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1 Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2 Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
Correspondence
Satoshi Nakagawa
nakasato{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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The DDBJ/EMBL/GenBank accession number for the 16S rDNA sequence of strain 29WT is AB086419.
Supplementary data are available in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Kryukov et al., 1984; Huber et al., 1992, 1998; Shima & Suzuki, 1993; Hugenholtz et al., 1998; L'Haridon et al., 1998; Reysenbach et al., 2000a, b, c; Marteinsson et al., 2001; Takacs et al., 2001; Takai et al., 2001, 2002). Members of the Aquificales are thought to have an impact on biogeochemical processes in these ecosystems (Kristjansson et al., 1985; Harmsen et al., 1997; Götz et al., 2002). In addition, the energy conversion systems and metabolism of Aquificales are interesting in relation to their antagonistic evolutionary traits inferred from rRNA gene trees (Burggraf et al., 1992) and from comparison of the genome sequences (Deckert et al., 1998).
A common property displayed by all of the cultivated strains of Aquificales is obligately or facultatively chemolithoautotrophic growth by oxidation of H2 or reduced sulfur compounds. The most recently constructed phylogenetic tree including the cultivated strains and environmental rDNA clones clearly indicated three distinct lineages (probably classified into Desulfurobacteriaceae, Aquificaceae and Hydrogenothermaceae) within the order Aquificales (Takai et al., 2002; Eder & Huber, 2002). The relatives of Desulfurobacterium are strictly anaerobic H2-oxidizing autotrophs using elemental sulfur, thiosulfate, polysulfide, sulfite or nitrate as electron acceptors (L'Haridon et al., 1998; Huber et al., 2002). Members of the genera Hydrogenobaculum (Shima & Suzuki, 1993; Stöhr et al., 2001), Aquifex (Huber et al., 1992), Thermocrinis (Huber et al., 1998) and Hydrogenobacter (Kawasumi et al., 1984) have been the most extensively studied. Although Thermocrinis ruber (Huber et al., 1998) is capable of chemo-organotrophic growth and Hydrogenobacter subterraneus (Takai et al., 2001) is an obligate aerobic heterotroph, all other members are strict chemolithoautotrophs using H2 and reduced sulfur compounds as electron donors, and O2 and nitrate as electron acceptors.
The last lineage largely consisted of a number of previously uncultivated environmental rDNA clones obtained from global terrestrial hot-spring environments such as in Yellowstone National Park (Hugenholtz et al., 1998; Reysenbach et al., 2000a), Iceland (Skirnisdottir et al., 2000; Takacs et al., 2001) and Japan (Yamamoto et al., 1998), and in subterranean hot springs (Marteinsson et al., 2001), and even in deep-sea hydrothermal vent systems (Reysenbach et al., 2000b, c). Recently, however, several representative strains of this lineage such as Hydrogenothermus (Stöhr et al., 2001), Persephonella (Reysenbach et al., 2000a; Götz et al., 2002) and a potentially new genus of strains (Takai et al., 2002) have been successfully isolated from shallow and deep marine hydrothermal vent environments and from a subsurface hot aquifer environment. Based on their characterization, the newly identified lineage of Aquificales shared similar physiological and metabolic properties with the members of the family Aquificaceae. However, many phylotypes of bacteria within this lineage remain uncultivated and unidentified, and further exploration of uncultivated members will provide important insights. In this study, we sought to cultivate anoxic H2-oxidizing thermophiles using nitrate as an electron acceptor from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc, Japan.
Sample collection, enrichment and purification
Samples from black smoker vents were obtained from the hydrothermal field at the Suiyo Seamount in the Izu-Bonin Arc, Japan (28° 34·287' N, 140° 38·663' E), at a depth of 1385 m, by means of the manned submersible Shinkai 2000 in a dive (dive no. 1237) performed in November 2000. A bulk of chimney having 310·8 °C of vent emission was brought to the sea surface in a sample box supplied with the submersible and immediately subsampled into four different parts (top part, surface layer, inside structure and vent surface) as described by Takai et al. (2001). The chimney was mainly formed from anhydrite (top part), barite (surface layer), pyrite and chalcopyrite (inside structure), and pyrite or chalcopyrite crystals were observed at the passage of hot fluid (vent surface). Each of the subsamples (approx. 10 g) was suspended with 20 ml sterilized MJ synthetic seawater (Sako et al., 1996) containing 0·05 % (w/v) sodium sulfide in a 100 ml glass bottle (Schott Glaswerke) tightly sealed with a butyl rubber cap under a N2 atmosphere. These suspended portions of the subsamples were used to inoculate a series of media including HNW medium (described below) on board.
The enrichment was performed in test tubes (Pyrex; 180x18 mm) containing 5 ml of the medium with 80 % H2+20 % CO2 (300 kPa) tightly sealed with butyl rubber caps and the cultures were incubated at 70 and 85 °C. The tubes of HNW medium inoculated only with a subsample of surface layer became turbid after 1 day incubation at 70 °C and the other tubes did not provide positive enrichments after 5 days incubation at 70 and 85 °C. The enrichment cultures grown at 70 °C contained highly motile cocci. To obtain a pure culture of cocci grown at 70 °C, an extinction-to-dilution method was employed and repeated at least five times. The first pure culture was designated strain 29WT (=DSM 15103T=JCM 11663T) and investigated in detail. The purity was confirmed routinely by microscopic examination and by repeated partial sequencing of the 16S rRNA gene using several PCR primers.
Morphology
Cells were routinely observed with a differential interference microscope (UFX; Nikon). Negative staining of cells for electron microscopy was done with 2 % (v/v) phosphotungstic acid. The cells grown under static culture were Gram-negative cocci about 0·9–1·0 µm in diameter (Fig. 1a). Despite a body of reports on the isolation and characterization of hydrogen-oxidizing thermophiles within the order Aquificales including Persephonella species (Kawasumi et al., 1984; Huber et al., 1992, 1998; Shima & Suzuki, 1993; L'Haridon et al., 1998; Takai et al., 2001, 2002; Götz et al., 2002; Huber et al., 2002), the coccoid morphology is first described here. When grown with agitation, each cell exhibited a straight rod shape with a mean length of 2·3–2·7 µm and a width of approximately 0·4–0·5 µm (Fig. 1b). Both coccoid and rod types of the cells appeared to have a single polar flagellum as observed by electron microscopy and to be highly motile by observation using light microscopy. Pili-like filaments and internal stacked membranes reported in Persephonella marina (Götz et al., 2002) were not observed in thin sections (data not shown). Cells occurred singly or in pairs in all phases of growth and no sporulation was observed.
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) per litre of DMJ synthetic seawater (2/3-fold-diluted MJ synthetic seawater; Sako et al., 1996). To prepare HNW medium, all the components of DMJ seawater were dissolved in 1 litre distilled deionized water, and the pH was adjusted to around 7·0 with NaOH at room temperature prior to autoclaving, unless otherwise noted. After autoclaving, filter-sterilized NaNO3 solution (100 g l-1), NaHCO3 solution (100 g l-1), Na2S solution (100 g l-1; pH 7·5) and trace vitamin solution were added. The tubes were then tightly sealed with butyl rubber stoppers under a gas phase of 80 % H2+20 % CO2 (300 kPa).
Growth of the new isolate under varying conditions was determined by direct cell counts after staining with 4',6-diamidino-2-phenylindole (DAPI) (Porter & Feig, 1980) using a Nikon Eclipse E800 microscope equipped with colour chilled 3 CCD camera system (C5810; Hamamatsu Hokutonikusu). To determine the temperature, pH and NaCl range for growth, duplicate cultures were grown in 100 ml glass bottles (Schott Glaswerke) containing 20 ml medium in an air-batch rotary shaker (RGS-32.TT; Sanki Seiki) and were shaken at 100 r.p.m. in all cases. The isolate grew over a temperature range of about 50–72·5 °C, showing optimum growth at 70 °C, and the generation time at 70 °C was about 40 min. No growth was observed at 75 and 45 °C (supplementary Fig. 1a
in IJSEM Online). Effects of pH and NaCl concentration on growth of the isolate were determined at 70 °C. When the pH optimum was determined, the pH of the pre-heated medium was readjusted immediately before inoculation with H2SO4 or NaOH by using a compact pH meter (Horiba B-212). The pH was found to be stable during the cultivation period. Growth of the new isolate occurred between pH 5·5 and 7·6, with optimum growth at about pH 7·2. No growth was detected below pH 5·5 or above pH 7·6 (supplementary Fig. 1b in IJSEM Online). NaCl requirements were determined with varying concentrations (from 0 to 6 %, w/v) of NaCl in DMJ synthetic seawater. The isolate required NaCl for growth, and grew over a concentration range of about 1·5–5·0 % NaCl, with optimum growth at around 2·5 % NaCl. No growth was observed below 1·5 % NaCl or above 5·0 % NaCl (supplementary Fig. 1c in IJSEM Online).
O2 tolerance and requirements were determined by injecting defined volumes of O2 into culture tubes of HNW medium without Na2S and NaNO3, and O2 final concentrations from 0 to 5 % (v/v) were tested. A very limited amount of O2 (below 1·2 %; optimum 0·6–0·8 %, v/v) supported growth in place of nitrate (supplementary Fig. 2 in IJSEM Online). However, microaerobic growth with the optimal O2 concentration produced a lower growth rate and yield than anaerobic growth with nitrate.
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) in addition to ion chromatography, and Nessler's reagent was employed to measure the ammonia concentration in the medium (Allen et al., 1974). The concentration of N2 in the gas phase was increased with decreasing concentration of nitrate during growth (supplementary Fig. 3 in IJSEM Online). The accumulation of potential end products such as nitrite, N2O and ammonium ions was not detected. These results suggest that the new isolate was a hydrogen-oxidizing chemolithoautotroph preferring anaerobic denitrifying growth to microaerobic growth. In an attempt to examine the alternative electron acceptors which could support growth, 0·1 % (w/v) NaNO2, Na2S2O3.5H2O, Na2S4O6.2H2O and Na2SO3, 3 % (w/v) S0 and 100 mM ferrihydrite were tested in place of nitrate in HNW medium. At the time of analysing ferrihydrite as electron acceptor, Na2S was omitted from the medium. None of these electron acceptors could support growth of the isolate. For the test of the alternative energy sources, 0·1 % (w/v) Na2S2O3, 3 % (w/v) S0 and organic substrates (described below) were used instead of H2 in HNW medium under a gas phase of 80 % N2+20 % CO2 (300 kPa). This test was also conducted under a gas phase of 80 % N2+19 % CO2+1 % O2 (300 kPa) in HNW medium without NaNO3 and Na2S. The isolate could not utilize any of these alternative energy sources. To our knowledge, the isolate is the first example of a hydrogen-dependent, facultatively anaerobic, and chemolithoautotrophic thermophile isolated from deep-sea hydrothermal environments.
When growth of the new isolate was tested in HNW medium (nitrate provided as a sole electron acceptor) in the absence of NaHCO3, no growth was observed at 70 °C even under 80 % H2+20 % CO2 (300 kPa). The results suggested that bicarbonate was the preferred carbon source for the autotrophic growth of the isolate as observed in a mesophilic hydrogen-oxidizer, Ralstonia eutropha (Repaske et al., 1971).
In an attempt to examine organotrophic growth of 29WT, experiments were conducted using HNW medium replacing NaHCO3 and CO2 with various organic carbon sources. Each of the following substrates was added at concentrations of 0·01 and 0·1 % (w/v): L-cystine, L-phenylalanine, L-proline, Casamino acids, D-(+)-glucose, lactose, maltose, chitin, starch, cellulose, formate, acetate, citrate, pyruvate, propionate, 2-propanol, methanol, tryptone peptone (Difco) and yeast extract (Difco). Two gas phases (100 % H2, and 80 % N2+20 % CO2; 300 kPa) were used. These tests were run in duplicate. The isolate was unable to grow under any of the organotrophic conditions tested in this study.
Cellular fatty acid composition
Cellular fatty acid composition was analysed using cells grown in HNW medium at 70 °C with agitation in the late-exponential growth phase. Cellular fatty acid composition analysis was performed at the NCIMB Japan Corporation, Shizuoka, Japan. Fatty acids were converted to methyl esters by treatment with anhydrous methanolic HCl. Fatty acid methyl esters (FAMEs) were analysed by GC using a non-polar capillary column and flame-ionization detection. The major cellular fatty acids were C20 : 1 (40·8 %), C18 : 0 (24·7 %), C18 : 1 (23·8 %) and C16 : 0 (4·2 %). The presence of C18 : 0 and C20 : 1 as the major components is a common feature of members of the order Aquificales. The percentages of each fatty acid of the isolate are particularly similar to those of Persephonella guaymasensis and Hydrogenothermus marinus (Stöhr et al., 2001; Götz et al., 2002). However, the new isolate can be distinguished by the presence of a relatively high amount of C16 : 0.
Isolation and base composition of DNA
Genomic DNA was prepared as described by Lauerer et al. (1986). The G+C content (mol%) of the genomic DNA was determined by direct analysis of the deoxyribonucleotides using HPLC with a DNA-GC kit (Yamasa Shouyu) after digestion of the DNA with nuclease P1 (Tamaoka & Komagata, 1984). The G+C content of the genomic DNA of strain 29WT was found to be 37·3 mol%, which was similar to that of Persephonella species (Table 1).
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). The sequence of the 1·5 kb PCR product was directly determined in both strands using the dideoxynucleotide chain-termination method with the ABI 373A DNA sequencer (Applied Biosystems). The almost complete sequence (1506 bp) of the 16S rDNA of strain 29WT has been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession number AB086419. In order to determine the phylogenetic position of the isolate, the sequence was manually aligned with a subset of 16S rDNA sequences obtained from the DNA Database of Japan (DDBJ) and the Ribosomal Database Project II (RDP-II) (Maidak et al., 2001) by using CLUSTAL X (Thompson et al., 1997). Phylogenetic analyses were restricted to nucleotide positions that were unambiguously alignable in all sequences (Takai & Horikoshi, 1999; Takai & Sako, 1999). Neighbour-joining analysis (Saitou & Nei, 1987) of 914 bases of sequence from each organism was accomplished using CLUSTAL X software. Bootstrap analysis was used for 1000 trial replications to provide confidence estimates for the phylogenetic tree topologies. The phylogenetic tree demonstrated that the new isolate was a member of the genus Persephonella (Fig. 2). However, 16S rDNA sequence similarities between strain 29WT and P. guaymasensis, P. marina and Aquificales strain CIR30126 were 95·6, 95·0 and 96·7 %, respectively. These similarity values indicated that the new isolate was a potentially new species of the genus Persephonella based on the evolutionary distance (97 %) of differentiation on the species level (Stackebrandt & Goebel, 1994).
DNA–DNA hybridization analysis was performed at 30 °C for 3 h and was measured fluorometrically using photobiotin according to the method of Ezaki et al. (1989). P. marina EX-H1T (=DSM 14530T =OCM 794T), P. guaymasensis EX-H2T (=DSM 14351T =OCM 975T) and Aquificales strain CIR30126 (Van Dover et al., 2001) were used as reference strains. Although the phylogenetic analyses based on the 16S rRNA gene sequence indicated that the new isolate was closely related to two known species of the genus Persephonella, the mean hybridization value for the isolate and P. guaymasensis EX-H2T was 5·4 %, the value for the new isolate and P. marina EX-H1T was 4·0 %, and the value for the isolate and Aquificales strain CIR30126 was 10·3 %. These results indicated that the new isolate could be genotypically differentiated from previously described species of the genus Persephonella.
Comparison with related species
Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain 29WT is closely related to the genus Persephonella. Cellular fatty acid composition analysis also supports the new isolate being a member of the genus. However, the isolate differs from previously described Persephonella species in many physiological properties (Table 1). The isolate is coccoid when grown under static culture conditions. This morphological feature is for the first time observed within the order Aquificales. The isolate grows significantly faster (40 min) than P. marina (5·02 h) and P. guaymasensis (7·8 h) when compared under their optimum growth conditions. The temperature range for growth of the new isolate is slightly lower than that of the two species of Persephonella. The new isolate is unable to utilize reduced sulfur compounds as energy sources (Table 1). In addition, DNA–DNA hybridization analysis clearly indicated that the new isolate could be genotypically differentiated from previously described species of the genus Persephonella. On the basis of these physiological and genetic properties, we propose a new species of the genus Persephonella, designated Persephonella hydrogeniphila; the type strain is strain 29WT (=JCM 11663T=DSM 15103T).
Recovery of Persephonella hydrogeniphila only from the surface zone of the chimney structure provides an important clue into delineating the ecological niche of the facultatively anaerobic hydrogen-oxidizing thermophiles. Nitrate is known to be unstable under high temperature and highly reducing environments such as deep-sea hydrothermal vents (Blöchl et al., 1992; Völkl et al., 1993). However, nitrate can be supplied from seawater with oxygen. Accordingly it might be possible that a population of facultatively anaerobic hydrogen-oxidizing thermophiles such as P. hydrogeniphila occurs in the surface area of the chimney, where nitrate and oxygen can be present and hydrogen is provided both from geochemical (gas propagation from the superheated vent fluids) and microbiological (indigenous microbial H2 production by abundant fermentative thermophiles such as Thermococcales and Thermotogales members) processes. In fact, the predominant occurrence of the Thermococcales population is specifically observed in the surface layer of the same chimney structure as observed in a black smoker chimney from the Manus Basin (Takai et al., 2001; K. Takai and others, unpublished results). We also succeeded in isolating both the obligately aerobic thermophiles Marinithermus hydrothermalis and Rhodothermus marinus and the strictly anaerobic nitrate- or sulfur-reducing thermophile Deferribacter sp. from the same subsample used in this study (Sako et al., 2003; Takai et al., 2003). The ecological potential of primary production by facultatively anaerobic, thermophilic chemolithoautotrophs in relatively low temperature and oxidative microhabitats in deep-sea hydrothermal vent environments is still unclear. This is a focus of our future investigations.
Description of Persephonella hydrogeniphila sp. nov.
Persephonella hydrogeniphila (hy.dro.ge.ni.phi'la. N.L. neut. n. hydrogenum hydrogen; Gr. adj. philos loving; N.L. fem. adj. hydrogeniphila hydrogen-loving, because growth depends on hydrogen).
Cells are motile cocci about 0·9–1·0 µm in diameter under static culture. Cells grown with agitation become straight rods 2·3–2·7 µm long and 0·4–0·5 µm wide. Gram-negative. Growth occurs at temperatures between 50 and 72·5 °C (optimum 70 °C), at pH 5·5–7·6 (optimum pH 7·2), and in the presence of 1·5–5·0 % NaCl (optimum 2·5 %). The optimum doubling time is about 40 min. The major cellular fatty acids are C20 : 1 (40·8 %), C18 : 0 (24·7 %), C18 : 1 (23·8 %) and C16 : 0 (4·2 %). Obligately chemolithoautotrophic. Utilizes only H2 as electron donor and nitrate and O2 as electron acceptors. Growth is inhibited by O2 concentrations above 1·2 % (v/v). The G+C content is 37·3 mol% (HPLC). The DNA–DNA relatedness to P. marina EX-H1T and P. guaymasensis EX-H2T is low.
The type strain is 29WT (=JCM 11663T=DSM 15103T), which was isolated from a deep-sea hydrothermal vent chimney at Suiyo Seamount in the Izu-Bonin Arc, Japan (28° 34·287' N, 140° 38·663' E; depth 1385 m).
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ACKNOWLEDGEMENTS |
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