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International Journal of Systematic and Evolutionary Microbiology (2004), 54, 41–45 Caminibacter profundus sp. nov., a novelthermophile of Nautiliales ord. nov. within the class‘Epsilonproteobacteria’, isolated from a deep-seahydrothermal vent M. L. Miroshnichenko,1 S. L’Haridon,2 P. Schumann,3 S. Spring,3E. A. Bonch-Osmolovskaya,1 C. Jeanthon2 and E. Stackebrandt3 Institute of Microbiology, Russian Academy of Sciences, Prospekt 60-letiya Oktyabrya 7/2, 2UMR 6539, Centre National de la Recherche Scientifique and Universite´ de Bretagne Occidentale, Institut Universitaire Europe´en de la Mer, 29280 Plouzane´, France 3DSMZ – German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, A novel moderately thermophilic, microaerobic to anaerobic, chemolithoautotrophic bacterium,designated strain CRT, was isolated from a deep-sea hydrothermal vent site at 366N on theMid-Atlantic Ridge. Cells were Gram-negative, non-motile rods. The organism grew at 45–65 6Cand pH 6?5–7?4, with optimum growth at 55 6C and pH 6?9–7?1. The NaCl range for growth was5–50 g l”1 (optimum 30 g l”1). Strain CRT was an obligate chemolithoautotroph, growing withH2 as energy source, sulfur, nitrate or oxygen as electron acceptors and CO2 as carbon source.
Hydrogen sulfide and ammonium were the respective products of sulfur and nitrate reduction.
The G+C content of the genomic DNA was 32?1 mol%. Based on 16S rRNA gene sequenceanalysis, this organism was most closely related to Caminibacter hydrogeniphilus (94?9 %similarity). On the basis of phenotypic and phylogenetic data, it is proposed that the isolaterepresents a novel species, Caminibacter profundus sp. nov. The type strain is CRT (=DSM15016T=JCM 11957T). The phylogenetic data also correlate well with the significant phenotypicdifferences between the lineage encompassing the genera Nautilia and Caminibacter andother members of the class ‘Epsilonproteobacteria’. The lineage encompassing the genera Nautiliaand Caminibacter is therefore proposed as a new order, Nautiliales ord. nov., represented bya single family, Nautiliaceae fam. nov.
The class ‘Epsilonproteobacteria’ represents a recently recog- et al., 2003). All the above-mentioned genera, except for nized line of descent within the Proteobacteria that encom- Thiovulum and Sulfurospirillum, which thrive in aquatic pass two families within the single order ‘Campylobacterales’ habitats, have been found associated with animals.
(Garrity & Holt, 2001). The family Campylobacteraceaecontains the genera Campylobacter, Arcobacter, Sulfurospir- Assessment of microbial diversity using molecular phylo- illum and Thiovulum, whereas the family ‘Helicobacteraceae’ genetic approaches has revealed that members of the is formed by the genera Helicobacter and Wolinella. These ‘Epsilonproteobacteria’ dominate various deep-sea hydro- bacteria are mesophiles adapted to environments that are thermal habitats such as microbial mats of Loihi Seamount low in oxygen. Most of them are oxidase-positive micro- (Moyer et al., 1995), surfaces of invertebrates (Haddad et al., aerophiles, but numerous members also grow in the absence 1995; Polz & Cavanaugh, 1995; Cary et al., 1997) and sulfides of oxygen (Vandamme et al., 1991). Among them, Sulfuro- from the Mid-Atlantic Ridge (Reysenbach et al., 2000; Corre spirillum halorespirans and Sulfurospirillum multivorans et al., 2001) and southern East Pacific Rise (Longnecker & have been described recently as obligate anaerobes (Luijten Reysenbach, 2001). Recently, thermophilic representativesof the ‘Epsilonproteobacteria’ have been isolated from tube Published online ahead of print on 20 June 2003 as DOI 10.1099/ fragments of Alvinella pompejana, an annelid polychaete endemic to chimney walls of the East Pacific Rise hydro-thermal vents. Both Nautilia lithotrophica and Caminibacter The GenBank accession number for the 16S rDNA sequence ofCaminibacter profundus strain CRT is AJ535664.
hydrogeniphilus are strictly anaerobic hydrogen-oxidizers able to grow chemolithoautotrophically with sulfur as tested on BM medium, with oxygen added to the H2/CO2 electron acceptor (Miroshnichenko et al., 2002; Alain et al., mixture (80 : 20, v/v, 200 kPa); the final concentration of 2002). Other organisms that are phylogenetically closely oxygen varied from 0?25 to 20 %. Carbon source utiliza- related and phenotypically similar to these species have been tion was determined using substrates at a concentration of partially characterized by Campbell et al. (2001). All these 0?05 %; in this case, the headspace was filled with 100 % H2 thermophilic isolates, along with a number of environ- (atmospheric pressure). Inoculated tubes were incubated at mental sequences retrieved from hydrothermal systems, 55 uC. The cell density was determined by direct cell count- form a deep monophyletic unit within the ‘Epsilon- ing using a light microscope. Gaseous and liquid fermenta- proteobacteria’. Very recently, many novel phylogenetically tion products, as well as the products of nitrate reduction, diverse representatives of the ‘Epsilonproteobacteria’ have were detected as described previously (Miroshnichenko been isolated from the hydrothermal fields of the Okinawa et al., 1994, 2003). Hydrogen sulfide was measured by a Trough and Central Indian Ridge and partially described colorimetric method (Tru¨per & Schlegel, 1964). The (Takai et al., 2003). Here, a second species in the genus sensitivity of strain CRT to rifampicin, chloramphenicol, Caminibacter, Caminibacter profundus sp. nov., isolated from vancomycin, penicillin, streptomycin and tetracycline a hydrothermal vent of the Mid-Atlantic Ridge, is described.
(Sigma) was tested at a concentration of 100 mg ml21.
Determination of the DNA G+C content was performed as Strain CRT was isolated from material collected using a vent described elsewhere (Miroshnichenko et al., 2003). DNA cap at the Rainbow hydrothermal vent field (36u169N; extraction, PCR amplification of the 16S rRNA gene and 33u549W; 2400 m depth) on the Mid-Atlantic Ridge during determination of the sequence followed described methods the Iris cruise in May 2001. An in situ growth chamber or (Rainey et al., 1996). The 16S rRNA sequences were aligned vent cap (Reysenbach et al., 2000), designed to concentrate with published sequences of the DSMZ database using the micro-organisms discharged by hydrothermal emis- the ae2 editor (Maidak et al., 1999) and sequences retrieved sions, was deployed using the hydraulic arm of the remotely from EMBL. Evolutionary distances were calculated by the operated vehicle Victor. After incubation in situ for 2 days, method of Jukes & Cantor (1969). Distance analysis dendro- the vent cap was closed by the hydraulic arm of the remotely grams were reconstructed by the neighbour-joining algo- operated vehicle before transportation to the surface. Once rithm. Bootstrap analysis was used to evaluate the tree on board, the vent cap content was immediately transferred topology by performing 500 resamplings (Felsenstein, 1988).
to 50 ml glass vials and flooded with a sterile solution of3 % (w/v) sea salts (Sigma). The vials were then closed Enrichment was performed in Bellco tubes filled with 5 ml tightly with butyl rubber stoppers (Bellco), pressurized with BM medium. A H2/CO2 mixture (80 : 20, 200 kPa) served as the energy and carbon source, and elemental sulfur was the 2 (100 kPa), reduced with sodium sulfide and stored at 4 uC until further processing in the laboratory.
electron acceptor. After inoculation of BM medium with0?5 ml material recovered from the vent cap content and For enrichment, the following basal medium (BM) was used inner surfaces and incubation of the tubes for 3 days at 55 uC without shaking, growth of non-motile rods was observed, accompanied by the formation of hydrogen sulfide. Transfer 2PO4, 0?33; CaCl2.2H2O, 0?33; MgCl2.6H2O, 0?33; NaCl, 25?0; yeast extract, 0?1; trace elements (Balch et al., 1979), of the enrichment culture into BM medium without yeast 10 ml l21; vitamins (Wolin et al., 1963), 10 ml l21. The extract did not affect its growth. A pure culture, CRT, was medium was prepared anaerobically and dispensed into isolated by serial dilutions in liquid mineral medium. Purity Bellco tubes; the headspace (25 ml) was filled with H of the culture was checked by the absence of growth in a (80 : 20, 200 kPa). No reducing agents were added to the non-selective glucose- and peptone-containing medium medium. Elemental sulfur was added to a final concentra- tion of 10 g l21. The pH of the medium was adjusted with Cells of strain CRT were rod-shaped (approximately 1?2– 2?5 M H2SO4 to 6?8–7?0. When substrates other than mole- 1?560?5 mm) and motile in the exponential phase of cular hydrogen were tested, the headspace was filled with growth. One polar flagellum was present on negatively N2/CO2 (8 : 2, v/v, atmospheric pressure). A pure culture stained whole-cell preparations (Fig. 1a). Formation of was obtained on the same basal medium without yeast spores was not observed. Thin sectioning revealed the extract using a serial tenfold dilution technique. Morpho- Gram-negative structure of the cell wall (Fig. 1b).
logy of the novel isolate was examined using an OlympusBX-60 microscope. The ultrastructure of whole cells and Strain CRT grew anaerobically with molecular hydrogen as thin sections was studied as described elsewhere (Bonch- the energy source and elemental sulfur or nitrate as the Osmolovskaya et al., 1990). For physiological studies, the electron acceptors. The only product detected during isolate was grown on BM medium containing MOPS growth with S0 was H2S. Ammonium was the only product (10 mM) as a buffer. The pH of the medium was adjusted to of nitrate reduction. Strain CRT was also able to grow 7?0 with 5 M NaOH before autoclaving. Potential growth microaerobically at low oxygen concentrations (up to 2 %, substrates and electron acceptors were added at concentra- optimal at 0?5 %). With hydrogen, S0 and CO2 as electron tions of 0?3 and 0?2 % (w/v), respectively. The ability of the donor, electron acceptor and carbon source, respectively, isolate to grow microaerobically and/or aerobically was the isolate grew at 45–65 uC, with optimum growth around International Journal of Systematic and Evolutionary Microbiology 54 100 mg ml21). It grew in the presence of chloramphenicoland tetracycline (both at 100 mg ml21). The G+C contentof the DNA of isolate CRT was 32?1 mol%.
Comparison of the 16S rRNA gene sequence (1414 bases)with those of members of the domain Bacteria indicatedthat strain CRT belonged to the class ‘Epsilonproteobacteria’and was moderately related to C. hydrogeniphilus (94?9 %similarity) and N. lithotrophica (91?2 % similarity), both ofwhich were isolated from 13uN on the East PacificRise. Strain CRT showed higher sequence similarity(92?3–96?1 %) to a group of clone sequences retrievedfrom material from deep-sea hydrothermal vents on theMid-Atlantic Ridge (VC2.1Bac7, VC2.1Bac17, VC2.1Bac8, VC2.1Bac30; Reysenbach et al., 2000). Slightly lowersimilarities (91?4–93?7 %) were found to clone sequencesretrieved from South-East Pacific vents (S17sBac14,S17sBac3, S17sBac5; Longnecker & Reysenbach, 2001) andto isolate AM1115 (Alain et al., 2002).
The phylogenetic relatedness of strain CRT to C. hydro-geniphilus is consistent with shared physiological character-istics and the DNA G+C content (Table 1). Both strains aremoderately thermophilic chemolithoautotrophs, growingwith hydrogen as electron donor and elemental sulfur ornitrate as electron acceptors. However, C. hydrogeniphilushas been described as a strictly anaerobic micro-organism,whereas strain CRT is able to grow anaerobically and micro-aerobically at an oxygen concentration of up to 2 %. Theisolate has a narrow pH growth optimum of 6?9–7?1,whereas C. hydrogeniphilus grows optimally at pH 5?5–6?5.
In contrast to C. hydrogeniphilus, which is capable of poorheterotrophic growth on complex organic substrates, strainCRT is a strictly lithotrophic micro-organism. Thus, onthe basis of phylogenetic, morphological and physiological features, it is proposed that CRT (=DSM 15016T=JCM 11957T) is the type strain of a novel species of Camini-bacter, for which the name Caminibacter profundus sp. nov.
Fig. 1. Electron micrographs of strain CRT. Negatively stained cell showing polar flagellum (a) and ultrathin section of the cell(b). Bars, 0?5 mm.
The class ‘Epsilonproteobacteria’ (Garrity & Holt, 2001) isrepresented by a single tentative order, ‘Campylobacterales’.
The order presently contains the family Campylobacteraceae (Vandamme & De Ley, 1991) and the as-yet tentative family pH 6?9–7?0). Optimal NaCl concentration for growth was ‘Helicobacteraceae’ (Garrity & Holt, 2001). Levels of 16S 30 g l21; no growth was observed in media containing less rRNA gene sequence similarity between the lineage encom- than 5 or more than 50 g NaCl l21. Under optimal passing Nautilia and Caminibacter and the ‘Campylo- conditions, the doubling time was about 40 min and the cell bacterales’ are about 83 % (Fig. 2). Phenotypic and genomic yield reached 76108 cells ml21. A slightly higher cell yield features also clearly distinguish the two phylogenetic (about 1?56109 cells ml21) was obtained under 0?5 % lineages (Table 1). It is therefore proposed that members oxygen. Acetate, formate, butyrate, propionate, malate, of the genera Nautilia and Caminibacter form a new order, succinate, methanol, ethanol, pyruvate, lactate, fumarate, Nautiliales ord. nov., represented by the single family methylamine, glucose, sucrose, starch, peptone and yeast extract did not support growth. Strain CRT did not growwhen sulfate, sulfite or thiosulfate were provided as alter- Description of Nautiliales ord. nov.
native electron acceptors. To examine possible carbon sources other than CO2, acetate, pyruvate, formate,methylamine, methanol and malate were tested; none of Nautiliales (Nau.ti9li.a.les. N.L. fem. n. Nautilia the type them supported growth. Strain CRT was sensitive to genus of the order; N.L. -ales ending denoting an order; N.L.
rifampicin, vancomycin, penicillin and streptomycin (all at fem. pl. n. Nautiliales the order of Nautilia).
Table 1. Differentiating characteristics of the families Nautiliaceae fam. nov., Campylobacteraceae and ‘Helicobacteraceae’ Data for Nautiliaceae were taken from Alain et al. (2002), Miroshnichenko et al. (2002) and this study. The family Campylobacteraceae con-tains the genera Campylobacter, Arcobacter, Sulfurospirillum and Thiovulum (data from Vandamme & De Ley, 1991; Vandamme et al., 1991;La Riviere & Schmidt, 1992; Schumacher et al., 1992; Luijten et al., 2003). The tentative family ‘Helicobacteraceae’ contains the generaHelicobacter and Wolinella (data from Tanner et al., 1981; Vandamme et al., 1991).
Helical, curved, S-shaped, spiral rods or ovoid Microaerobic, some strains aerobic or anaerobic Chemo-organotrophic; Sulfospirillum, mixotrophic; *Sulfur is reduced by Sulfurospirillum species.
Order of the ‘Epsilonproteobacteria’ separate and distinct sulfur or nitrate are used as electron acceptors. Chemo- from the ‘Campylobacterales’. Segregation of these organ- lithoautotrophs; mixotrophy occurs. Positive for H2 oxida- isms into a new order is justified by (i) their distinct tion. DNA G+C content of 29–35 mol%. Type genus: phylogenetic position and (ii) their thermophilic way of Nautilia Miroshnichenko et al. 2002.
life. Marine thermophilic rod-shaped bacteria, mean cellsize of 0?561?3 mm, non-spore-forming. Gram-negative.
Description of Nautiliaceae fam. nov.
Obligately anaerobic or microaerobic. For anaerobic growth, Nautiliaceae (Nau.ti9li.a.ce.ae. N.L. fem. n. Nautilia the typegenus of the family; N.L. -aceae ending denoting a family;N.L. fem. pl. n. Nautiliaceae the family of Nautilia).
Description is the same as that for the order. Type genus:Nautilia Miroshnichenko et al. 2002.
Description of Caminibacter profundus sp. nov.
Caminibacter profundus (pro.fun9dus. L. masc. adj. profun-dus of the depths of the ocean).
Cells are motile, rod-shaped (1?2–1?560?5 mm) with singlepolar flagellum. Gram-negative cell wall structure. Anaero-bic to microaerobic. Spores absent. Moderate thermophile,growing at 45–65 uC (optimum 55 uC). Neutrophile, grow-ing at pH 6?5–7?4 (optimum pH 6?9–7?1). Grows in 5–50 gNaCl l21 (optimum around 30 g NaCl l21). Utilizes H2 asenergy source, elemental sulfur, nitrate or oxygen as electronacceptors and CO2 as carbon source. Nitrate and sulfur arerespectively reduced to ammonium and hydrogen sulfide inthe course of growth. Growth is not supported by acetate, Fig. 2. Neighbour-joining dendrogram based on 16S rDNA formate, butyrate, propionate, malate, succinate, methanol, sequences showing the position of strain CRT in relation to its ethanol, pyruvate, lactate, fumarate, methylamine, glucose, phylogenetic neighbours, members of the genera Caminibacter sucrose, starch, peptone or yeast extract. Acetate, pyruvate, and Nautilia, ‘Epsilonproteobacteria’ and as-yet uncultured bac-teria from vents of the Pacific and Atlantic. Percentages of 500 formate, methylamine, methanol and malate cannot replace bootstrap resamplings that support branching points above CO2 as carbon source. Sulfate, sulfite and thiosulfate are not 70 % confidence are indicated. Bar, 10 nt substitutions per utilized as electron acceptors. Grows in the presence of 100 sequence positions. The tree was rooted with 16S rDNA chloramphenicol and tetracycline (both at 100 mg ml21).
sequences of members of the class ‘Gammaproteobacteria’.
DNA G+C content of the type strain is 32?1 mol%.
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