in both 16S rRNA gene libraries (RNA and DNA) constructed with symbiont-specific primers. This result can be attributed to either a low symbiont cell concentration in the sample or to the absence of sulfur-oxidizing activity at the time of sampling. Alternatively, the symbionts may be in a dormant form, or may have a different metabolism, before they are trapped in the gill cells of Bathymodiolus sp.

Oxidation of reduced sulfur compounds as a possible prevailing microbial metabolism in the mats was also consistent with the finding of sequences clustering among the Epsilonproteobacteria, Alphaproteobacteria and Bacteroidetes groups in the 16S rRNA gene library (Table 3, Figs S2 and S3). Several recent molecular studies have demonstrated the presence and the dominance of the Epsilonproteobacteria as both free-living organisms or in association with metazoans at deep-sea hydrothermal vents (Corre et al., 2001; Campbell et al., 2006; Zbinden et al., 2008). Many sequences from the 16S rRNA gene library were affiliated with Epsilonproteobacteria, including OTUs previously identified from vent epibionts and environmental sequences from deep-sea cold seeps and hydrothermal vents (Corre et al., 2001). The phylogenetic analysis of the rTCA gene library also revealed OTUs related to known Epsilonproteobacteria. Surprisingly, no sequences in the soxB clone library matched known Epsilonproteobacteria sequences, although sulfur oxidizers of this group are known to use the SOX pathway and were well represented in the aclB gene library. This may indicate PCR biases or the predominance of Gammaproteobacteria in sulfur-oxidizer populations. Indeed, despite the fact that the Epsilonproteobacteria are known for their metabolic and thermal versatility, they appear to be well adapted to microaerobic or anaerobic conditions and also to high sulfide concentrations (Engel et al., 2003; Inagaki et al., 2004a; Campbell et al., 2006; Takai et al., 2006). The low sulfide (0.8–2 μM) and high oxygen (230–240 μM) concentrations measured in the mats might actually inhibit Epsilonproteobacteria and generally favor Gammaproteobacteria. In addition, it is possible that the Epsilonproteobacteria are active below the seafloor, where conditions are more suitable, and become inactive when washed out with the hydrothermal fluids and being exposed to microbial mat conditions.

The Alphaproteobacteria and the Bacteroidetes groups are ubiquitous in diverse marine environments, including surface and deep waters, sediments (Lopez-Garcia et al., 2001; Kirchman, 2002) and deep-sea hydrothermal vents (Reysenbach et al., 2000; Alain et al., 2002; Lopez-Garcia et al., 2002; Huber et al., 2003). The presence of sequences belonging to Alphaproteobacteria in the 16S rRNA gene library as well as in the soxB and form II RuBisCO gene libraries (Fig. 4a and c) seems to demonstrate its importance in LS mats, even if no transcript sequences were obtained. The Bacteroidetes bacteria are usually aerobic chemoorganotrophic or lithotrophic microorganisms and exhibit diverse metabolic capabilities (Kirchman, 2002) such as oxidation of reduced sulfur compounds, although they do not depend on this reaction for growth (Teske et al., 2000).

Presence of active methane oxidizers

Methane concentrations in LS smokers were 0.68 mM and 0.5–40 μM in the mussel assemblages (Table S1). Methane oxidation-based metabolism is thus a possible pathway utilized by species in our mat samples (Fig. 5). Few sequences in the 16S rRNA gene library were affiliated with the orders Methylobacterales and Methylococcales (Gammaproteobacteria) (Fig. S2). In addition, one sequence in the 16S rRNA gene transcript library was related to a methanotrophic uncultured clone, perhaps representative of aerobic methane-oxidizing activity. The analysis of the pmoA gene-based libraries provides support for this interpretation, as eight OTUs, including three in RNA transcript library, were detected (Fig. 3b). There are therefore potential active methane oxidizers in our mats such as Gammaproteobacteria, including Methylococcales. The presence (but not the activity) of methanotrophic B. azoricus symbionts among the mat microbial community is also confirmed. As surmised above for the thiotrophic symbiont, this may indicate that the symbiont-like microorganisms are not active in their free-living state and may be in a dormant form.

Other metabolic pathways in LS mats

Apart from methane and sulfur oxidizers, the geochemistry of the vent fluids at LS provides favorable conditions for other chemoautotrophic metabolisms based on ammonium, hydrogen, metals, etc. (Salerno et al., 2005; Duperron et al., 2007a, b; Sarradin et al., 2009). Our study did not focus on these pathways, but analysis of the 16S rRNA gene libraries provided some information (Fig. 5). A phylotype related to N. maritimus dominated the archaeal 16S clones and constituted probably the main active Archaea present in the collected mats (Fig. 2). These ubiquitous, low-temperature Thaumarchaeota grow chemolithoautotrophically via the aerobic oxidation of ammonium to nitrite (Konneke et al., 2005). Their presence in LS microbial mats, as the presence of other marine archaeal group 1-related sequences, may reflect aerobic ammonium-oxidizing activity (Walker et al., 2010), which is consistent with the ammonia concentrations observed in the LS hydrothermal fluid (i.e. 8–10 μmol L−1) and in the mussel bed environment (<2 μmol L−1) (Sarradin et al., 1999). Nevertheless, the recent discovery of giant Thaumarchaeota species (Muller et al., 2010) with 16S rRNA gene sequences closely related to N. maritimus, but morphologically and metabolically different (as they do not seem to have AmoA genes), makes it difficult to conclude that the Thaumarchaeota detected in this study are ammonia oxidizers at all.

Heterotrophs also seemed to be present, with a significant number of OTUs affiliated with phylum Bacteroidetes in the 16S rRNA gene library (Table 3 and Fig. S3). Their ability to degrade diverse organic molecules also explains the diversity of this group in rich organic material systems such as microbial mats (Kirchman, 2002; Stevens et al., 2005).

Other sequences in this library were related to members of the Deltaproteobacteria with species affiliated with Myxococcales and Desulfobacterales (Table 3 and Fig. S2). Myxococcales including various aerobic heterotrophs and Desulfurobacterales have been described as being able to reduce sulfate (Rabus et al., 2006). However, despite the fact that APS reductase is used in both the reductive and the oxidative pathways of sulfur metabolism, we did not identify any sequence affiliated with sulfate reducers (Fig. 3c). However, we cannot reject the possibility of anaerobic microenvironments within the mats where sulfate reducers could be active. For example, form II RuBisCO is only functional under anaerobic conditions (Haygood, 1996; Elsaied et al., 2007) and we detected substantial taxonomic diversity for this gene. Biases in PCR and the possibility that environmental free-living microorganisms – some of them being anaerobic – may aggregate in mats may also partially explain these results.

Identification of the filamentous bacteria and possible interactions with mussels

While our data demonstrate the existence of a highly diverse microbial community within microbial mats, the combination of light micrograph observations (Fig. 1) and molecular results indicates the dominance of a large filamentous phylotype, probably involved in the sulfur cycle. The 16S rRNA gene transcript sequences were affiliated with those of a large marine, sulfur-oxidizing Beggiatoa species (order Thiotrichales, class Gammaproteobacteria) isolated in the shallow White Point hydrothermal system, confirming the morphological observations (Jacq et al., 1989; Kalanetra et al., 2004). Although these sequences dominated our RNA transcript library, only a few copies were present in our gene library, suggesting possible biases in lysis efficiency, low 16S rRNA gene copy number, difficulties in PCR amplification and other factors already mentioned in the literature (Heijs et al., 2005). Beggiatoa-like filaments may also have proportionally more RNA compared with other cells.

It is difficult to affiliate the other observed morphologies (cells 2–6 μm in diameter or smaller filaments) with clone sequences. However, preliminary FISH results (Fig. 1) showed that they mostly belonged to Gammaproteobacteria. In addition, the presence of one clone closely affiliated (99%) with an uncultured Leucothrix sp. in our 16S rRNA gene library suggested that at least another member of the order Thiotrichales may be present (Fig. S2).

The relationships between mussels and mats are still poorly understood and studied, but the results obtained here lend themselves to several hypotheses. It has been demonstrated previously that dense clusters of B. thermophilus can disperse the hydrothermal fluids laterally over distances of several meters. This dispersion increases the redox transition zone area, where both dissolved oxygen and hydrogen sulfide are available. As a result, faunal communities can occupy areas that would not otherwise provide adequate reduced substrates (Johnson et al., 1994). Large filamentous bacteria composing mats may benefit from this extended redox transition zone for growth. The relationship does not seem mutual because only scarce bacterial populations have been observed in B. azoricus digestive tracts (V. Crépeau, unpublished data), suggesting low bacterial consumption. Competition for hydrogen sulfide and methane or a detoxication role does not seem plausible either. Healthy mussel assemblages covered by microbial mats coexist with mussel assemblages free of microbial mats, and to date, there is no significant physiological or toxicological evidence that the associated sulfur-oxidizing filamentous bacteria affect the mussels in the LS area (Cuvelier et al., 2009; Martins et al., 2009). A commensal relationship is the most convincing scenario: sulfur and methane oxidizers benefit from fluid dispersion by mussels and numerous heterotrophs such as Bacteroidetes can degrade the organic material that is released.

Are mats implicated in symbiont transmission?

Two sequences in our 16S DNA library and one in our pmoA DNA library were affiliated with genes from B. azoricus methanotrophic symbionts. Moreover, using domain-specific primers, we obtained clone sequences closely associated (>99.5%) with both thiotrophic and methanotrophic symbionts of B. azoricus (Fig. 3a and b). In addition, the presence of 16S RNA transcript clones indicated that, in addition to the presence of these symbionts, they may be metabolically active. However, the absence of sequences related to methane or sulfur oxidizers from B. azoricus symbionts in the pmoA and aprA transcript libraries may indicate that even if free-living symbionts are present in the environment, their methane- or sulfur-oxidation activity only occurs inside the mussel bacteriocytes.

Mats may be involved in the symbiont transmission process. Their structure and localization in diffuse-flow areas offer a favorable environment for maintaining both B. azoricus symbionts. This is consistent with an optimal larval strategy involving the local retention of larvae within environments where symbionts can thrive and contribute to larval and or postlarval infection and then promote host growth (Van Dover et al., 2001). Mats may help mussels retain their symbionts and postlarvae and may therefore participate in the maintenance of mussel assemblages. Hence, our work supports the assumption of the environmental acquisition of thiotrophic endosymbionts by vent mussels from the MAR (Won et al., 2003) and may indicate a similar transmission for methanotrophic symbionts.

Taken together, our results suggest that there may be mutualisms between mats and Bathymodiolus mussel patches, whereby the mats facilitate larval and postlarval infection, as well as larval settlement. In return, Bathymodiolus sp. may furnish organic matter, ammonium and promote fluid diffusion, resulting in a long-term association.


  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 captain and the crew of the R/V L'Atalante and Pourquoi Pas?, chief scientists and ROV Victor 6000 team during EXOMAR, MoMARETO and MoMAR-08 cruises. We gratefully acknowledge Isabelle Boutet and Sébastien Duperron for providing 16S rRNA gene primers specific to Bathymodiolus sp. symbionts. We also thank Daniel Prieur and Erwan Roussel for their suggestions and advice. This work was conducted with the financial support of the ANR Deep Oases and the GDR ECCHIS.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  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. Rarefaction curves of the diversity determined for the bacterial and archaeal 16S rRNA gene and transcript libraries defined at 97% similarity.

Fig. S2. Neighbor-joining phylogenetic tree showing the relationship of 16S rRNA gene sequences from Proteobacteria obtained with universal 16S rRNA gene primers specific to Bacteria.

Fig. S3. Neighbor-joining phylogenetic tree showing the relationship of 16S rRNA genes sequences obtained with universal 16S rRNA gene primers specific to Bacteria.

Table S1. Mean value of temperature (T), sulfur total concentrations (σS), methane concentrations (CH4), and pH and their SDs measured or estimated on different habitats identified at the Eiffel Tower hydrothermal edifice: assemblage 1: dense Bathymodiolus azoricus beds (the mussels are of the large size class, in general >4 cm), occasionally patches of microbial mats are present; assemblage 2b: Bathymodiolus azoricus clumps (in this assemblage, the mussels are almost always <4 cm in length) separated by visible microbial mats; substratum 1b has bare dark brownish, sometimes slightly reddish, surfaces with visible microbial mats.

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FEM_1070_sm_suppl-mat.doc4936KSupporting info item

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