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Vulcanibacillus modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea hydrothermal vents

S. L'Haridon¹, M. L. Miroshnichenko², N. A. Kostrikina², B. J. Tindall³, S. Spring³, P. Schumann³, E. Stackebrandt³, E. A. Bonch-Osmolovskaya² and C. Jeanthon^1,

¹ UMR 6197, Centre National de la Recherche Scientifique, IFREMER and Université de Bretagne Occidentale, Institut Universitaire Européen de la Mer, 29280 Plouzané, France
² Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabraya 7/2, 117811 Moscow, Russia
³ German Collection of Microorganisms and Cell Cultures (DSMZ), Mascheroder Weg 1b, 38124 Braunschweig, Germany

Correspondence
C. Jeanthon
jeanthon{at}sb-roscoff.fr

	ABSTRACT

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A novel anaerobic, moderately thermophilic, spore-forming bacterium, designated strain BR^T, was isolated from deep-sea hydrothermal core samples collected at the Rainbow vent field on the Mid-Atlantic Ridge (36° 14' N 33° 54' W). The cells were found to be rod-shaped, non-motile, Gram-positive and spore-forming. The organism grew in the temperature range 37–60 °C, with an optimum at 55 °C, and at pH values in the range 6–8.5, with an optimum around pH 7. NaCl concentrations for growth were in the range 10–40 g l^–1, with an optimum at 20–30 g l^–1. Strain BR^T grew chemo-organoheterotrophically with carbohydrates, proteinaceous substrates and organic acids with nitrate as electron acceptor. The novel isolate was not able to ferment. The G+C content of the genomic DNA was 34.5 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed strain BR^T in the Bacillaceae within the class ‘Bacilli’. On the basis of the phenotypic and phylogenetic data, this isolate should be described as a member of a novel genus, for which the name Vulcanibacillus gen. nov. is proposed. The type species is Vulcanibacillus modesticaldus sp. nov., with the type strain BR^T (=DSM 14931^T=JCM 12998^T).

Electron micrographs and details of the polar lipid and cellular fatty acid compositions of strain BR^T are available as supplementary material in IJSEM Online.

Present address: UMR 7144, Equipe Phytoplancton Océanique, Station Biologique, Place Georges-Teissier, 29680 Roscoff Cedex, France.

	MAIN TEXT

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Deep-sea hydrothermal vents encompass diverse microbial habitats inhabited by unique microbial communities. Traditional culture-based approaches have led to the characterization of numerous microbes that include mesophiles, thermophiles and hyperthermophiles from diverse trophic groups (Jeanthon, 2000). Of the thermal classes isolated from deep-sea vents, less attention has been paid to mesophiles and moderate thermophiles in comparison with hyperthermophiles. In particular, mesophilic and moderately thermophilic micro-organisms belonging to the Epsilonproteobacteria, a prevalent group in culture-independent molecular analyses, have only recently been obtained in culture (Miroshnichenko et al., 2002, 2004; Takai et al., 2003).

Of the thermophiles isolated from these environments, only a few belong to the Gram-positive bacteria. To date, thermophilic Gram-positive isolates from deep-sea hydrothermal vents comprise aerobic bacilli (Marteinsson et al., 1996) and strict anaerobes of the orders ‘Thermoanaerobacteriales’ (Sokolova et al., 2001; Fardeau et al., 2004) and Clostridiales. The latter include recently described species of the genera Caloranaerobacter (Wery et al., 2001), Clostridium (Brisbarre et al., 2003) and Tepidibacter (Slobodkin et al., 2003; Urios et al., 2004).

In this paper, we describe a strictly anaerobic, moderately thermophilic, Gram-positive, spore-forming bacterium that was isolated from a deep-sea hydrothermal vent sediment sample; this novel organism has phenotypic, chemotaxonomic and phylogenetic characteristics that allow its assignment to a novel genus within the family Bacillaceae.

Chimney and core samples were collected at the Rainbow hydrothermal vent field on the Mid-Atlantic Ridge (36° 16' N 33° 54' W; 2300 m depth) by the remotely operated vehicle Victor during the Iris Cruise. A brief description of the hydrothermal site and the sampling procedures has been reported elsewhere (Nercessian et al., 2005). Once on board, the samples were aseptically transferred in 50 ml glass vials under N₂. The vials were closed tightly with butyl rubber stoppers (Bellco), pressurized with N₂ (100 KPa), reduced with sodium sulfide, if required, and stored at 4 °C until needed.

The enrichment medium (EM) contained the following (g l^–1 unless indicated otherwise): NH₄Cl, 0.33; KCl, 0.33; KH₂PO₄, 0.33; CaCl₂.2H₂O, 0.33; MgCl₂.6H₂O, 0.33; NaCl, 25.0; NaNO₃, 3.0, sodium acetate, 2.0; yeast extract, 0.1; trace element solution (Balch et al., 1979), 10 ml l^–1; vitamin solution (Wolin et al., 1963), 10 ml l^–1. The medium was prepared anaerobically (Balch et al., 1979) and dispensed in 15 ml Hungate tubes; the headspace (5 ml) was filled with N₂ (atmospheric pressure). No reducing agents were added to the medium. The pH of the medium was 6.5. Single colonies were isolated using EM in which acetate had been replaced by tryptone (2 g l^–1) and which had been solidified with 1.5 % agar (Difco), using a serial 10-fold dilution technique in agar shake tubes. During routine cultivation of strain BR^T in the laboratory, it was shown that the addition of FeSO₄.7H₂O (0.1 g l^–1) and yeast extract (0.5 g l^–1) to the medium significantly improved the stability and reproducibility of cell growth. An Olympus BH-2 microscope equipped with an Olympus OM-2 camera was used routinely to observe and count the bacteria. Gram staining was carried out as described by Murray et al. (1994). Staining of intracellular inclusions was done with Sudan black B, according to the protocol of Murray et al. (1994), and with Nile blue A, as described by Spring et al. (2005). Cells of Malikia granosa DSM 15619^T that accumulate polyhydroxyalkanoates served as the control. The ultrastructure of whole cells and thin-section preparations was studied using a model JEM-100 electron microscope. Cells were prepared as described previously (Bonch-Osmolovskaya et al., 1990).

The influence of pH on growth was assessed using 10 mM acetate/acetic acid buffer, MES, PIPES, HEPES and Tris between pH 4 and 8.5 and without buffer above pH 8.5. The pH of the medium was adjusted after sterilization of the medium. The effect of NaCl on growth was determined on the same medium containing increasing NaCl concentrations. The effects of pH and of NaCl concentrations were determined at the optimal temperature for growth. Physiological tests were performed in EM from which sodium acetate and sodium nitrate had been omitted. Proteinaceous potential growth substrates were added at a concentration of 1 g l^–1 to the EM medium without acetate; carbohydrates, sodium salts of organic acids and alcohols were tested at a concentration of 3 g l^–1. When molecular hydrogen served as substrate, the headspace (10 ml) was filled with a H₂/CO₂ mixture (4 : 1, v/v). Possible electron acceptors, such as elemental sulfur (5 g l^–1), thiosulfate (10 mM), sulfate (10 mM) and nitrite (10 mM), were tested. The ability to reduce amorphous Fe(III) oxide (90 mM) or Fe(III) citrate (20 mM) was tested as described previously (Slobodkin et al., 1999). The ability to grow aerobically was determined in 100 ml flasks containing the same medium (10 ml). To check for microaerophilic growth, air was added to the headspace (10 ml) of tubes filled with EM (5 ml) lacking sodium nitrate. The final oxygen concentration varied from 0.5 to 9 %.

Growth was determined by measuring changes in turbidity at 600 nm by inserting culture tubes directly in a Spectronic 20D spectrophotometer (Bioblock). All growth experiments were performed in duplicate. NO, N₂O and N₂ were tested by GC with a Porapak-Q column at 70 °C and at flow rates of 4 ml min^–1 (the carrier gas was argon). For quantitative nitrite analysis, 0.1 ml culture medium was added to a mixture containing 0.9 ml deionized water, 0.5 ml 0.6 % solution of sulfanilic acid in 20 % HCl and 0.5 ml (60 mg per 50 ml) solution of N-naphthylethylenediamine. The absorbance at 548 nm was measured after 15 min (the time necessary for colour development). For quantitative nitrate analysis, the method of Cataldo et al. (1975) was used. Oxidase activity was assayed using discs impregnated with dimethyl-p-phenylenediamine (bioMérieux). Catalase activity was assayed by mixing a pellet of fresh culture with a drop of H₂O₂ (10 %, v/v).

EM medium was inoculated with different samples collected at the Rainbow hydrothermal site. After incubation at 55 °C, long thin rods were observed in enrichments from one chimney sample and two core samples. The cultures were diluted and transferred into EM agar. After 5 days incubation at 60 °C, single cream-coloured colonies about 2 mm in diameter were visible in the agar shake tubes. The pure culture obtained after regrowth in liquid EM medium was designated strain BR^T. Cells of strain BR^T consisted of long thin rods, 4–5 µm in length and 0.2–0.3 µm in diameter (see Supplementary Fig. S1 in IJSEM Online). Cells generally occurred singly and stained Gram-positive. In the early phase of growth, inclusions of reserve polymers were frequently detected, and caused the cells to swell. The reserve compound was highly refractile under phase-contrast microscopy and stained black with the lipophilic stain Sudan black B. In contrast, the affinity for the stain Nile blue A, which is known to be highly specific for polyhydroxyalkanoates, was quite low, such that no red fluorescence could be detected by epifluorescence microscopy. Hence, we presume that the reserve compound might represent an unknown storage lipid. Flagella were not seen on negatively stained electron preparations, and no swimming motility was observed. Spore-forming cells could be observed in the early stage of propagation of the strain after its isolation. Cells lysed rapidly at the beginning of the stationary phase of growth. Colonies showed a negative reaction for oxidase and catalase.

Strain BR^T grew at temperatures between 37 and 60 °C, with an optimum at 55 °C; no growth was observed at 30 or 65 °C. The pH range for growth was between 6 and 8.5, with an optimum at pH 7; no growth was observed at pH 5.5 or 9. Strain BR^T grew with NaCl concentrations ranging from 10 to 40 g l^–1, with an optimum at 20–30 g l^–1; no growth was observed without NaCl or in the presence of 50 g NaCl l^–1.

Strain BR^T was strictly anaerobic: it was unable to grow under aerobic or microaerophilic conditions. Nitrate was essential for growth and was reduced to nitrite. Nitrate was not further reduced to ammonia or N₂. Elemental sulfur, thiosulfate, sulfate, nitrite, amorphous Fe(III) oxide (90 mM) and Fe(III) citrate (20 mM) could not be used as alternative electron acceptors. The substrates used for growth in the presence of nitrate as electron acceptor were acetate, yeast extract, tryptone, Bio-Trypticase, Bacto-peptone, sucrose, glucose, fructose, starch, cellobiose, ethanol and pyruvate. Malate, succinate, methanol, formate, lactate, galactose, arabinose, lactose, butyrate and propionate were not used. No fermentative growth was observed.

Cell-wall preparations were obtained by boiling cells in 10 % (w/v) aqueous trichloroacetic acid solution for 20 min, washing them with water and subsequently treating them with trypsin at 37 °C, as described by Schleifer & Kandler (1972). Diaminopimelic acid isomers were detected in cell-wall hydrolysates (4 M HCl, 100 °C, 16 h) by TLC on cellulose sheets (Merck), using the solvent system of Rhuland et al. (1955).

Respiratory lipoquinones and polar lipids were extracted from 100 mg freeze-dried cell material using the two-stage method described by Tindall (1990a, b). Fatty acids were analysed as the methyl ester derivatives prepared from 10 mg dry cell material. Cells were subjected to differential hydrolysis in order to detect ester-linked and non-ester-linked (amide-bound) fatty acids (B. J. Tindall, unpublished). Fatty acid methyl esters were analysed by GC using a 0.2 µmx25 m non-polar capillary column and flame-ionization detection. The run conditions were as follows: injection and detector port temperature, 300 °C; inlet pressure, 60 kPa, split ratio, 50 : 1; injection volume, 1 µl; temperature program, 130–310 °C at a rate of 4 °C min^–1.

The peptidoglycan of strain BR^T contained meso-diaminopimelic acid as the diagnostic diamino acid, which is consistent with the occurrence of peptidoglycan type A1 according to Schleifer & Kandler (1972). The predominant menaquinone was MK-7. The fatty acids comprised a mixture of straight-chain and branched (iso- and anteiso-) fatty acids (see Supplementary Table S1); i-15 : 0 predominates among the fatty acids (>69 %), but ai-15 : 0, 16 : 0, i-17 : 0 (4.8–8.1 %) and 14 : 0, 15 : 0, ai-16 : 0, ai-17 : 0, 18 : 19 and 18 : 0 (1.0–2.6 %) are also present. The polar lipids comprised a comparatively simple pattern of phospholipids, including phospholipids and aminophospholipids. The major polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and an unidentified aminophospholipid (see Supplementary Fig. S2).

The presence of predominantly iso- and/or anteiso-fatty acids, the absence of hydroxy fatty acids and the presence of menaquinones as the major respiratory quinones are features of organisms associated with ‘Bacillus and relatives’. Although most of the members of this group produce MK-7, there is evidence that shorter (MK-6) or longer (MK-8 or MK-9) menaquinones may also predominate, providing a limited degree of differentiation within the group. With regard to the fatty acids, i-/ai-15 : 0 or i-/ai-17 : 0 usually predominate, although the relative proportions of the fatty acids, together with the presence/absence of other fatty acids, may indicate that there are larger groupings within the bacilli. The resolution of such data, however, are not usually sufficient to allow clear definition of groupings that might correlate with genera (Kämpfer, 1994, 2002). With regard to the polar lipids, the chemical diversity of various Bacillus species is well documented (Minnikin & Minnikin, 1981; O'Leary & Wilkinson, 1988), although there have been no serious attempts to use such data, together with physiological, morphological and genetic data, to define groupings within the bacilli that might be regarded as genera.

In describing a novel taxon that, through 16S rRNA gene sequence analysis, shows a degree of similarity to the type species of the genus Bacillus (Bacillus subtilis), it is important not only to develop a concept of what constitutes the novel genus, but also to consider how the genus Bacillus should be defined in the future. Although great emphasis has been placed on the use of 16S rRNA gene sequences (with the definition of RNA groups), this has still not led to a new definition of the genus Bacillus. Given the well-documented chemical diversity of various species previously or currently assigned to the genus Bacillus, it would appear opportune to consider the differences between the chemical composition of the type species of the genus (B. subtilis), as the nomenclatural type of the genus, and the new taxon. Although both strain BR^T and B. subtilis DSM 10^T share MK-7 and iso-/anteiso-branched fatty acids, there are some differences in the fatty acid patterns (Supplementary Table S1), as well as in the polar lipid profiles. In particular, B. subtilis DSM 10^T produces phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, a glycolipid and an aminophospholipid with an R_F value different from that found in strain BR^T (Supplementary Fig. S2). The similarities between the two organisms are clearly indicative of the fact that they both belong to the bacilli, but the differences indicate that strain BR^T should not be included in the genus Bacillus.

DNA was isolated after disruption of cells using a French pressure cell (Thermo Spectronic) and purified by hydroxyapatite chromatography (Cashion et al., 1977). The DNA was hydrolysed with P1 nuclease and the nucleotides were dephosphorylated with bovine alkaline phosphatase (Mesbah et al., 1989). The DNA G+C content was determined by using the HPLC method described by Tamaoka & Komagata (1984).

Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and sequencing of PCR products were carried out as described by Rainey et al. (1996). The sequence reaction mixtures were electrophoresed using a model 373A automated DNA sequencer (Applied Biosystems).

The 16S rRNA gene sequence of strain BR^T was aligned with published sequences obtained from the EMBL nucleotide sequence database and the Ribosomal Database Project using the ae2 editor (Maidak et al., 2001) and the ARB program (Ludwig et al., 2004). Evolutionary distance calculations were based on the algorithms of Jukes & Cantor (1969), DeSoete (1983) and Felsenstein (1993), using neighbour-joining, maximum-likelihood and parsimony methods of tree reconstruction.

The G+C content of the DNA of strain BR^T was shown to be 34.5 mol%. The 16S rRNA gene sequence of strain BR^T had only similarity values below 93 % with those of its closest relatives. Its phylogenetic position differed slightly according to the algorithm used and the selection of sequences included in the analysis. In no case, however, did strain BR^T cluster specifically with one of the established taxa (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree of strain BRT and selected members of Bacillus and related taxa based on neighbour-joining analysis. Numbers (%) at branch points are bootstrap values greater than 70 %. Bar, 10 % sequence divergence.

View this table: [in this window] [in a new window]   Table 1. Salient characteristics of genera of the family Bacillaceae (Garrity et al., 2003) that contain thermophilic, endospore-forming rods Data were taken from Nystrand (1984), Nazina et al. (2001) and Fortina et al. (2001a, b). V, Variable; ND, not determined.

Cells are rod-shaped and produce spores. Cells occur mostly singly and are not motile. The cell-wall structure is Gram-positive. Moderately thermophilic, neutrophilic and adapted to the salinity of sea water. Strictly anaerobic. Chemo-organotrophic, using nitrate as the sole electron acceptor, this being reduced to nitrite. Nitrate is not further reduced to ammonia or N₂. No fermentation occurs. Oxidase- and catalase-negative. Produces MK-7 as the major respiratory lipoquinone. The predominant fatty acid is i-15 : 0, but 14 : 0, ai-15 : 0, 15 : 0, ai-16 : 0, 16 : 0, i-17 : 0, ai-17 : 0, 18 : 19 and 18 : 0 are also present. The polar lipids comprise phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine, as well two unidentified phospholipids (PL1 and PL2) and a single unidentified amino-phospholipid. The DNA G+C content is 34.5 mol%. 16S rRNA gene sequence analysis places Vulcanibacillus in the class ‘Bacilli’. The type species of the genus is Vulcanibacillus modesticaldus.

Description of Vulcanibacillus modesticaldus sp. nov.
Vulcanibacillus modesticaldus (mo.des'ti.cal.dus. L. adj. modestus moderate; L. adj. caldus warm, hot; N.L. masc. adj. modesticaldus moderately hot).

In addition to having the characteristics given in the genus description (above), cells are rods, 4–5 µm in length and 0.2–0.3 µm in diameter. Colonies of strain BR^T are lens-shaped and cream in colour. The temperature range for growth is 37–60 °C, with an optimum at 55 °C. Neutrophilic. The optimum NaCl concentration for growth is 2–3 %. Substrates used for growth in the presence of nitrate as electron acceptor include acetate, yeast extract, tryptone, Bio-Trypticase, Bacto-peptone, sucrose, glucose, fructose, starch, cellobiose, ethanol and pyruvate. Elemental sulfur, thiosulfate, sulfate, nitrite, amorphous Fe(III) oxide (90 mM) and Fe(III) citrate (20 mM) cannot be used as alternative electron acceptors. Malate, succinate, methanol, formate, lactate, galactose, arabinose, lactose, butyrate and propionate are not used. The chemical composition of the species is identical to that given for the genus.

The type strain, BR^T (=DSM 14931^T=JCM 12998^T), was isolated from a core sample collected at the Rainbow vent field on the Mid-Atlantic Ridge (36° 14' N).

	ACKNOWLEDGEMENTS

 
We thank Yves Fouquet (Chief Scientist), for inviting us to participate in the Iris Cruise (2001), and the crews of R/V L'Atalante and pilots of ROV Victor. This work was supported by a PRIR (Conseil Régional de Bretagne) grant, a CNRS/Rhône-Poulenc grant, INTAS grant 99-1250, RFBR grant 00-04-4894, Programs ‘Molecular and Cell Biology’ and ‘Origin and Evolution of the Biosphere’ of the Russian Academy of Sciences and Program ‘Biodiversity’ of the Russian Ministry of Industry, Science and Technology. M. L. M. was supported by the Ministère de l'Education Nationale during her stay in France.

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