HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Miroshnichenko, M. L. Articles by Bonch-Osmolovskaya, E. A. Search for Related Content PubMed PubMed Citation Articles by Miroshnichenko, M. L. Articles by Bonch-Osmolovskaya, E. A. Agricola Articles by Miroshnichenko, M. L. Articles by Bonch-Osmolovskaya, E. A.

Caldithrix abyssi gen. nov., sp. nov., a nitrate-reducing, thermophilic, anaerobic bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent, represents a novel bacterial lineage

¹ Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, 117811 Moscow, Russia
² AN Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia
³ Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig, Germany

Correspondence
Margarita L. Miroshnichenko
alfamirr{at}mail.ru

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
A novel, moderately thermophilic, strictly anaerobic, mixotrophic bacterium, designated strain LF13^T, was isolated from a deep-sea hydrothermal chimney sample that was collected at a vent site at 14° 45' N, 44° 59' W on the Mid-Atlantic Ridge. Cells were Gram-negative, thin, non-motile rods of variable length. Strain LF13^T grew optimally at pH 6·8–7·0 and 60 °C with 2·5 % (w/v) NaCl. It grew chemo-organoheterotrophically, fermenting proteinaceous substrates, pyruvate and Casamino acids. The strain was able to grow by respiration, utilizing molecular hydrogen (chemolithoheterotrophically) or acetate as electron donors and nitrate as an electron acceptor. Ammonium was formed in the course of denitrification. One-hundred milligrams of yeast extract per litre were required for growth of the strain. The G+C content of the genomic DNA of strain LF13^T was 42·5 mol%. Neither 16S rDNA sequence similarity values nor phylogenetic analysis unambiguously related strain LF13^T with members of any recognized bacterial phyla. On the basis of 16S rDNA sequence comparisons, and in combination with physiological and morphological traits, a novel genus, Caldithrix, is proposed, with strain LF13^T (=DSM 13497^T =VKM B-2286^T) representing the type species, Caldithrix abyssi.

The GenBank accession number for the 16S rDNA sequence of Caldithrix abyssi LF13^T is AJ430587.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Due to sharp physical and chemical gradients, deep-sea hydrothermal vents provide a variety of microniches that can potentially be habitats for physiologically diverse thermophilic and hyperthermophilic micro-organisms (Prieur et al., 1995). The application of molecular biological approaches to submarine hydrothermal vents has confirmed the presence of phylogenetically variable microbial communities in submarine hydrothermal vents (Moyer et al., 1995; Harmsen et al., 1997; Reysenbach et al., 2000; Jeanthon, 2000). Up until now, many novel micro-organisms residing in hydrothermal microbial communities have been enriched for and have been isolated by classical microbiological methods (i.e. culturing). Most of these novel organisms have been anaerobic, chemolithotrophic or chemo-organotrophic representatives of the Archaea. Bacteria isolated from hydrothermal habitats have been found to possess versatile metabolic pathways. Chemolithoautotrophic prokaryotes inhabiting deep-sea hot vents are represented by the anaerobic sulfur-reducing bacteria Desulfurobacterium thermolithotrophum (L'Haridon et al., 1998) and Nautilia lithotrophica (Miroshnichenko et al., 2002), the sulfur- and nitrate-reducing bacterium Caminibacter hydrogeniphilus (Alain et al., 2002a), the sulfate-reducing bacterium Thermodesulfobacterium hydrohenophilum (Jeanthon et al., 2002) and two microaerophilic bacteria of the recently described genus Persephonella (Götz et al., 2002), which are also able to reduce sulfur and nitrate. Chemo-organoheterotrophic species include aerobic thermophilic bacteria of the genera Bacillus and Thermus (Marteinsson et al., 1995, 1999) and the anaerobes Thermosipho melanesiensis (Antoine et al., 1997), Thermosipho japonicus (Takai & Horikoshi, 2000), Marinitoga camini (Wery et al., 2001), Marinitoga piezophila (Alain et al., 2002b) and Caminicella sporogenes (Alain et al., 2002c). All anaerobic organotrophic bacteria isolated so far from the deep-sea hydrothermal environment possess a fermentative type of metabolism; for some of these species, the ability to reduce sulfur or thiosulfate in the course of fermentation has been reported (Wery et al., 2001; Antoine et al., 1997). In this report, we present the description of a novel obligately anaerobic organism, strain LF13^T, which was isolated from a hydrothermal vent of the Mid-Atlantic Ridge. This novel bacterium is capable of fermentation and nitrate respiration.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Sampling.
A sample was obtained during the 41st cruise of the Russian scientific vessel A. Mstislav Keldish in 1998 from the Logatchev hydrothermal field (14°45'N, 44°59'W) on the Mid-Atlantic Ridge. A sample of a sulfidic chimney wall was collected, during a dive by the submersible Mir, at a depth of 3000 m. Immediately after being taken on board the ship, the sample was placed in a sterile container with Ar gas, where it was stored at between 3 and 5 °C.

Enrichments and isolation.
For the enrichment, the following basal medium (BM) was used (gl^-1): NH₄Cl, 0·33; KCl, 0·33; KH₂PO₄, 0·33; CaCl₂.2H₂O, 0·33; MgCl₂.6H₂O, 0·33; NaCl, 25·0; yeast extract, 0·1; Na₂S.9H₂O, 0·7; NaHCO₃, 0·3; resazurin, 0·002; 1 ml trace elements l^-1 (Balch et al., 1979); 1 ml vitamins l^-1 (Wolin et al., 1963). The BM was supplemented with 3 g sodium acetate l^-1 and 2 g sodium nitrate l^-1. The medium was prepared anaerobically (Balch et al., 1979), dispensed into 15 ml Hungate tubes and inoculated with pieces of the chimney sample (5 %, v/v). The head space was filled with a N₂ mixture (atmospheric pressure). The pH of the medium was adjusted with 6 M HCl to pH 6·8–7·0. Colonies were obtained on BM agar (1·5 % agar; Difco) using the agar-shake method. Tubes were incubated at 60 °C for 5 days.

Morphological and ultrastructure studies.
Morphology of strain LF13^T was studied using a light microscope (Mikmed-1; LOMO). The ultrastructure of whole cells and thin-section preparations were studied using a model JEM-100 electron microscope. Cells were prepared as described previously (Bonch-Osmolovskaya et al., 1990).

Physiological studies.
Potential growth substrates were added to BM to a concentration of 0·3 % (w/v). When molecular hydrogen served as the substrate, the head space (10 ml) was filled with a H₂/CO₂ mixture (4 : 1, v/v). Possible electron acceptors were tested at concentrations of 0·2 % (w/v) except for elemental sulfur, which was added at 1 %. All experiments were performed in triplicate. The pH range for growth was determined in the culture medium with the various buffers at a concentration of 10 mM (MES for pH 5·0–6·0; PIPES for pH 6·5 and 7·0; HEPES for pH 7·5; Tris for pH 8·0 and 8·5). To determine the requirement of strain LF13^T for NaCl, BM was prepared with different concentrations of NaCl. Bacterial growth was followed by phase-contrast microscopy.

Analytical methods.
Cell density was determined by direct cell counting under a light microscope. Gaseous and liquid fermentation products were detected by GLC (Miroshnichenko et al., 1994). NO, N₂O and N₂ were detected using a GC apparatus with a Porapak-Q column at 70 °C and flow rates of 4 mlmin^-1 (the carrier gas was Ar). For the quantitative determination of ammonium, 0·05 ml culture medium was added to 0·1 ml Nessler reagent and mixed with 2 ml deionized water. The formation of a yellow colour indicated the presence of ammonium, which was quantified colorimetrically by measurement of the optical density at 410 nm. For quantitative nitrite analysis, 0·1 ml culture medium was added to a mixture containing 0·9 ml deionized water, 0·5 ml of a 0·6 % solution of sulfanilic acid in 20 % HCl, and 0·5 ml of a solution (60 mg per 50 ml) of N-naphthylethylenediamine (NEDA). Absorbance at 548 nm was measured after a 15 min incubation, necessary for colour development. For quantitative nitrate analysis, the method of Cataldo et al. (1975) was used.

Determination of DNA G+C content.
DNA was isolated and purified from lysozyme- and SDS-treated cells by the method of Marmur (1961). The G+C content was determined by the denaturation method (Owen & Lapage, 1976).

16S-rDNA-based phylogenetic analysis.
Extraction of genomic DNA, PCR-mediated amplification of the 16S rDNA and direct sequencing of the purified PCR product were carried out according to Rainey et al. (1996). The sequence reaction mixtures were electrophoresed using a model 373A automated DNA sequencer (Applied Biosystems). The 16S rDNA sequence of strain LF13^T was aligned with published sequences obtained from the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk) and the Ribosomal Database Project (RDP; http://rdp.cme.msu.edu/html/) using the AE2 editor (Maidak et al., 2001) and the ARB program (Ludwig & Strunk, 1997). Evolutionary distances were calculated based on the algorithms of Jukes & Cantor (1969), DeSoete (1983) and Felsenstein (1993), using neighbour-joining, maximum-likelihood and parsimony methods of tree reconstruction.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Enrichments and isolation
Anaerobic BM supplemented with 2 g sodium acetate l^-1, 1 g sodium nitrate l^-1 and 200 mg yeast extract l^-1 was inoculated with the chimney sample and incubated at 60 °C for 5 days. In the primary enrichment culture, the growth of morphologically diverse micro-organisms was observed, among them cocci and rods of various length, sometimes with sheaths. The culture was diluted and transferred into BM agar. After 5 days incubation at 60 °C, single colonies were visible in the tubes inoculated with the highest dilutions. Colonies were round and white, with their diameter varying from 0·3 to 1 mm. Colonies were transferred into BM broth; after 3 days incubation at 60 °C two types of micro-organisms were found growing in the liquid cultures – thin, long rods and coccoid cells. For the rod-shaped organism, the purification procedure was repeated twice; after the second purification, the culture was considered to be pure. Purity was confirmed by microscopic examination of the culture growing in medium containing 3 g glucose l^-1, 3 g pyruvate l^-1 and 3 g yeast extract l^-1. The micro-organism obtained was designated strain LF13^T and chosen for detailed studies.

Morphology and ultrastructure of strain LF13^T
Cells of strain LF13^T were thin rods of 0·2–0·35 µm in diameter and 4–20 µm in length (Fig. 1a). Young cells stained Gram-negative. Flagella were not seen on negatively stained electron preparations, though tumbling motility was sometimes observed in the exponential phase of growth. In the stationary phase, spherical empty bodies protruding from different parts of the cells were often observed (Fig. 1c). When they were part of the cell, the spherical bodies were covered by common envelopes, but they could be seen separately as well. Thin-section preparations revealed strain LF13^T to have a Gram-negative type of cell wall (Fig. 1b). Formation of spores by the strain was never observed.

View larger version (116K): [in this window] [in a new window]   Fig. 1. (a) Electron micrograph of a negatively stained cell of strain LF13T. Bar, 0·5 µm. (b) Electron micrograph of thin-section of a cell of strain LF13T, exhibiting cell-wall structure. CM, cytoplasmic membrane; OM, outer membrane. Bar, 0·5 µm. (c) Electron micrograph of thin-section of a cell of strain LF13T, exhibiting formation of a spherical body. Bar, 0·5 µm.

Nutrition
Strain LF13^T grew well with complex proteinaceous substrates, such as beef extract, soy bean, peptone and yeast extract. Fermentation products detected during growth of the strain with yeast extract were molecular hydrogen, acetate and propionate. Strain LF13^T also utilized Casamino acids and pyruvate, forming molecular hydrogen and acetate as the growth products in the latter case. Yeast extract (0·01%) was obligately required for growth of the strain. Strain LF13^T was found to be capable of anaerobic respiration, with acetate used as the electron donor and nitrate as the electron acceptor (Fig. 2). The only detected product of nitrate reduction was ammonium. Other possible products of nitrate reduction (NO₂^-, N₂, NO, N₂O) were not detected. The novel strain was also able to grow lithotrophically with molecular hydrogen as the energy source and nitrate as the electron acceptor. Yeast extract (0·01%) was necessary for lithotrophic growth. Electron acceptors other than nitrate (i.e. sulfate, elemental sulfur, thiosulfate, nitrite) did not support growth of strain LF13^T. No growth of the strain was detected with starch, cellobiose, dextrin, sucrose, glucose, galactose, xylose, maltose, ethanol, methanol, mannitol, propionate, butyrate or lactate, both in the presence and absence of nitrate.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Growth of strain LF13T at 60 °C in BM supplemented with 2 g sodium acetate l-1, 1 g sodium nitrate l-1 and 200 mg yeast extract l-1. , Cell number; , acetate consumption; , nitrate consumption; , ammonium production.

16S rDNA sequence analyses
As a first step in the analyses, the almost-complete 16S rDNA sequence (1504 nt) of strain LF13^T was analysed by BLAST, revealing it to be most similar to an environmental partial sequence retrieved from a shallow-water hydrothermal vent near the Isle of Milos, Greece (uncultured vent bacterium ML-5, accession no. AF209001). In the overlapping region of both sequences (Escherichia coli positions 358–906), the sequences displayed 98·2 % similarity, indicating a close phylogenetic relationship between the unknown hydrothermal vent bacterium and strain LF13^T, at least at the genus level. Moorella thermacetica, a member of the Clostridium–Bacillus line of descent, was reported by BLAST as the cultured organism most closely related to strain LF13^T, but the sequence similarity was only 82·3 %. Phylogenetic analyses resulted in different affiliations of strain LF13^T, depending upon the algorithm used and the reference organisms included in the analyses. Using a broad selection of 85 reference sequences from different bacterial phyla, maximum-likelihood analysis (using FASTDNAML implemented in the ARB package) placed strain LF13^T at the root of the Deferribacter and Nitrospina lines of descent. Neighbour-joining analysis based on a selection of reference sequences using the AE2 editor (Maidak et al., 2001) or ARB (PHYLIP) placed strain LF13^T at the root of the phylum Cytophaga–Flavobacterium–Bacteroides or close to the Deferribacter, Nitrospina and Thermodesulfobacterium lines of descent, respectively. The maximum-parsimony tool of ARB, allowing comparison of the novel sequence with an almost-complete dataset of available full-length 16S rRNA gene sequences, placed strain LF13^T at the root of the Green Sulfur Bacteria division (phylum Chlorobium). Analysis by the algorithm of DeSoete (1983) indicated strain LF13^T to form an individual lineage comparable to phylum status. Detailed pairwise analyses of sequences indicated that except for the values obtained with clone sequences retrieved from environmental samples no similarity value was higher than 82·3 % (Moorella thermacetica). Representatives of different phyla showed similarity values of less than 80 % with the 16S rDNA sequence of strain LF13^T. Consequently, the phylogenetic tree shown in Fig. 3, based on an analysis of more than 100 sequences, should be judged as a tentative model used to illustrate the isolated position of strain LF13^T among other phyla of the Bacteria. Bootstrapping analyses of the tree topology revealed very low confidence values for most branches. A common origin for the Deferribacter and strain LF13^T line of descent is supported by a bootstrap value of only 21 %, which is statistically insignificant, whereas the clustering of strain LF13^T with the sequence of the uncultured vent bacterium ML-5 is supported by a bootstrap value of around 90 %.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Global phylogenetic tree based on 16S rRNA gene sequences showing the position of Caldithrix abyssi among the main lines of descent of the Bacteria. The tree is derived from a distance matrix constructed from a selection of over 100 sequences representing the major phyla of the Bacteria and Archaea. The neighbour-joining method of Saitou & Nei (1987) was used to reconstruct the matrix and the algorithm described by Felsenstein (1982) was used to calculate phylogenetic distance values. The partial sequence (553 nt) of the uncultured vent bacterium ML-5 was included in the tree by using an option of the parsimony method implemented in the ARB package that allows incorporation of sequences in existing trees without changing their topology. Bar, estimated base exchanges. Accession numbers for the sequences neighbouring the sequence of Caldithrix abyssi are as follows: ML-5, AF209001; Nitrospina gracilis, L35504; Thermodesulfobacterium hveragerdense, X96725. The phylum Deferribacteres is represented by the sequences of Deferribacter thermophilus (U75602) and Geovibrio ferrireducens (X95744). Phyla have been named according to Garrity & Holt (2001).

Thus, depending on the environmental conditions, strain LF13^T could play different roles in the hydrothermal ecosystem: it could either ferment organic molecules, or use molecular hydrogen or acetate for nitrate reduction. Its only close phylogenetic relative was an uncultured hydrothermal bacterium (ML-5; Sievert et al., 2000). The 16S rDNA fragment of ML-5 was detected by denaturing-gradient gel electrophoresis (DGGE); the sample containing this fragment was taken at a distance of 1·23 m away from the centre of a hydrothermal vent. The respective DGGE band was most intense in the sample obtained at a sediment depth of 10–20 mm. In earlier work, Sievert et al. (1999) reported that oxygen is absent in deeper sediment layers of hydrothermal vents and that temperature increases rapidly with depth. The physicochemical conditions in the habitat of ML-5 may, therefore, be comparable to the isolation site of strain LF13^T at the deep-sea hydrothermal vent with the difference that the water depth was only 8 m for ML-5, in contrast to 3000 m for strain LF13^T. As described by Sievert et al. (2000), it can be claimed that the retrieved sequence ML-5 represents a dominant fraction of the microbial population in the studied habitat.

At present, the affiliation of strain LF-13^T to one of the main lines of descent within the domain Bacteria is a moot point, as neither physiological nor morphological characteristics would support clustering of the strain with recognized phyla. Inclusion of this Gram-negative bacterium within the phylum of Gram-positive bacteria cannot be excluded, as more than 20 Gram-negative genera are affiliated to this phylum, e.g. Heliobacterium, Dialister and Megasphaera (Stackebrandt & Hippe, 2001). Sequences of broader phylogenetic depth and width of this newly recognized taxon are needed to eventually stabilize its phylogenetic position. However, the lack of convincing relatedness of strain LF13^T to any described genus, supported by metabolic and cultural properties, facilitates the proposal to describe a novel genus, Caldithrix, for the sole species Caldithrix abyssi (strain LF13^T). The depth of the branching point of the Caldithrix lineage indicates that this genus represents an even higher taxon, at least at the order level. It is, however, prudent not to describe a higher taxon before a broader range of organisms have been affiliated to the Caldithrix line of descent.

Description of Caldithrix gen. nov.
Caldithrix (Cal.di'thrix. L. fem. adj. caldus hot; Gr. fem. n. thrix thread; N.L. fem. n. Caldithrix a thread existing in a hot environment).

Cells are Gram-negative, long rods. Moderately thermophilic and neutrophilic. Adapted to the salinity of the marine environment. Anaerobe. Chemo-organoheterotroph capable of fermenting proteinaceous substrates. Capable of anaerobic respiration with molecular hydrogen or acetate as electron donors and nitrate as an electron acceptor. Ammonium is the only product of denitrification. 16S rDNA sequence analyses do not place the genus Caldithrix in any of the recognized phyla of the domain Bacteria. Isolated from a deep-sea hydrothermal-vent chimney. The type species of the genus is Caldithrix abyssi.

Description of Caldithrix abyssi sp. nov.
Caldithrix abyssi (a.bys'si. L. fem. gen. n. abyssi of immense depths, living in the depth of the ocean).

Cells are Gram-negative rods of 0·2–0·35 µm in width; their length is variable. Temperature range for growth is 40–70 °C, with optimum growth at 60 °C. pH range for growth is between pH 5·8 and 7·8, with optimum growth between pH 6·8 and 7·0. Optimum salinity is 2·5 %; growth occurs between 0·1 and 5·0 % NaCl. Able to ferment pyruvate, Casamino acids and proteinaceous substrates. Capable of lithoheterotrophic growth with molecular hydrogen or acetate, in the presence of nitrate. In the course of denitrification, ammonium is produced. Yeast extract is required for growth. DNA G+C content is 42·5 mol%. Isolated from a deep-sea hydrothermal-vent chimney at 14° 45' N, 44° 59' W on the Mid-Atlantic Ridge. The type strain is LF13^T (=DSM 13497^T =VKM B-2286^T).

	ACKNOWLEDGEMENTS

 
This work was supported by INTAS grant no. 99-1250, RFBR grant no. 00-04-48924 and the ‘Biodiversity’ Programme of the Russian Ministry of Industry, Science and Technology.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Alain, K., Querellou, J., Lesongeur, F., Pignet, P., Crassous, P., Raguenes, G., Cueff, V. & Cambon-Bonavita, M.-A. (2002a). Caminibacter hydrogeniphilus gen. nov., sp. nov., a novel thermophilic hydrogen-oxidizing bacterium isolated from an East Pacific Rise hydrothermal vent. Int J Syst Evol Microbiol 52, 1317–1323.[Abstract]

Alain, K., Marteinsson, V. T., Miroshnichenko, M. L., Bonch-Osmolovskaya, E. A., Prieur, D. & Birrien, J.-L. (2002b). Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 52, 1331–1339.[Abstract]

Alain, K., Pignet, P., Zbinden, M. & 8 other authors (2002c). Caminicella sporogenes gen. nov., sp. nov., a novel thermophilic spore-forming bacterium isolated from an East-Pacific Rise hydrothermal vent. Int J Syst Evol Microbiol 52, 1621–1628.[Abstract]

Antoine, E., Cilla, V., Meunier, J. R., Guezennec, J., Lesongeur, F. & Barbier, G. (1997). Thermosipho melanesiensis sp. nov., a new thermophilic anaerobic bacterium belonging to the order Thermotogales, isolated from deep-sea hydrothermal vents in the southwestern Pacific Ocean. Int J Syst Bacteriol 47, 1118–1123.[Abstract/Free Full Text]

Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R. & Wolfe, R. S. (1979). Methanogens: reevaluation of a unique biological group. Microbiol Rev 43, 260–296.[Free Full Text]

Blöchl, E., Rachel, R., Burggraf, S., Hafenbradl, D., Jannasch, H. W. & Stetter, K. O. (1997). Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113 °C. Extremophiles 1, 14–21.[CrossRef][Medline]

Bonch-Osmolovskaya, E. A., Sokolova, T. G., Kostrikina, N. A. & Zavarzin, G. A. (1990). Desulfurella acetivorans gen. nov. and sp. nov. – a new thermophilic sulfur-reducing eubacterium. Arch Microbiol 153, 151–155.[CrossRef]

Caldwell, D. E., Caldwell, S. J. & Laycock, J. P. (1976). Thermothrix thiopara gen. et sp. nov., a facultatively anaerobic facultative chemolithotroph living at neutral pH and high temperature. Can J Microbiol 22, 1509–1517.[Medline]

Cataldo, D. A., Haroon, M., Schrader, L. E. & Youngs, V. L. (1975). Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun Soil Sci Plant Anal 6, 71–80.

DeSoete, G. (1983). A least squares algorithm for fitting additive trees to proximity data. Psychometrika 48, 621–626.[CrossRef]

Felsenstein, J. (1982). Numerical methods for inferring phylogenetic trees. Q Rev Biol 57, 379–404.[CrossRef]

Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.

Garrity, G. M. & Holt, J. G. (2001). Taxonomic outline of the Archaea and Bacteria. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 155–166. Edited by D. R. Boone & R. W. Castenholz. New York: Springer-Verlag.

Götz, D., Banta, A., Beveridge, T. J., Rushdi, A. I., Simoneit, B. R. T. & Reysenbach, A.-L. (2002). Persephonella marina gen. nov., sp. nov. and Persephonella guaymasensis sp. nov., two novel, thermophilic, hydrogen-oxidizing microaeropholes from deep-sea hydrothermal vents. Int J Syst Evol Microbiol 52, 1349–1359.[Abstract]

Hafenbradl, D., Keller, M., Dirmeier, R., Rachel, R., Roßnagel, P., Burggraf, S., Huber, H. & Stetter, K. O. (1996). Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe²⁺ at neutral pH under anoxic conditions. Arch Microbiol 166, 308–314.[CrossRef][Medline]

Harmsen, H. J. M., Prieur, D. & Jeanthon, C. (1997). Distribution of microorganisms in deep-sea hydrothermal vent chimneys investigated by whole-cell hybridization and enrichment culture of thermophilic subpopulations. Appl Environ Microbiol 63, 2876–2853.[Abstract]

Huber, R., Wilharm, T., Huber, D. & 7 other authors (1992). Aquifex pyrophilus gen. nov., sp. nov., represents a novel group of marine hyperthermophilic hydrogen-oxidizing bacteria. Syst Appl Microbiol 15, 340–351.

Huber, H., Rossnagel, P., Woese, C. R., Rachel, R., Langworthy, T. A. & Stetter, K. O. (1996). Formation of ammonium from nitrate during chemolithoautotrophic growth of the extremely thermophilic bacterium Ammonifex degensii gen. nov. sp. nov. Syst Appl Microbiol 19, 40–49.[Medline]

Huber, H., Diller, S., Horn, C. & Rachel, R. (2002). Thermovibrio ruber gen. nov., sp. nov., an extremely thermophilic, chemolithoautotrophic, nitrate-reducing bacterium that forms a deep branch within the phylum Aquificae. Int J Syst Evol Microbiol 52, 1859–1865.[Abstract]

Jeanthon, C. (2000). Molecular ecology of hydrothermal vent microbial communities. Antonie van Leeuwenhoek 77, 117–133.[CrossRef][Medline]

Jeanthon, C., L'Haridon, S., Cueff, V., Banta, A., Reysenbach, A.-L. & Prieur, D. (2002). Thermodesulfobacterium hydrogeniphilum sp. nov., a thermophilic, chemolithoautotrophic, sulfate-reducing bacterium isolated from a deep-sea hydrothermal vent at Guaymas Basin, and emendation of the genus Thermodesulfobacterium. Int J Syst Evol Microbiol 52, 765–772.[Abstract]

Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.

L'Haridon, S., Cilia, V., Messner, P., Raguenes, G., Gambacorta, A., Sleytr, U. B., Prieur, D. & Jeanthon, C. (1998). Desulfurobacterium thermolithotrophum gen. nov., sp. nov., a novel autotrophic, sulfur-reducing bacterium isolated from a deep-sea hydrothermal vent. Int J Syst Bacteriol 48, 701–711.[Abstract/Free Full Text]

Ludwig, W. & Strunk, O. (1997). The ARB project (http://www.arb-home.de) (last accessed 5 June 2002).

Maidak, B. L., Cole, J. R., Lilburn, T. G. & 7 other authors (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res 29, 173–174.[Abstract/Free Full Text]

Marmur, J. (1961). A procedure for the isolation of DNA from microorganisms. J Mol Biol 3, 208–218.

Marteinsson, V. T., Birrien, J. L., Kristjansson, J. K. & Prieur, D. (1995). First isolation of thermophilic non-sporulating heterotrophic bacteria from deep-sea hydrothermal vents. FEMS Microbiol Ecol 18, 163–174.[CrossRef]

Marteinsson, V. T., Birrien, J. L., Raguenes, G., da Costa, M. S. & Prieur, D. (1999). Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent. Extremophiles 3, 247–251.[CrossRef][Medline]

Miroshnichenko, M. L., Gongadze, G. M., Lysenko, A. M. & Bonch-Osmolovskaya, E. A. (1994). Desulfurella multipotens sp. nov., a new sulfur-respiring thermophilic eubacterium from Raoul Island (Kermadec archipelago). Arch Microbiol 161, 88–93.[CrossRef]

Miroshnichenko, M. L., Kostrikina, N. A., L'Haridon, S., Jeanthon, C., Hippe, H., Stackebrandt, E. & Bonch-Osmolovskaya, E. A. (2002). Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 52, 1299–1304.[Abstract]

Moyer, C. L., Dobb, F. C. & Karl, D. M. (1995). Phylogenetic diversity of the bacterial community from a microbial mat at an active, hydrothermal vent system. Appl Environ Microbiol 61, 1555–1562.[Abstract]

Nazina, T. N., Tourova, T. P., Poltaraus, A. B. & 8 other authors (2001). Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51, 433–446.[Abstract]

Owen, R. J. & Lapage, S. P. (1976). The thermal denaturation of partly purified bacterial deoxyribonucleic acid and its taxonomic applications. J Appl Bacteriol 41, 335–340.[Medline]

Prieur, D., Erauso, G. & Jeanthon, C. (1995). Hyperthermophilic life at deep-sea hydrothermal vents. Planet Space Sci 43, 115–122.

Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.[Abstract/Free Full Text]

Reysenbach, A.-L., Longnecker, K. & Kirshtein, J. (2000). Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent. Appl Environ Microbiol 66, 3798–3806.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sievert, S. M., Brinkhoff, T., Muyzer, G., Ziebis, W. & Kuever, J. (1999). Spatial heterogeneity of bacterial populations along an environmental gradient at a shallow submarine hydrothermal vent near Milos Island (Greece). Appl Environ Microbiol 65, 3834–3842.[Abstract/Free Full Text]

Sievert, S. M., Kuever, J. M. & Muyzer, G. (2000). Identification of 16S ribosomal DNA-defined bacterial populations at a shallow submarine hydrothermal vent near Milos Island (Greece). Appl Environ Microbiol 66, 3102–3109.[Abstract/Free Full Text]

Slobodkin, A. I., Zavarzina, D. G., Sokolova, T. G. & Bonch-Osmolovskaya, E. A. (1999). Dissimilatory reduction of inorganic electron acceptors by anaerobic thermophilic prokaryotes. Microbiology (English translation of Mikrobiologiya) 68, 522–542.

Stackebrandt, E. & Hippe, H. (2001). Taxonomy and systematics. In Clostridia. Biotechnology and Medical Applications, pp. 19–48. Edited by H. Bahl & P. Dürre. Weinheim: Wiley-VHC.

Takai, K. & Horikoshi, K. (2000). Thermosipho japonicus sp. nov., an extremely thermophilic bacterium isolated from a deep-sea hydrothermal vent in Japan. Extremophiles 4, 9–17.[Medline]

Völkl, P., Huber, R., Drobner, E., Rachel, R., Burggraf, S., Trincone, A. & Stetter, K. O. (1993). Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum. Appl Environ Microbiol 59, 2918–2926.[Abstract/Free Full Text]

Wery, N., Lesongeur, F., Pignet, P., Derennes, V., Cambon-Bonavita, M.-A., Godfroy, A. & Barbier G. (2001). Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 51, 495–504.[Abstract]

Williams, R. A. D. & da Costa, M. S. (1992). The genus Thermus and related microorganisms. In The Prokaryotes, 2nd edn, vol. 1, pp. 3746–3751. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer-Verlag.

Wolin, E. A., Wolin, M. J. & Wolfe, R. S. (1963). Formation of methane by bacterial extracts. J Biol Chem 238, 2882–2888.[Free Full Text]

This article has been cited by other articles:

				K. Mori, M. Sunamura, K. Yanagawa, J.-i. Ishibashi, Y. Miyoshi, T. Iino, K.-i. Suzuki, and T. Urabe First Cultivation and Ecological Investigation of a Bacterium Affiliated with the Candidate Phylum OP5 from Hot Springs Appl. Envir. Microbiol., October 15, 2008; 74(20): 6223 - 6229. [Abstract] [Full Text] [PDF]

				J. W. Voordeckers, V. Starovoytov, and C. Vetriani Caminibacter mediatlanticus sp. nov., a thermophilic, chemolithoautotrophic, nitrate-ammonifying bacterium isolated from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge Int J Syst Evol Microbiol, March 1, 2005; 55(2): 773 - 779. [Abstract] [Full Text] [PDF]

				M. L. Miroshnichenko, S. L'Haridon, P. Schumann, S. Spring, E. A. Bonch-Osmolovskaya, C. Jeanthon, and E. Stackebrandt Caminibacter profundus sp. nov., a novel thermophile of Nautiliales ord. nov. within the class 'Epsilonproteobacteria', isolated from a deep-sea hydrothermal vent Int J Syst Evol Microbiol, January 1, 2004; 54(1): 41 - 45. [Abstract] [Full Text] [PDF]

				C. Vetriani, M. D. Speck, S. V. Ellor, R. A. Lutz, and V. Starovoytov Thermovibrio ammonificans sp. nov., a thermophilic, chemolithotrophic, nitrate-ammonifying bacterium from deep-sea hydrothermal vents Int J Syst Evol Microbiol, January 1, 2004; 54(1): 175 - 181. [Abstract] [Full Text] [PDF]

				M. L. Miroshnichenko, A. I. Slobodkin, N. A. Kostrikina, S. L'Haridon, O. Nercessian, S. Spring, E. Stackebrandt, E. A. Bonch-Osmolovskaya, and C. Jeanthon Deferribacter abyssi sp. nov., an anaerobic thermophile from deep-sea hydrothermal vents of the Mid-Atlantic Ridge Int J Syst Evol Microbiol, September 1, 2003; 53(5): 1637 - 1641. [Abstract] [Full Text] [PDF]

				M. L. Miroshnichenko, S. L'Haridon, O. Nercessian, A. N. Antipov, N. A. Kostrikina, B. J. Tindall, P. Schumann, S. Spring, E. Stackebrandt, E. A. Bonch-Osmolovskaya, et al. Vulcanithermus mediatlanticus gen. nov., sp. nov., a novel member of the family Thermaceae from a deep-sea hot vent Int J Syst Evol Microbiol, July 1, 2003; 53(4): 1143 - 1148. [Abstract] [Full Text] [PDF]

				M. L. Miroshnichenko, S. L'Haridon, C. Jeanthon, A. N. Antipov, N. A. Kostrikina, B. J. Tindall, P. Schumann, S. Spring, E. Stackebrandt, and E. A. Bonch-Osmolovskaya Oceanithermus profundus gen. nov., sp. nov., a thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium from a deep-sea hydrothermal vent Int J Syst Evol Microbiol, May 1, 2003; 53(3): 747 - 752. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Miroshnichenko, M. L. Articles by Bonch-Osmolovskaya, E. A. Search for Related Content PubMed PubMed Citation Articles by Miroshnichenko, M. L. Articles by Bonch-Osmolovskaya, E. A. Agricola Articles by Miroshnichenko, M. L. Articles by Bonch-Osmolovskaya, E. A.