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Applied and Environmental Microbiology, September 2005, p. 5440-5450, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5440-5450.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Chemoautotrophic Endosymbiosis between a Member of the Epsilonproteobacteria and the Hydrothermal-Vent Gastropod Alviniconcha aff. hessleri (Gastropoda: Provannidae) from the Indian Ocean

Yohey Suzuki,^1* Takenori Sasaki,² Masae Suzuki,¹ Yuichi Nogi,¹ Tetsuya Miwa,¹ Ken Takai,¹ Kenneth H. Nealson,^1,3 and Koki Horikoshi¹

Frontier Research System for Extremophiles, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan,¹ Department of Historical Geology and Paleontology, The University Museum, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,² Department of Earth Sciences, University of Southern California, 3651 Trousdale Parkway, Los Angeles, California 90089-0740³

Received 8 December 2004/ Accepted 21 March 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The hydrothermal-vent gastropod Alviniconcha aff. hessleri from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage of the epsilon subdivision of the Proteobacteria. Fluorescence in situ hybridization analysis with an oligonucleotide probe targeting the specific epsilonproteobacterial subgroup showed the bacterium densely colonizing the gill filaments. Carbon isotopic homogeneity among the gastropod tissue parts, regardless of the abundance of the endosymbiont cells, suggests that the carbon isotopic composition of the endosymbiont biomass is approximately the same as that of the gastropod. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all ¹³C enriched relative to the gastropod biomass and that the monounsaturated C₁₆ fatty acid that originates from the endosymbiont is as ¹³C enriched relative to the gastropod biomass as that of the epsilonproteobacterial cultures grown under chemoautotrophic conditions. This fractionation pattern is most likely due to chemoautotrophy based on the reductive tricarboxylic-acid (rTCA) cycle and subsequent fatty acid biosynthesis from ¹³C-enriched acetyl coenzyme A. Enzymatic characterization revealed evident activity of several key enzymes of the rTCA cycle, as well as the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the gill tissue. The results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses all support the inference that a novel nutritional strategy relying on chemoautotrophy in the epsilonproteobacterial endosymbiont is utilized by the hydrothermal-vent gastropod from the Indian Ocean. The discrepancies between the data of the present study and those of previous ones for Alviniconcha gastropods from the Pacific Ocean imply that at least two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host animal type.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nutritionally mutualistic endosymbioses between marine invertebrates and chemoautotrophic sulfur-oxidizing bacteria (thioautotrophs) are widespread in natural habitats ranging from shallow-water sulfide-rich environments such as mangrove swamps, rotting whale carcasses, and sewage outfalls to deep-sea hydrothermal vents and seeps (9, 12). Such symbioses occur in diverse animal phyla (50). In sharp contrast, all known thioautotrophic endosymbionts belong to the gamma subdivision of the Proteobacteria (-Proteobacteria), despite of the phylogenetically diverse occurrence of thioautotrophic metabolism in prokaryotes.

Although few members of the - and -Proteobacteria occur inside host organisms, together with -proteobacterial endosymbionts (10, 34), a primary endosymbiont not affiliated to the -Proteobacteria has been unknown in marine invertebrates thus far. Some members within a subgroup of the -Proteobacteria, referred to as -proteobacterial group F (28), form intimate associations with marine invertebrates externally (18, 37). Some free-living members of group F have been isolated and characterized recently as chemolithoautotrophs using hydrogen and/or reduced sulfur compounds as electron donor(s) (20, 35, 46). It is now recognized, based on molecular ecological surveys, that members of -proteobacterial group F, as well as of -proteobacterial group B, represent the most predominant bacterial components and the potential primary producers in the low-temperature habitats of global deep-sea hydrothermal-vent environments (28, 29, 37, 43, 48). Considering the higher abundance of -proteobacterial thioautotrophy relative to -proteobacterial thioautotrophy in these habitats, deep-sea hydrothermal-vent animals are widely expected to have the potential to adopt a nutritional strategy based on -proteobacterial endosymbionts. However, such a symbiosis has not yet been described.

Alviniconcha gastropods belonging to the family Provannidae inhabit deep-sea hydrothermal fields in the margins of the Western and Southwestern Pacific Ocean and on the Central Indian Ridge (54). It has been previously shown that Alviniconcha hessleri from the Mariana Trough harbors a thioautotrophic endosymbiont in its gill (42) and that the endosymbiont utilizes the Calvin-Benson cycle and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for carbon-dioxide fixation (42). The gastropod biomass is highly ¹³C depleted relative to the CO₂ emitted from the hydrothermal vents in the same field (13, 52). Unlike A. hessleri from the Western Pacific, the carbon isotopic composition of A. aff. hessleri from the Central Indian Ridge is nearly equal to that of hydrothermal CO₂ (14, 45, 51). This isotopic difference suggests that the carbon metabolism of the bacterial endosymbionts in the two hosts may be different. The reductive tricarboxylic acid (rTCA) cycle, which is one of the alternative carbon-fixation pathways to the Calvin-Benson cycle, typically results in relatively small isotopic fractionation during the conversion of CO₂ into biomass (23, 49). In addition, recent genomic analysis has revealed that some members of -proteobacterial group F, which are the epibionts of the polychete Alvinella pompejana, potentially mediate the rTCA cycle for CO₂ fixation (4). Thus, it seems possible that A. aff. hessleri from the Indian Ocean forms an endosymbiotic relationship with a member of -proteobacterial group F, which fixes CO₂ through the rTCA cycle for host nutrition.

In the present study, we conducted anatomic, molecular phylogenetic, fatty acid profile, bulk and compound-specific carbon isotopic, and enzymatic analyses in order to (i) reveal the occurrence, distribution, and identity of the bacterial endosymbiont in A. aff. hessleri from the Indian Ocean and (ii) determine the carbon metabolism of the bacterial endosymbiont and the nature of the gastropod/bacterial endosymbiosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gastropod specimens and anatomy.
A. aff. hessleri was collected in February 2001 at the base of black-smoker complexes in the Kairei field on Hakuho Knoll in the Central Indian Ridge, the Indian Ocean (25°19.24'S, 70°02.40'E), at a depth of 2,420 m, utilizing the manned submersible Shinkai 6500 (JAMSTEC Scientific Cruises YK01-15). A map showing the location of the sampling site is available elsewhere (53). The in situ temperature of the deep seawater around the gastropod habitat was 2 to 4°C. For the anatomic examination, the specimens were immediately fixed with 10% formalin in surface seawater filtered through a membrane filter with a pore size of 0.2 µm and stored in 70% ethanol at room temperature.

The animal was observed and dissected into various organ systems under a binocular microscope. The gill filaments were removed from the mantle and observed with a scanning electron microscope (Hitachi S-2400) upon coating with platinum vanadium. The samples were dehydrated with a graded series of ethanol, transferred into t-butyl alcohol, and dried with a freeze dryer (Hitachi ES-2030).

DNA analysis.
Genomic DNA was extracted from the dissected gill tissues by using a Soil DNA Kit (Mo Bio Laboratories, Inc., Solana Beach, CA) and magnetically purified by using a MagExtractor Kit (TOYOBO, Osaka, Japan) in accordance with the manufacturer's instructions. The 16S rRNA gene sequences were amplified through the PCR using LA Taq polymerase (TaKaRa, Tokyo, Japan) with the oligonucleotide primers Bac27F and Uni1492R (26). Thermal cycling was performed by using a GeneAmp 9700 Thermal Cycler, with 30 cycles of denaturation at 96°C for 20 s, annealing at 53°C for 45 s, and extension at 72°C for 120 s. The amplified 16S rRNA gene sequence products were either cloned or sequenced directly with an ABI 3100 capillary sequencer and a dRhodamine sequencing kit according to the manufacturer's recommendations (Perkin-Elmer/Applied Biosystems, Foster City, CA). Bacterial clone libraries were constructed by using the Original TA Cloning Kit (Invitrogen, Carlsbad, CA). The sequence similarity among all of the partial sequences, which were 500 nucleotides long, was analyzed by using the FASTA program equipped with the DNASIS software (Hitachi Software, Tokyo, Japan). A single phylogenetic clone type (phylotype) was obtained from the clone type analysis, and the partial sequence was extended and manually aligned according to the secondary structures by using ARB (a software environment for sequence data [31]). Evolutionary analysis was performed by the distance, parsimony, and maximum-likelihood methods using PAUP (44) based on 1,351 nucleotide positions (positions 34 to 1465 [Escherichia coli numbering]), which have >80% homology across the -proteobacterial sequences.

The accession numbers for the bacterial 16S rRNA gene sequences from the gill tissue and the three -proteobacterial isolates are available at DDBJ under the accession numbers AB205405, AB091292, AB175500, and AB091295.

Fluorescence in situ hybridization (FISH) analysis.
Using ARB (31), an rRNA-targeted oligonucleotide probe for a single dominant phylotype from the gill filaments was designed. The probe, hereafter referred to as EPF93, is 17 bases long, which corresponds to E. coli positions 93 to 109 (5'-TCCGCCACTTAGCTGAC-3'). The specificity of the probe was checked in part by using the Gapped-BLAST search algorithm (2) and by check probe analysis from the RDP-II project (5).

For whole-cell hybridization, dissected gill filaments from three individuals were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 2 h and dehydrated in an ethanol series (50, 75, and 100% [vol/vol]), followed by three washes in xylene and infiltration with paraffin wax. The wax-embedded specimens were then sectioned (thickness, 3 µm) and mounted on 3-aminopropyltriethyloxysilane (APTS)-coated slides. For the deparaffinized specimens, hybridization was conducted at 46°C in a solution containing 20 mM Tris-HCl (pH 7.4), 0.9 M NaCl, 0.1% sodium dodecyl sulfate, 30% (vol/vol) formamide, and 50 ng of the EPF93 probe and the "universal" bacterial probe EUB338 (16)/µl, which were labeled at the 5' end with Cy-3 and fluorescein, respectively. After hybridization, the slide was washed at 48°C in a solution lacking the probe and formamide at the same stringency, adjusted by NaCl concentration (27), and subsequently stained with DAPI (4',6'-diamidino-2-phenylindole) at 0.4 µg/ml. The slides were examined by using either an Olympus BX51 microscope or an Olympus FV5000 confocal laser-scanning microscope. A negative control probe for EPF93, in which one mismatch was introduced in the middle (5'-TCCGCCTCTTAGCTGAC-3'), was used for testing unspecific labeling.

-Proteobacterial isolates and cultivation.
Three strictly chemoautotrophic members of the -Proteobacteria used in the present study were previously isolated from the Iheya North deep-sea hydrothermal field in the Mid-Okinawa Trough, Western Pacific Ocean (20, 35, 46). The carbon isotopic fractionations for the cultures grown under chemoautotrophic conditions between the inorganic carbon source and the total biomass and between the total biomass and fatty acids were measured. One strain was found to fall into -proteobacterial group B and is referred to as "Sulfurimonas paralvinella" strain GO25 (8, 46). Nitratifractor salsuginis strain E9I37-1 (35, 46) and Sulfurovum lithotrophicum strain 42BKT (20) are affiliated with -proteobacterial group F (8).

For the cultivation of the strains E9I37-1 and GO25, MJ synthetic seawater (3), which is composed of 30.0 g of NaCl, 0.14 g of CaCl₂ · 2H₂O, 3.40 g of MgSO₄ · 7H₂O, 4.18 g of MgCl₂ · 6H₂O, 0.14 g of K₂HPO₄, 0.33 g of KCl, 0.25 g of NH₄Cl, 0.5 mg of NiCl₂ · 6H₂O, 0.5 mg of Na₂SeO₃ · 5H₂O, 0.01 g of FeSO₄, and 10 ml of trace mineral solution (3) per liter, was supplemented with 3% (wt/vol) elemental sulfur, 0.1% (wt/vol) Na₂S₂O₃ · 5H₂O, 0.1% (wt/vol) NaNO₃, and a 0.1% (vol/vol) vitamin mixture (3). Some medium not containing the vitamin mixture was autoclaved at 95°C for 3 h without being chemically reduced. Strain 42BKT was chemoautotrophically grown in MJ synthetic seawater containing 0.15% NaHCO₃ and Na₂S₂O₃ · 5H₂O and a 0.01% (vol/vol) vitamin mixture. The inoculated culture media (200 ml) were incubated in a 1-liter glass bottle sealed with a butyl rubber stopper at 25°C with continuous shaking. The gas phases of H₂-CO₂ (8:2; 250 kPa) and N₂-CO₂-O₂ (77:16:6; 150 kPa) were used for the cultivation of the two hydrogen-oxidizing strains, E9I37-1 and GO25, and the sulfur-oxidizing strain, 42BKT, respectively. The pH of the first medium was adjusted to 7.5 with 2 N NaOH, whereas the second medium included a bicarbonate-CO₂ buffer, which was necessary to promote the growth of strain 42BKT. The cell concentrations were determined by counting the cells under a light microscope.

Bulk carbon isotopic analyses.
Each of the three -proteobacterial cultures was harvested at the early stage of their exponential growth phase by centrifugation (10,000 x g, 20 min). The harvested culture was rinsed twice with PBS, frozen, and then lyophilized, after which the dry weight was determined. A small portion of the lyophilized culture was acid fumed for 6 h (53), and the untreated rest was stored at –80°C for fatty acid extraction. Three gastropod individuals were dissected into gill, mantle, and foot tissues, and the dissected tissues were lyophilized. A small portion of each lyophilized tissue was powdered and then acid fumed. The rest of the untreated lyophilized tissue was stored at –80°C for fatty acid extraction. The carbon isotopic compositions of the cultures and the gastropod tissues were analyzed by a Thermo Electron DELTA^plus Advantage mass spectrometer connected to an elemental analyzer (EA1112) through a ConFlo III interface. The measured isotopic composition was expressed as ¹³C, which can be defined as follows:

(1)

where (¹³C/¹²C)_sample is the ¹³C/¹²C abundance ratio for the sample and (¹³C/¹²C)_standard is the ¹³C/¹²C abundance ratio for the Pee Dee Belemnite carbonate standard. The values of ¹³C therefore represent the difference, in parts per thousand (pro mille []), between the ¹³C/¹²C value of the sample and that of the standard.

For measurements of ¹³C in CO₂, triplicate 5-ml gas samples were collected from the headspaces of the culture bottles and transferred to evacuated 10-ml vials at the beginning and end of the cultivation of strains GO25 and E9I37-1. The cryogenically purified CO₂ was analyzed with a Thermo Electron DELTA^plus XL mass spectrometer through a dual inlet. The measurement errors were <1 for all bulk carbon isotopic analyses.

Analysis of FAME profiles.
For the extraction of cellular fatty acids, a method described previously (25) was used. Approximately 20 mg of the lyophilized -proteobacterial cultures and gastropod tissues were incubated in 1 ml of anhydrous methanolic hydrochloric acid at 100°C for 3 h. After the addition of 1 ml of deionized, distilled water to the cooled aliquots, the fatty acid methyl esters (FAMEs) were extracted three times with 3 ml of n-hexane. The n-hexane fractions were washed with an equal volume of deionized, distilled water and dehydrated with anhydrous Na₂SO₄. The concentrated FAMEs were stored at –20°C for subsequent carbon isotopic analyses. Although this extraction method degrades the cyclopropyl FAME (33), it does enable minimum loss of material during extraction.

The identities of the FAMEs were determined by comparison of the retention times and spectra to those of known FAME standards by gas chromatography-mass spectrometry, using a Shimadzu GCQ gas chromatography-mass spectrometry system. The oven temperature was set to 140°C for 3 min and then increased to 250°C at a rate of 4°C/min with He at a constant flow of 1.1 ml/min through a DB-5MS column (30 m by 0.25 µm by 0.25 mm; J&W Scientific). The standard nomenclature for fatty acids was used. Fatty acids are designated X:Y, where X is the number of carbon atoms and Y is the number of double bonds. Mid-chain methyl branches are designated by the position from the carboxyl end followed by "Me."

Compound-specific carbon isotopic analysis.
The ¹³C values of the FAMEs were determined by the GC-carbon-isotope ratio MS using a Thermo Electron DELTA^plus Advantage mass spectrometer connected to a GC (Agilent 6890) through a GC/C/C/III interface. The oven temperature was set to 120°C for 3 min and then increased to 300°C at a rate of 4°C/min with He at a constant flow of 1.1 ml/min through an HP-5 column (30 m by 0.25 µm by 0.25 mm; Agilent). The isotopic compositions of the FAMEs were measured with an internal isotopic standard (19:0; ¹³C = –29.80), and the additional carbon atom from the methanol-derivatizing reagent (¹³C = –39.04) was corrected. The internal isotopic standard produced measurement errors within 1 for all isotopic analyses.

Enzymatic characterization of the carbon-fixation pathways.
The same dissected gill tissue as that used for DNA extraction (1 g [wet weight]) was suspended in 3 ml of 100 mM Tris-HCl (pH 7.8) and 1 mM dithiothreitol (DTT). The tissue suspension was thoroughly disrupted by ultrasonification on ice and centrifuged at 20,000 x g for 20 min at 4°C. Solid ammonium sulfate was added to the cell extract (supernatant) to 80% saturation. After stirring for 30 min, the extract was centrifuged at 20,000 x g for 20 min at 4°C. The pellet was dissolved in 100 mM Tris-HCl (pH 7.8) and 1 mM DTT and dialyzed against the same buffer. All of the procedures were anaerobically performed under an N₂-gas atmosphere. The dialyzed fraction after ammonium-sulfate precipitation was used as the cell extract and used in the characterization of the enzyme activity. The protein concentration was estimated by using the NanoOrange protein quantification kit (Molecular Probe, Inc., Eugene, OR) in accordance with the manufacturer's manual.

All of the enzyme activities described below were measured at 25°C. The reactions in the absence of the substrate and in the presence of heat-denatured cell extract were used as the negative controls. Positive controls were prepared by using the chemolithoautotrophic isolates among the -Proteobacteria. ATP-citrate lyase (ACL), a key enzyme of the rTCA cycle, was assayed spectrophotometrically as described previously (47). The reaction mixture contained 200 mM Tris-HCl (pH 7.8), 2 mM DTT, 2 mM MgCl₂, 2.5 mM ATP, 10 mM sodium citrate, 0.25 mM NADH, and 10 U of malate dehydrogenase/ml from the porcine heart (Sigma) as a coupling enzyme. Pyruvate-acceptor oxidoreductase (POR) and 2-oxoglutarate-acceptor oxidoreductase (OGOR), both reversible CO₂ assimilation enzymes of the rTCA cycle, were assayed spectrophotometrically as described previously (40). The reaction mixture contained 200 mM Tris-HCl (pH 7.8), 2 mM DTT, 2 mM MgCl₂'6H₂O, 0.25 mM coenzyme A (CoA), 5 mM methyl viologen, and 10 mM sodium pyruvate for POR or 10 mM sodium oxoglutarate for OGOR. Isocitrate dehydrogenase, a reversible CO₂ assimilation enzyme of the rTCA cycle, was assayed spectrophotometrically as described previously (41). The reaction mixture contained 200 mM Tris-HCl (pH 7.8), 2 mM DTT, 2 mM MgCl₂, 10 mM sodium oxoglutarate, 10 mM NaHCO₃, and 0.25 mM NADH. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PC), both anaplerotic CO₂ assimilation enzymes related to the rTCA cycle, were assayed spectrophotometrically as described previously (40). The reaction mixture contained 200 mM Tris-HCl (pH 7.8), 2 mM DTT, 2 mM MgCl₂ · 6H₂O, 10 mM NaHCO₃, 0.25 mM NADH, 10 U of malate dehydrogenase/ml from the porcine heart (Sigma) as a coupling enzyme, and 10 mM sodium phosphoenolpyruvate for PEPC or 10 mM sodium pyruvate for PC. Rubisco, a key CO₂ assimilation enzyme of the Calvin-Benson cycle, was assayed by spectrophotometry and high-pressure liquid chromatography in accordance with references 11 and 32, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anatomy.
The gastropod A. aff. hessleri from the Kairei deep-sea hydrothermal field in the Indian Ocean consists chiefly of a pallial cavity, a head-foot and visceral mass. The head-foot is provided with a snout (Fig. 1), cephalic tentacles, a neck region with a deep neck furrow, and a massive foot with an opercular lobe. The pallial cavity is very deep and attains more than one volution and is mostly occupied by the ctenidium. The visceral mass is divisible into the gonad, intestine, and digestive gland externally. The circulatory system is hypertrophied, and the pallial cavity and adjacent body wall are highly vascularized. The digestive system is characterized by radular teeth showing little signs of wear, an enlarged esophagus, and a greatly reduced stomach relative to its body size. The intestine often contains fine grains of black substance.

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FIG. 1. A. aff. hessleri, seen from the right anterior side. Part of the mantle is cut, and the pallial cavity is opened to show the well-developed ctenidial lamellae (gill filaments). The arrowhead indicates the posterior end of the pallial cavity and ctenidial lamellae (gill filaments). acv, afferent ctenidial vessel; clb, ctenidial lamellae (lacking basal attachment); clm, ctenidial lamellae (attached to the mantle); ct, cephalic tentacle; dg, digestive gland; ecv, efferent ctenidial vessel; f, foot; g, gonad; i, intestine; mm, mantle margin; nf, neck furrow; ol, opercular lobe; r, rectum; sn, snout.

Similarly to the A. hessleri collected from the Mariana Trough (42), the A. aff. hessleri from the Kairei field on the Central Indian Ridge has an unusually enlarged ctenidium for a gastropod. The ctenidial lamellae (gill filaments) are attached to the mantle on the left side of the pallial cavity (Fig. 1), and the right side of each lamella is projected into the cavity as free tips. Each lamella is composed of skeletal rods (Fig. 2A) functioning as a supporting structure, lateral cilia possibly used for ventilation between the lamellae, and blood vessels emptying from the afferent into the efferent sides though vertical ridges. The surface of the vertical ridges is distinctly roughened and tubercular (Fig. 2B). The vertical sections of the ctenidial lamellae show densely packed bacterium-like cells, which are coccoid and 1 µm in diameter (Fig. 2C and D). They are mainly contained within the host cells around the afferent vessel (Fig. 2A) and never attached to the surface of the ctenidial lamellae (Fig. 2B).

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FIG. 2. Scanning electron micrographs of the ctenidial lamella (gill filaments) of A. aff. hessleri. (A) The surface of the ctenidial lamella, seen from the posterior side of the animal. (B) Enlarged oblique view of the ridge surfaces of the ctenidial lamellae. (C and D) Internal bacterium-like cells on the vertical sections of the ctenidial lamellae. av, vessel on the afferent side; ev, vessel on the efferent side; lc, lateral cilia; rcl, ridges of the ctenidial lamellae; sr, skeletal rods.

Phylogenetic analysis.
The phylogenetic affiliation of the microbial cells in the gill filaments was determined based on the 16S rRNA gene sequences. The examination of 32 clones generated from a 16S rRNA gene-sequence library from the gill filaments of a single gastropod showed only one phylotype. The occurrence of a single endosymbiont in the gill filaments was also supported by the results of the direct sequencing of the 16S rRNA gene sequence of the phylotype from two other gastropods. As shown in Fig. 3, phylogenetic analysis placed the phylotype within -proteobacterial group F, which includes the epibionts of the polychete A. pompejana (18), the vent tubeworm Riftia pachyptila (30), and the swarming vent shrimp Rimicaris exoculata (37, 39), as well as free-living bacteria in various deep-sea hydrothermal environments (28, 29, 37, 43, 48). The closest relatives of the phylotype were epibionts attached to the iron-sulfide exoskeleton of another vent gastropod in the same habitat (17) and bacterial clones associated with microcolonizers deployed on the Mid-Atlantic Ridge (29). Among the cultivated species, Sulfurovum lithotrophicum strain 42BKT was found to be the closest relative of the Alviniconcha endosymbiont.

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FIG. 3. Distance tree of the members of the -Proteobacteria, including the Alviniconcha endosymbiont and the culture organisms used in the present study (in boldface), based on near-complete 16S rRNA gene sequences (1,315 nucleotides). Bootstrap values (in percent) are based on 1,000 replicates (distance and parsimony) and are shown for branches with more than 50% bootstrap support.

FISH.
In order to ensure that the dominant phylotype revealed by direct sequencing and 16S rRNA gene sequence clone library analysis originates from the endosymbiont in the gill filaments, we conducted FISH analysis with the probe EPF93. The specificity was checked against the databases, and the probe was found to be fully complementary to the rRNA sequence of the endosymbiont and many other members belonging to -proteobacterial group F. Except for the rRNA sequence of some members of an -proteobacterial group represented by Arcobacter spp. (Arcobacter group), which has a one-base mismatch near the 3' end, the other available rRNA sequences were found to have more than three mismatches. Sulfurovum lithotrophicum strain 42BKT was used for the positive control experiment, and Nitratifractor salsuginis strain E9I37-1 (group F) and "S. paralvinella" strain GO25 (group B) were used for negative control experiments. In addition, a negative control probe, in which a one-base mismatch was introduced in the middle of the EPF93 probe, precluded the possibility of unspecific labeling. Although the hybridization condition described above discriminated the one-base mismatch in the middle, as confirmed by the negative control probe, the rRNA sequence of the negative control strain E9I37-1, which has a mismatch identical to that of Arcobacter species, was hybridized with the probe EPF93. Thus, the probe EPF93 was bound specifically to the rRNA of the bacteria that phylogenetically fall into -proteobacterial group F and the Arcobacter group.

The sections of the gill filaments from three gastropods were hybridized with the EUB338 and EPF93 probes, followed by DNA staining with DAPI. Representative epifluorescence micrographs are shown in Fig. 4. The presence of dense aggregates of bacterial cells, as well as host nuclei (labeled "N" in Fig. 4A) in the gill filaments was confirmed by FISH with the EUB338 probe, together with DAPI staining (Fig. 4A and B). Hybridization with the EPF93 probe specific to the two -proteobacterial groups gave a signal pattern almost identical to that of the EUB338 probe (Fig. 4B and C), indicating that most of the bacterial cells are affiliated to the -proteobacterial groups.

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FIG. 4. Epifluorescence micrographs of the endosymbiotic bacteria associated with the gill filaments of A. aff. hessleri from the Central Indian Ridge. (A) DNA staining of the section of the gill filaments with DAPI. In addition to the bacterium-like cells, the host nuclei are stained and labeled "N." (B) FISH performed with the fluorescein-labeled EUB338 probe (same microscopic field). (C) FISH performed with the Cy-3-labeled EPF93 probe specific for two -proteobacterial groups (-proteobacterial group F and the Arcobacter group).

Cultivation and bulk carbon isotopic analyses.
The three -proteobacterial isolates were all grown under chemoautotrophic conditions. The average cell concentrations in the triplicate cultures (cells per ml) at the harvesting of the cells were 8.3 x 10⁸, 4.2 x 10⁷, and 5.1 x 10⁸ for strains E9I37-1, GO25, and 42BKT, respectively. The standard errors were <10%. Closed-system isotope effects were not encountered during the growth of the stains E9I37-1 and GO25, because the carbon isotopic composition of the remaining CO₂ was unchanged at –28.9. Similarly, although the change in the isotopic composition of CO₂ was not monitored for the growth of the strain 42BKT, the effect of the carbon pool is considered to have been minimal due to the excess supply of CO₂ in addition to bicarbonate. On a dry-weight basis, ca. 1.2, 1.8, and 2.4% of the total inorganic carbon pool was incorporated into the biomass during the growth of the strains E9I37-1, GO25, and 42BKT, respectively. The ¹³C values of the biomass were –34.8, –35.5, and –32.8 for E9I37-1, GO25, and 42BKT, respectively (Tables 1 and 2). The strains E9I37-1 and GO25 grown under chemoautotrophic conditions had ¹³C-depleted biomasses relative to CO₂ by 5.9 and 6.6, respectively (see also Fig. 5).

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TABLE 1. Carbon isotopic compositions of total biomass and FAMEs of pure cultures of -Proteobacteria

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TABLE 2. Carbon isotopic compositions of total biomass and FAMEs of gastropod tissues

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FIG. 5. Carbon-isotope fractionation patterns of CO2 relative to the total biomass, and of the total biomass relative to the monounsaturated C16 fatty acid, of the -proteobacterial cultures (left). The measured 13C values of the total biomass and monounsaturated C16 fatty acid of the gastropod are inferred to be identical to those for the endosymbiont (right). Carbon isotopic compositions of the biomass and monounsaturated C16 fatty acid of the endosymbiont are estimated based on the 13C value of CO2 emitted from the hydrothermal vents on the Central Indian Ridge and the established carbon isotopic relationship between biomass and monounsaturated C16 fatty acid of the -proteobacterial cultures (right). The isotopic value of the carbon source for Sulfurovum lithotrophicum strain 42BKT is undetermined.

The ¹³C values of the gastropod gill, mantle, and foot tissues were measured. In good agreement with the carbon isotopic data from a previous study in which 10 A. aff. hessleri individuals from the Kairei field were investigated (51), all of the tissues of the three gastropods had ¹³C values of ca. -11 as shown in Table 1. Thus, there appears to be no variance in the carbon isotopic composition among the individuals. Despite the abundance of endosymbiont cells in the gill, the carbon isotopic compositions of the symbiont-free tissues are nearly identical to that of the gill, indicating that the endosymbiont biomass was as ¹³C depleted as the symbiont-free gastropod tissues.

FAME profiles.
It is well established that the FAME profiles of marine mollusks are similar to those of the organisms they feed on (1, 15, 38). The analysis of the FAME profiles from the three -proteobacterial isolates showed high levels of the saturated C₁₆ fatty acids and one or both of the monounsaturated C₁₆ and C₁₈ fatty acids (Fig. S1 in the supplemental material). Although the symbiont-free mantle and foot tissues of the gastropod both contained abundant fatty acids (16:0 and 18:0, respectively), the proportion of the 16:1 fatty acid in the gill is much higher than that in both of the symbiont-free tissues, which indicates that the substantial proportion of the 16:1 fatty acid was derived from the endosymbiotic cells (Fig. S2 in the supplemental material).

Compound-specific carbon isotopic analysis.
The carbon isotopic compositions of some FAMEs from the -proteobacterial cultures and the gastropod tissues were measured; the ¹³C values of the FAMEs after correction for the methanol-derivatizing reagent are shown in Table 1. The FAMEs analyzed in the present study were all ¹³C-enriched relative to the biomass. The isotopic compositions of the total FAMEs were calculated for the cultures and the gastropod tissues on the basis of the FAME compositions (Table 1). The ¹³C enrichment of the fatty acids relative to the biomass was greater for the cultures (ca. 4 to 6) than for the gastropod tissues (ca. 1 to 2), except for the gill tissue (4). The saturated and monounsaturated C₁₆ fatty acids in the gill tissue were particularly ¹³C enriched, by 4.5 and 5.9, respectively, relative to the biomass (Table 1).

The enzyme activities of the carbon fixation pathways.
The potential activity of several key enzymes in carbon assimilation pathways such as the rTCA cycle and the Calvin-Benson cycle was examined in the cell extract from the gill tissue. The ACL activity was 13.6 ± 2.5 nmol of citrate/min/mg at 25°C. The activities of POR and OGOR were 5.6 ± 1.2 nmol of pyruvate/min/mg and 1.7 ± 0.8 nmol of oxoglutarate/min/mg at 25°C, respectively. Isocitrate dehydrogenase, PEPC, and PC activity was not detected. Rubisco activity could not be detected by either spectrophotometric or high-pressure liquid chromatography assays. Although the POR and OGOR reactions are reversible and thus do not constitute direct evidence for the operation of the rTCA cycle, the ACL activity could indicate the operation of the rTCA cycle. The enzymatic characterization strongly suggests that the endosymbiotic bacterium in the gill tissue could serve as the primary producer assimilating CO₂ via the rTCA cycle.

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Within the gill filaments, the gastropod harbors an endosymbiont that phylogenetically falls into -proteobacterial group F. Most members of the F group are free living (28, 29, 37, 43, 48) or externally associated with marine invertebrates (18, 30, 37, 39). Based on several anatomical features such as the enlargement of the gill, the reduced digestive tract, and the radula without wear, we assume that the gastropod derives its nutrition from the endosymbiont. The internal occurrence of members of the -Proteobacteria, which was confirmed by FISH and scanning electron microscopy in the present study, is rare. Thus, further investigations are required to determine whether or not the symbiotic interactions are mutualistic.

rTCA cycle in the chemoautotrophic isolates.
In order to establish the nature of the gastropod/-proteobacterial endosymbiosis, it is critical to determine whether the mode of growth of the endosymbiont is chemoautotrophic or heterotrophic. Previously, the physiologies of the members of -proteobacterial group F were largely unknown due to the lack of isolates resulting from their resistance to cultivation. However, a recent success in the isolation of some members belonging to -proteobacterial group F and subsequent physiological characterizations of the isolates revealed that they are strict chemolithoautotrophs using reduced sulfur compounds such as thiosulfate and elemental sulfur and/or hydrogen as the electron donor(s) and oxygen and/or nitrate as the electron acceptor(s) (20, 35, 45). All chemoautotrophic bacterial endosymbionts in marine invertebrates known to date are uncultivated, and the Alviniconcha endosymbiont is no exception. Thus, alternative methods must be applied in order to determine the physiology of the gastropod endosymbiont.

If an organism assimilates carbon from a small molecule, either CO₂, CH₄, or acetate, significant isotope fractionation is likely to accompany the carbon assimilation (19). The fractionation of carbon isotopes is also associated with the biosynthesis of acetyl-CoA, a precursor fatty acid synthesis (19, 36). The range of the fractionations varies with the pathways involved in carbon assimilation and acetyl-CoA synthesis. Therefore, the fractionation patterns of the carbon source relative to the biomass and of the biomass relative to the fatty acids of a given organism provide diagnostic information regarding the carbon metabolism of the organism (19). To our knowledge, little information is available on the carbon isotope fractionations for the cultured members of the -Proteobacteria; these patterns are essential for the interpretation of the complex isotopic signatures of gastropod/-proteobacterium endosymbiosis (55). In the present study, the carbon isotope fractionations were determined for one isolate from group B and two isolates from group F (Fig. 3) under chemoautotrophic growth conditions.

The two -proteobacterial strains, E9I37-1 and GO25, are ¹³C depleted relative to CO₂ (–28.9) by 5.9 and 6.6, respectively (Table 1). The ¹³C enrichment of the total fatty acids relative to the biomass ranges from 4.0 to 6.1 for the -proteobacterial isolates tested (Table 1). The fractionation pattern of the -proteobacterial isolates characterized by the ¹³C enrichment of the fatty acids relative to the biomass is distinguishable from that of heterotrophic organisms and chemoautotrophs using the Calvin-Benson cycle and Rubisco for CO₂ fixation, because the latter two produce fatty acids substantially ¹³C depleted relative to the biomass (19). To date, the biosynthesis of fatty acids through acetyl-CoA, which is biosynthesized from CO₂ through the rTCA cycle by chemoautotrophic bacteria, is the only known mechanism by which fatty acids are ¹³C enriched relative to the biomass (23, 49). Recent genomic analysis of the A. pompejana epibiont community demonstrated that dominant epibionts, which belong phylogenetically to -proteobacterial group F (Fig. 3), possess two key genes in the rTCA cycle, ACL and OGOR (4), which is consistent with the fractionation pattern obtained for the -proteobacterial cultures in the present study. Taken together, these results suggest that the -proteobacterial isolates grow chemoautotrophically via the rTCA cycle.

Chemoautotrophy in the -proteobacterial endosymbiont.
Previous studies have shown that the ¹³C value of CO₂ emitted from hydrothermal vents in the Kairei field ranges from –6.0 to –6.2 (14, 45). The carbon isotopic composition of the endosymbiont biomass is interpreted as equal to that of the gastropod, because there is no significant variance in the carbon isotopic composition between the tissue parts, despite the abundance of endosymbiont cells in the gill tissue. The monounsaturated C₁₆ FA in the gill tissue is inferred to originate mostly from the -proteobacterial endosymbiont based on two facts: it is the major fatty acid of all of the -proteobacterial cultures analyzed, and it occurred substantially only in the symbiont-bearing gill tissue.

Carbon isotopic data on the CO₂ from the hydrothermal vents, together with the measured fractionations for the -proteobacterial cultures grown under chemolithoautotrophic conditions, can be used to construct a model for describing the expected isotopic compositions of the endosymbiont biomass and the 16:1 fatty acid. The fractionation patterns measured for the laboratory cultures, the ¹³C values of the gastropod tissues, the 16:1 fatty acid in the gill, and the resulting model for the endosymbiont are schematically presented in Fig. 5. The conversion of CO₂ with ¹³C values of ca. -6.0 into biomass, associated with a ¹³C depletion ranging from 5.9 to 6.6, results in an endosymbiont biomass with a ¹³C range of –11.9 to –12.6 (Fig. 5). The range is very close to the ¹³C values of the gastropod tissues (ca. –11). By using the estimated ¹³C range of –11.9 to –12.8 for the endosymbiont biomass and the fractionation range of 4.9 to 7.3 between the biomass and the 16:1 fatty acid of the laboratory cultures, the endosymbiont is expected to synthesize the 16:1 fatty acid with a ¹³C value ranging from –4.6 to –7.9 (Fig. 5). The ¹³C value of the 16:1 fatty acid inferred to originate from the endosymbiont (–5.1) falls within the estimated ¹³C range of the 16:1 fatty acid (–4.6 to –7.9). Thus, it is suggested that, similarly to the -proteobacterial cultures, the endosymbiont most probably mediates the rTCA cycle for its chemoautotrophic growth. In addition to the carbon-isotope characteristics, enzymatic characterization has identified the activities of the enzymes involved in the prokaryotic rTCA cycle (21, 40, 41). The gill tissue containing the endosymbiont showed evident ACL, POR, and OGOR activity but no Rubisco activity. This strongly suggests that the endosymbiont might be able to operate the autotrophic rTCA cycle as its carbon source and even as the host's carbon source. The integrated results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses provide convincing evidence that the host nutrition is primarily based on the chemoautotrophy of the -proteobacterial endosymbiont.

The trophic relationships of the gastropod/-proteobacterial endosymbiosis.
In order to better understand the nutritional relationships, we attempted to compare our results obtained for the gastropod/-proteobacterial endosymbiosis to those of previous studies on relatively well-established endosymbioses between marine bivalves and some thioautotrophic or methanotrophic -proteobacteria (9). Marine bivalves, which nutritionally depend on intracellular thioautotrophic -Proteobacteria, have fatty acid profiles similar to those of their endosymbionts, indicating that the endosymbiont cells are digested by, and incorporated into, the host bivalves (6, 7, 38). As for a marine bivalve nutritionally dependent on an endosymbiotic methanotroph belonging to the -Proteobacteria, monounsaturated C₁₆ fatty acids, the dominant fatty acids in the symbiont-bearing tissue as well as in the free-living methanotrophic relatives of the endosymbiont (22, 24), are absent in the symbiont-free tissues, which implies that the fatty acids synthesized by the endosymbiont are not utilized by the host bivalve. The presence of methylsterols, known to be produced only by methanotrophic bacteria in symbiont-free tissue, suggests that the methylsterols are released from the endosymbiont and taken up by the host (24). Similarly to the methanotroph-harboring bivalve, the almost-absent utilization of the endosymbiont fatty acid by the host gastropod indicates that the endosymbiont might translocate organic material across the cell membrane, rather than the endosymbiont cells being digested by the host.

Symbiont heterogeneity among the Alviniconcha gastropods from distinct localities.
Several discrepancies do exist between the results of the present study and those of previous ones in which Alviniconcha gastropods from other localities were investigated. Although the phylogenetic affiliation of the endosymbiont of the Alviniconcha gastropod from the Mariana Trough in the Western Pacific is currently unknown, the endosymbiotic bacteria in the gill filaments of the gastropod actively catalyze CO₂ fixation using the Calvin-Benson cycle and Rubisco. The difference in the carbon fixation pathway is also supported by the ¹³C depletion in the gastropod biomass, which has a ¹³C value of –28.9 (52) relative to the CO₂ from the hydrothermal vents in the Mariana Trough with a ¹³C value of –4.3 (13). The Alviniconcha gastropod from the North Fiji basin in the Southwestern Pacific has monounsaturated FAs originating from the phylogenetically undetermined endosymbiont in the symbiont-free tissue, as well as the symbiont-bearing gill. These previous results for the Alviniconcha gastropods from the Pacific Ocean are consistent with those for the bivalves harboring thioautotrophic endosymbionts belonging to the -Proteobacteria. These disagreements suggest that at least two lineages of bacteria phylogenetically distinct at the subdivision level may occur as the primary endosymbiont in one host animal type.

 
We thank the captains and crews of the R/V Yokosuka and the Shinkai 6500 for their technical expertise. We also thank Yoshihiro Fujiwara, Fumio Inagaki, Satoshi Nakagawa, Shinji Tsuchida, Sumihiro Koyama, and Michinari Sunamura for their contributions.

 
* Corresponding author. Mailing address: Frontier Research System for Extremophiles, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan. Phone: 81-468-67-9710. Fax: 81-468-67-9715. E-mail: yohey{at}jamstec.go.jp.

Supplemental material for this article may be found at http://aem.asm.org/.

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1
1. Ackman, R. G., and S. N. Hooper. 1973. Non-methylene-interrupted fatty acids in lipids of shallow-water marine invertebrates: a comparison of two molluscs (Littorina littorea and Lunatia triseriata) with the sand shrimp (Crangon septemspinosus). Comp. Biochem. Phys. 46B:153-165.[CrossRef]
2
2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Basic local alignment search tool. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
3
3. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296.[Free Full Text]
4
4. Campbell, B. J., J. L. Stein, and S. C. Cary. 2003. Evidence of chemolithoautotrophy in the bacterial community associated with Allvinella pompejana, a hydrothermal vent polychaete. Appl. Environ. Microbiol. 69:5070-5078.[Abstract/Free Full Text]
5
5. Cole, J. R., B. Chai, T. L. Marsh, R. J. Farris, Q. Wang, S. A. Kulam, S. Chandra, D. M. McGarrell, T. M. Schmidt, G. M. Garrity, and J. M. Tiedje. 2003. The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res. 31:442-443.[Abstract/Free Full Text]
6
6. Conway, N., and J. M. Capuzzo. 1991. Incorporation and utilization of bacterial lipids in the Solemya velum symbiosis. Mar. Biol. 108:277-291.[CrossRef]
7
7. Conway, N. M., B. L. Howes, J. E. McDowell Capuzzo, R. D. Turner, and C. M. Cavanaugh. 1992. Characterization and site description of Solemya borealis (Bivalvvia: Solemyidae), another bivalve-bacteria symbiosis. Mar. Biol. 112:610-613.
8
8. Corre, E., A. L. Reysenbach, and D. Prieur. 2001. Epsilon-proteobacterial diversity from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge. FEMS Microbiol. Lett. 205:329-335.[Medline]
9
9. Distel, D. L. 1998. Evolution of chemoautotrophic endosymbioses in bivalves. Bioscience 48:277-286.[CrossRef]
10
10. Dubilier, N., C. Mulders, T. Ferdelman, D. de Beer, A. Pernthaler, M. Klein, M. Wagner, C. Erseus, F. Thiermann, J. Krieger, O. Giere, and R. Amann. 2001. Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete worm. Nature 411:298-302.[CrossRef][Medline]
11
11. Ezaki, S., N. Maeda, T. Kishimoto, H. Atomi, and T. Imanaka. 1999. Presence of a structurally novel type ribulose-bisphosphate carboxylase/oxygenase in the hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. J. Biol. Chem. 274:5078-5082.[Abstract/Free Full Text]
12
12. Fisher, C. R. 1990. Chemoautotrophic and methanotrophic symbioses in marine invertebrates. Rev. Aquat. Sci. 2:399-436.
13
13. Gamo, T. 1995. Wide variation of chemical characteristics of submarine hydrothermal fluids due to secondary modification processes after high temperature water-rock interaction: a review, p. 425-451. In H. Sakai and Y. Nozaki (ed.), Biogeochemical processes and ocean flux in the Western Pacific. Terra Scientific Publishing Company, Tokyo, Japan.
14
14. Gamo, T., H. Chiba, T. Yamanaka, T. Okudaira, J. Hashimoto, S. Tsuchida, J. Ishibashi, S. Kataoka, U. Tsunogai, K. Okamura, Y. Sano, and R. Shinjo. 2001. Chemical characteristics of newly discovered black smoker fluids and associated hydrothermal plumes at the Rodriguez Triple Junction, Central Indian Ridge. Earth Planet. Sc. Lett. 193:371-379.[CrossRef]
15
15. Gardner, D., and J. P. Riley. 1972. The component fatty acids of the lipids of some species of marine and freshwater molluscs. J. Mar. Biol. Assoc. UK 52:827-838.
16
16. Giovannoni, S. J., E. F. DeLong, G. J. Olsen, and N. R. Pace. 1988. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J. Bacteriol. 170:720-726.[Abstract/Free Full Text]
17
17. Goffredi, S. K., A. Waren, V. J. Orphan, C. L. Van Dover, and R. C. Vrijenhoek. 2004. Novel forms of structural integration between microbes and a hydrothermal vent gastropod from the Indian Ocean. Appl. Environ. Microbiol. 70:3082-3090.[Abstract/Free Full Text]
18
18. Haddad, A., F. Camacho, and P. Durand. 1995. Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychete Alvinella pompejana. Appl. Environ. Microbiol. 61:1679-1687.[Abstract]
19
19. Hayes, J. M. 2001. Fractionation of the isotopes of carbon and hydrogen in biosynthetic processes, p. 225-277. In J. Vally and D. Cole (ed.), Stable isotope geochemistry, vol. 43. Mineralogical Society of America, Washington, D.C.
20
20. Inagaki, F., K. Takai, K. H. Nealson, and K. Horikoshi. 2004. Sulfurovum lithotrophicum gen. nov. sp. nov., a novel sulfur-oxidizing chemolithoautotroph within the -Proteobacteria isolated from Okinawa Trough hydrothermal sediments. Int. J. Syst. Evol. Microbiol. 54:1477-1488.[Abstract/Free Full Text]
21
21. Ishii, M., T. Miyake, T. Satoh, H. Sugiyama, Y. Oshima, T. Kodama, and Y. Igarashi. 1997. Autotrophic carbon dioxide fixation in Acidianus brierleyi. Arch. Microbiol. 166:368-371.[CrossRef]
22
22. Jahnke, L. L., and K. Diggs. 1989. Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acids in Methylococcus capsulatus by the anaerobic pathway. FEMS Microbiol. Lett. 58:183-188.[CrossRef][Medline]
23
23. Jahnke, L. L., W. Eder, R. Huber, J. M. Hope, K. U. Hinrichs, J. M. Hayes, D. J. Des Marais, S. L. Cady, and R. E. Summons. 2001. Signature lipids and stable carbon isotope analyses of Octopus Spring hyperthermophilic communities compared with those of Aquificales representatives. Appl. Environ. Microbiol. 67:5179-5189.[Abstract/Free Full Text]
24
24. Jahnke, L. L., R. E. Summons, L. M. Dowling, and K. D. Zahiralis. 1995. Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis. Appl. Environ. Microbiol. 61:576-582.[Abstract]
25
25. Komagata, K., and K. Suzuki. 1987. Lipid and cell-wall analysis in bacterial systematics. Method. Microbiol. 19:161-207.
26
26. Lane, D. J. 1991. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Inc., New York, N.Y.
27
27. Lathe, R. 1985. Synthetic oligonucleotide probes deduced from amino acid sequence data: theoretical and practical considerations. J. Mol. Biol. 183:1-12.[CrossRef][Medline]
28
28. Longnecker, K., and A. L. Reysenbach. 2001. Expansion of the geographic distribution of a novel lineage of -Proteobacteria to a hydrothermal vent site on the Southern East Pacific Rise. FEMS Microbiol. Ecol. 35:287-293.[Medline]
29
29. Lopez-Garcia, P., S. Duperron, P. Philippot, J. Foriel, J. Susini, and D. Moreira. 2003. Bacterial diversity in hydrothermal sediment and epsilonproteobacterial dominance in experimental microcolonizers at the Mid-Atlantic Ridge. Environ. Microbiol. 5:961-976.[CrossRef][Medline]
30
30. Lopez-Garcia, P., F. Gail, and D. Moreira. 2002. Wide bacterial diversity associated with tubes of the vent worm Rifta pachyptila. Environ. Microbiol. 4:204-215.[CrossRef][Medline]
31
31. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A. W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R. Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K. H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32:1363-1371.[Abstract/Free Full Text]
32
32. Maeda, N., T. Kanai, H. Atomi, and T. Imanaka. 2002. The unique pentagonal structure of an archaeal Rubisco is essential for its high thermostability. J. Biol. Chem. 277:31654-31662.
33
33. Moss, C. W., M. A. Lambert, and W. Merwin. 1974. Comparison of rapid methods for analysis of bacterial fatty acids. Appl. Microbiol. 28:80-85.[Medline]
34
34. Naganuma, T., C. Kato, H. Hirayama, N. Moriyama, J. Hashimoto, and K. Horikoshi. 1997. Intracellular occurrence of -proteobacterial 16S rDNA sequences in the vestimentiferan trophosome. J. Oceanogr. 53:193-197.
35
35. Nakagawa, S., K. Takai, F. Inagaki, K. Horikoshi, and S. Sako. 2004. Nitratiruptor tergarcus gen. nov., sp. nov. and Nitratifractor salsuginis gen. nov., sp. nov., two nitrate-reducing chemolithoautotrophs within the epsilon-Proteobacteria isolated from a deep-sea hydrothermal system in the Mid-Okinawa Trough. Int. J. Syst. Evol. Microbiol. [Online.] http://dx.doi.org/10.1099/ijs.0.63480-0.
36
36. Pancost, R., and J. S. Sinninghe Damste. 2003. Carbon isotopic compositions of prokaryotic lipids as tracers of carbon cycling in diverse settings. Chem. Geol. 195:29-58.[CrossRef]
37
37. Polz, M. F., and C. M. Cavanaugh. 1995. Dominance of one bacterial phylotype at a Mid-Atlantic Ridge hydrothermal vent site. Pro. Natl. Acad. Sci. USA 92:7232-7236.[Abstract/Free Full Text]
38
38. Pranal, V., A. FialaMedioni, and J. Guezennec. 1997. Fatty acid characteristics in two symbiont-bearing mussels from deep-sea hydrothermal vents of the south-western Pacific. J. Mar. Biol. Assoc. UK 77:473-492.
39
39. Segonzac, M., M. de Saint Laurent, and B. Casanova. 1993. The enigma of the trophic behavior of Alvinocaridid shrimps from hydrothermal vent sites on the Mid-Atlantic Ridge. Cah. Biol. Mar. 34:535-571.
40
40. Shiba, H., T. Kawasumi, Y. Igarashi, T. Kodama, and Y. Minoda. 1985. The CO₂ assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus. Arch. Microbiol. 141:198-203.[CrossRef]
41
41. Shiba, H., T. Kawasumi, Y. Igarashi, T. Kodama, and Y. Minoda. 1982. The deficient carbohydrate metabolic pathways and the incomplete tricarboxylic acid cycle in an obligately autotrophic hydrogen-oxidizing bacterium. Agr. Biol. Chem. 46:2341-2345.
42
42. Stein, J. L., S. C. Cary, R. R. Hessler, S. Ohta, R. D. Vetter, J. J. Childress, and H. Felbeck. 1988. Chemoautotrophic symbiosis in a hydrothermal vent gastropod. Biol. Bull. 174:373-378.[Abstract/Free Full Text]
43
43. Sunamura, M., Y. Higashi, C. Miyako, J. Ishibashi, and A. Maruyama. 2004. Two bacteria phylotypes are predominant in the Suiyo Seamount hydrothermal plume. Appl. Environ. Microbiol. 70:1190-1198.[Abstract/Free Full Text]
44
44. Swofford, D. L. 1999. PAUP*: phylogenetic analysis using parsimony (* and other methods), version 4.02 ed. Sinauer Associates, Sunderland, Mass.
45
45. Takai, K., T. Gamo, U. Tsunogai, N. Nakayama, H. Hirayama, K. H. Nealson, and K. Horikoshi. 2004. Geochemical and microbiological evidence for a hydrogen-based, hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field. Extremophiles 8:269-282.[Medline]
46
46. Takai, K., F. Inagaki, S. Nakagawa, H. Hirayama, T. Nunoura, Y. Sako, K. H. Nealson, and K. Horikoshi. 2003. Isolation and phylogenetic diversity of members of previously uncultivated -Proteobacteria in deep-sea hydrothermal fields. FEMS Microbiol. Lett. 218:167-174.[Medline]
47
47. Takeda, Y., F. Suzuki, and H. Inoue. 1969. ATP citrate lyase (citrate-cleavage enzyme). Methods Enzymol. 13:153-160.
48
48. Teske, A., K. U. Hinrichs, V. Edgcomb, A. de Vera Gomez, D. Kysela, S. P. Sylva, M. L. Sogin, and H. W. Jannasch. 2002. Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities. Appl. Environ. Microbiol. 68:1994-2007.[Abstract/Free Full Text]
49
49. van der Meer, M. T. J., S. Schouten, and J. S. Sinninghe Damste. 1998. The effect of the reversed tricarboxylic acid cycle on the ¹³C contents of bacterial lipids. Org. Geochem. 28:527-533.
50
50. Van Dover, C. L. 2000. The ecology of deep-sea hydrothermal vents. Princeton University Press, Princeton, N.J.
51
51. Van Dover, C. L. 2002. Trophic relationships among invertebrates at the Kairei hydrothermal vent field (Central Indian Ridge). Mar. Biol. 141:761-772.[CrossRef]
52
52. Van Dover, C. L., and B. Fry. 1989. Stable isotopic compositions of hydrothermal vent organisms. Mar. Biol. 102:257-263.[CrossRef]
53
53. Van Dover, C. L., S. E. Humphris, D. Fornari, C. M. Cavanaugh, R. Collier, S. K. Goffredi, J. Hashimoto, M. D. Lilley, A. L. Reysenbach, T. M. Shank, K. L. Von Damm, A. Banta, R. M. Gallant, D. Gotz, D. Green, J. Hall, T. L. Harmer, L. A. Hurtado, P. Johnson, Z. P. McKiness, C. Meredith, E. Olson, I. L. Pan, M. Turnipseed, Y. Won, C. R. Young, and R. C. Vrijenhoek. 2001. Biogeography and ecological setting of Indian Ocean hydrothermal vents. Science 294:818-823.[Abstract/Free Full Text]
54
54. Waren, A., and P. Bouchet. 2001. Gastropoda and monoplacophora from hydrothermal vents and seeps: new taxa and records. Veliger 44:116-231.
55
55. Wirsen, C. O., S. M. Sievert, C. M. Cavanaugh, S. J. Molyneaux, A. Ahmad, L. T. Taylor, E. F. Delong, and C. D. Taylor. 2002. Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur. Appl. Environ. Microbiol. 68:316-325.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, September 2005, p. 5440-5450, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5440-5450.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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