Nitratiruptor tergarcus gen. nov., sp. nov. and ... - Semantic Scholar

International Journal of Systematic and Evolutionary Microbiology (2005), 55, 925–933

DOI 10.1099/ijs.0.63480-0

Nitratiruptor tergarcus gen. nov., sp. nov. and Nitratifractor salsuginis gen. nov., sp. nov., nitrate-reducing chemolithoautotrophs of the e-Proteobacteria isolated from a deep-sea hydrothermal system in the Mid-Okinawa Trough Satoshi Nakagawa,1 Ken Takai,2 Fumio Inagaki,2 Koki Horikoshi2 and Yoshihiko Sako1 1

Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Correspondence Satoshi Nakagawa [email protected]

2

Subground Animalcule Retrieval (SUGAR) Project, Frontier Research System for Extremophiles, Japan Agency for Marine–Earth Science and Technology, 2–15 Natsushima-cho, Yokosuka 237-0061, Japan

Two novel denitrifying bacteria, designated strains MI55-1T and E9I37-1T, were isolated from deep-sea hydrothermal vent chimney structures at the Iheya North hydrothermal field in the Mid-Okinawa Trough, Japan. Both isolates were strict chemolithoautotrophs growing by respiratory nitrate reduction with H2, forming N2 as a metabolic product. Oxygen (at low concentrations) could serve as an alternative electron acceptor for growth of the isolates. Growth of strain MI55-1T was observed at temperatures between 40 and 57 6C (optimum, 55 6C; doubling time, 2 h), at pH values between 5?4 and 6?9 (optimum, pH 6?4) and in the presence of between 1?5 and 4?0 % (w/v) NaCl (optimum, 2?5 %). Growth of strain E9I37-1T was observed at temperatures between 28 and 40 6C (optimum, 37 6C; doubling time, 2?5 h), at pH values between 5?6 and 7?6 (optimum, pH 7?0) and in the presence of between 1?5 and 3?5 % (w/v) NaCl (optimum, 3?0 %). The G+C contents of the genomic DNA of strains MI55-1T and E9I37-1T were 29?6 and 35?5 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains MI55-1T and E9I37-1T belonged to groups A and F of the e-Proteobacteria, but that they had distant phylogenetic relationships with any species, within the phylogenetic groups, that had validly published names (sequence similarities were less than 91 %). On the basis of the physiological and molecular characteristics of the novel isolates, it is proposed that they should each be classified in a novel genus: Nitratiruptor tergarcus gen. nov., sp. nov., with MI55-1T (=JCM 12459T=DSM 16512T) as the type strain, and Nitratifractor salsuginis gen. nov., sp. nov., with E9I37-1T (=JCM 12458T=DSM 16511T) as the type strain.

INTRODUCTION Members of the e-Proteobacteria have been found in a variety of microbial habitats (reviewed by On, 2001) including the gastrointestinal tracts of animals (Engberg et al., 2000), sulfurous springs (Angert et al., 1998; Rudolph Published online ahead of print on 12 November 2004 as DOI 10.1099/ijs.0.63480-0. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains MI55-1T and E9I37-1T are AB175499 and AB175500, respectively. Graphs showing growth rates for strains MI55-1T and E9I37-1T at various temperatures, pH values, NaCl concentrations and O2 concentrations are available as supplementary figures in IJSEM Online.

63480 G 2005 IUMS

Printed in Great Britain

et al., 2001), activated sludge (Snaider et al., 1997), oilfields (Gevertz et al., 2000), an Antarctic Ocean water-column (Bano & Hollibaugh, 2002) and deep-sea cold seep sediments (Li et al., 1998; Inagaki et al., 2002). They metabolize a variety of compounds such as sulfur, iron, arsenate and even man-made pollutants such as tetrachloroethene (Scholz-Muramatsu et al., 1995). Recently, culture-independent molecular analyses revealed the global predominance of members of the e-Proteobacteria in deep-sea hydrothermal environments (Polz & Cavanaugh, 1995; Reysenbach et al., 2000). On the basis of 16S rRNA gene sequences, the members of the e-Proteobacteria detected in extreme environments were very diverse and were classified into six subgroups (groups A to G) (Corre 925

S. Nakagawa and others

et al., 2001; Takai et al., 2003a). Some e-proteobacteria were found in episymbiotic association with deep-sea vent metazoans (Haddad et al., 1995; Lo´pez-Garcıá et al., 2002; Goffredi et al., 2004). These researches provided new insights into the ecological roles and phylogenetic diversity of previously unknown extremophiles (Takai et al., 2003a; Campbell et al., 2003). In terms of physiology, members of the e-Proteobacteria have been poorly understood because of their strong resistance to cultivation. There is some evidence for the involvement of these micro-organisms in the biogeochemical sulfur cycle in deep-sea hydrothermal environments (Wirsen et al., 1993; Lo´pez-Garcıá et al., 2003). However, none of these micro-organisms had been cultured until recently. Some members of the e-Proteobacteria have now been successfully isolated from deep-sea hydrothermal environments and then characterized (Alain et al., 2002; Miroshnichenko et al., 2002, 2004; Inagaki et al., 2003, 2004; Takai et al., 2003a, 2004, 2005; Nakagawa et al., 2005). Although these isolates have provided some physiological and taxonomic information about deep-sea eproteobacteria, they still represent only a tiny fraction of the phylogenetic diversity revealed by culture-independent analysis. The physiological diversity and ecological significance of these members of the e-Proteobacteria remain to be determined. Previously, we reported on the isolation and partial characterization of many e-proteobacterial strains, covering nearly all of the previously uncultivated phylogroups (Takai et al., 2003a). Among the isolates, Hydrogenimonas thermophila EP1-55-1%T and Sulfurovum lithotrophicum 42BKTT were recently reported as the first species within e-proteobacterial groups A and F, respectively (Takai et al., 2004; Inagaki et al., 2004). In addition to the species, strains MI55-1T and E9I37-1T were also identified as representatives of groups A and F, respectively, but were not described in detail (Takai et al., 2003a). In this paper, we report a detailed characterization and taxonomic study of these strains and propose the new genera Nitratiruptor and Nitratifractor.

METHODS

(Chiba et al., 2000): the Cl2 concentration of the vent fluids from North Big Chimney was similar to that of sea water (511 mM), whereas the vent fluids from Central Big Chimney had a brine-rich composition (864 mM). Subsamples of the chimney structures were individually suspended in sterilized MJ synthetic sea water (Sako et al., 1996) containing 0?05 % (w/v) sodium sulfide under a N2 atmosphere. The suspended slurries were used to inoculate MMJHS medium (Takai et al., 2003a). MMJHS medium contained 1 g NaHCO3, Na2S2O3.5H2O and NaNO3, 30 g S0 and 10 ml vitamin solution (Balch et al., 1979) per litre MJ synthetic sea water. The medium was prepared under a H2/CO2 (80 : 20) gas phase (300 kPa). The pH of the medium was adjusted to 6?7. To evaluate the abundance of culturable micro-organisms, a series of serial 1 : 7 dilution experiments were performed at 25, 37, 55, 70 and 85 uC. To obtain pure cultures from the highest positive dilutions, dilution to extinction was carried out at least five times at a temperature identical to that used for the enrichment. Purity was confirmed routinely under a phase-contrast Olympus BX51 microscope and by repeated partial sequencing of the 16S rRNA gene using several PCR primers (Lane, 1991). Optical and electron microscopy. Cells were routinely observed

with a phase-contrast microscope (BX51; Olympus) equipped with the SPOT RT Slider CCD camera system (Diagnostic Instruments). Negative staining of cells for transmission electron microscopy (JEM-1210 apparatus; JEOL) was achieved as described previously (Zillig et al., 1990). Measurement of growth. Growth of the novel isolates was deter-

mined using direct cell counts after staining with 49,6-diamidino-2phenylindole (Porter & Feig, 1980). All of the experiments described below were conducted in duplicate. The cultivation temperatures were 37 and 55 uC for strains E9I37-1T and MI55-1T, unless otherwise noted. To determine temperature, pH and NaCl ranges for growth, cultures of each isolate were grown in 100 ml glass bottles (Schott Glaswerke) containing 20 ml MMJHS medium in a temperature-controlled dry oven and were shaken at 100 r.p.m. in all cases. Temperatures were measured inside control flasks alongside the cultures, as described previously (Sako et al., 2003). When a pH optimum was being determined, the pH of the MMJHS medium was adjusted to various values with 10 mM acetate/acetic acid buffer (pH 4–5), MES (pH 5–6), PIPES (pH 6–7), HEPES (pH 7–7?5) and Tris (pH 8–9?5) at room temperature. If necessary, the pH of the medium was readjusted immediately before inoculation with H2SO4 or NaOH. The pH was found to be stable during the cultivation period. NaCl requirements were determined by using various concentrations of NaCl (0–7 %, w/v) in the medium.

Sample collection, enrichment and purification. Sample collec-

tion and subsampling procedures were as described elsewhere (Takai et al., 2003a). Samples of two chimney structures called North Big Chimney and Central Big Chimney were collected at the summits of the sulfide mounds in the sediment-hosted back-arc hydrothermal system Iheya North (27u 479 N 126u 539 E), at a depth of approximately 1000 m. The distance between the two sulfide mounds was approximately 50 m. The chimney fraction from North Big Chimney, approximately 10 cm in length and up to 15 cm in width, was subsampled into four sections as previously described (Takai et al., 2001). Likewise, the chimney fraction from Central Big Chimney, approximately 6 cm in length and up to 3 cm in width, was subsampled into three sections. The temperatures of vent fluids from North Big Chimney and Central Big Chimney were 311 and 247 uC, respectively. As a consequence of subseafloor phase-separation (boiling/distillation of hydrothermal fluids), the geochemical composition of the two sets of vent fluids differed 926

Each isolate was tested for the ability to grow on combinations of a single electron donor and acceptor. MJ synthetic sea water supplemented with 0?1 % (w/v) NaHCO3 and vitamin solution (Balch et al., 1979) was used as the basal medium. In an attempt to examine growth on hydrogen as an electron donor, a H2/CO2 (80 : 20) gas phase (300 kPa) was used. Electron acceptors were provided at final concentrations of 0?1 % (w/v) (Na2S2O3.5H2O and NaNO3), 0?01–0?1 % (w/v) (Na2SO3 and NaNO2), 5 mM (arsenate, arsenite, selenate and selenite), 3 % (w/v) (S0) or 0?09–20 % (v/v) (O2). For testing growth on Na2S2O3.5H2O (0?1 %, w/v), S0 (3 %, w/v) or arsenite and selenite (5 mM) as electron donor, N2/CO2 (80 : 20) was used as the gas phase (300 kPa). NaNO3 (0?1 %, w/v) or O2 (0?09– 20 %, v/v) was provided as an electron acceptor; the latter was provided by injecting a defined volume of O2 into the culture bottles as previously described (Nakagawa et al., 2003). The presence or absence of cell growth was determined by microscopic observation. International Journal of Systematic and Evolutionary Microbiology 55

Two novel genera of the e-Proteobacteria To clarify the metabolic characteristics of the isolates, gas composition and anion concentrations were monitored during growth. Gas- and ion-chromatography were used as described previously (Nakagawa et al., 2004). Qualitative ammonium determination was done spectrophotometrically using Nessler’s reagent on a UV-1600 spectrophotometer (Shimadzu). In an attempt to examine heterotrophic growth, experiments were conducted using MMJHS medium without NaHCO3 under a gas phase of 100 % H2 (300 kPa). Each of the following potential carbon sources was tested 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, formaldehyde, formamide, acetate, citrate, pyruvate, propionate, 2-propanol, methanol, tryptone peptone (Difco) and yeast extract (Difco). In addition, to assess the utilization of these organic compounds as an energy source, substrates were added to MMJHS medium under a N2/CO2 (80 : 20) gas phase (300 kPa). To determine the nitrogen source for growth of the isolates, 0?025 % (w/v) NaNO2, NH4Cl or NaNO3 was added to MMJHS medium lacking all nitrogen sources, under a H2/CO2/O2 (80 : 19?5 : 0?5) gas phase (300 kPa). Likewise, utilization of N2 was examined under a H2/N2/CO2/O2 (60 : 20 : 19?5 : 0?5) gas phase (300 kPa). Susceptibility to antibiotics (ampicillin, kanamycin, rifampicin, streptomycin and chloramphenicol) was determined using MMJHS medium. Antibiotic concentrations from 50 to 300 mg ml21 were tested. Fatty acid methyl ester analysis. Cellular fatty acid composi-

tions were analysed using cells grown in MMJHS medium under a H2/CO2 (80 : 20) gas phase (300 kPa) at optimum growth temperatures in the late-exponential growth phase. Lyophilized cells (100 mg) were placed in a Teflon-lined, screw-capped tube (Corning) containing 3 ml anhydrous methanolic HCl and heated at 100 uC for 3 h. The extraction and analysis of fatty acid methyl esters were as described previously (Takai et al., 2003b). 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 deoxyribonucleotides using HPLC with a DNA-GC kit (Yamasa Shouyu) after digestion of the DNA with nuclease P1 (Tamaoka & Komagata, 1984).

chimney structures. The pure cultures were obtained by dilution-to-extinction methods from the highest positive dilutions. Strain MI55-1T was isolated from the middle intermediate part (4–15 mm from the exterior surface) of the chimney called North Big Chimney. The cultivation temperature for the enrichment and purification of strain MI55-1T was 55 uC. The culturable population determined by using MMJHS medium at 55 uC was between 5?36102 and 3?76103 cells (g wet weight)21. Strain E9I37-1T was isolated from the interior part (20–30 mm from the exterior surface) of the other chimney, Central Big Chimney. The cultivation temperature for the enrichment and purification of strain E9I37-1T was 37 uC. The culturable population was between 2?36102 and 1?66103 cells (g wet weight)21. Cell morphology Cells of both strains (MI55-1T and E9I37-1T) were short rods (Fig. 1). Gram staining was negative for both isolates. In both cases, the cells appeared to be non-motile under any cultivation conditions, although strain MI55-1T had bipolar flagella, as shown in Fig. 1(a). Cells occurred singly or in pairs. No sporulation was apparent under any laboratory conditions. Growth characteristics of strain MI55-1T The optimum growth temperature of strain MI55-1T was similar to those of other thermophilic species of the e-Proteobacteria (Table 1). However, strain MI55-1T had

(a)

16S rRNA gene sequence analysis. The 16S rRNA gene was

amplified by using a PCR with primers Eubac27F and 1492R (Lane, 1991). The sequence of the 1?5 kb PCR product was directly determined in both strands using a dideoxynucleotide chain-termination method with a DNA sequencer (model 3100; Perkin Elmer/Applied Biosystems). The sequences were aligned with a subset of 16S rRNA gene sequences obtained from DDBJ by the FastAligner utility of ARB software (Ludwig et al., 2004). The resulting alignment was verified against known secondary regions, and only unambiguously aligned nucleotide positions (1105 bases) were used for phylogenetic analyses with PAUP* 4.0 beta 10 (Swofford, 2000). A phylogenetic tree was inferred by using neighbour-joining analysis (Saitou & Nei, 1987) with the Jukes–Cantor correction (Jukes & Cantor, 1969). Bootstrap analysis was done using 100 replicates to provide confidence estimates for tree topologies.

(b)

RESULTS AND DISCUSSION Enrichment and purification Serial dilutions were performed using MMJHS medium and inocula from different portions of two spatially separated http://ijs.sgmjournals.org

Fig. 1. Electron micrographs of negatively stained cells of strain MI55-1T (a) and strain E9I37-1T (b). Bars, 1?0 mm. 927

Strains: 1, Nitratiruptor tergarcus MI55-1T; 2, H. thermophila EP1-55-1%T (data from Takai et al., 2004); 3, Nautilia lithotrophica 525T (Miroshnichenko et al., 2002); 4, Caminibacter hydrogeniphilus AM1116T (Alain et al., 2002); 5, Nitratifractor salsuginis E9I37-1T; 6, Sulfurovum lithotrophicum 42BKTT (Inagaki et al., 2004); 7, Thioreductor micantisoli BKB25Ts-YT (Nakagawa et al., 2005); 8, Sulfurimonas autotrophica OK10T (Inagaki et al., 2003). ND, Not determined. Characteristic Motility Morphology

International Journal of Systematic and Evolutionary Microbiology 55

Temperature range (uC) Temperature optimum (uC) pH range pH optimum NaCl range (%, w/v) NaCl optimum (%, w/v) Microaerobic growth Electron donor(s)

1

2

3

4

5

6

7

8

2 Rod

+* Rod-shaped or spherical 35–65 55 4?9–7?2 5?9 1?6–5?6 3?2 + H2

+ Rod

+ Rod

2 Rod

2 Coccoid to oval

+ Rod

+ Rod

30–40 37 5?6–7?6 7?0 1?5–3?5 3?0 + H2

10–40 28–30 5?0–9?0 6?5–7?0 0?5–6?0D 4?0D + S0, S2 O2{ 3

20–42 32 5?0–6?5 6?0 2?0–4?0 2?5 2 H2

10–40 23–26 5?0–9?0 6?5 1?6–6?0d 4?0d + S0, S2 O2{ 3

40–55 55 5?4–6?9 6?4 1?5–4?0 2?5 + H2

37–68 50–70 53 60 6?4–7?4 5?5–7?5 6?8–7?0 5?5–6?0 0?8–5?0 1?0–4?0 3?0 2?0–2?5 2 2 H2, H2, organic formate substratesd 0 S0, SO2{ NO{ 3 3 , S , cystined – NHz 4

NO{ NO{ 3 , O2 (up to 3 , O2 0 0?7 %, v/v), S § (up to 2 %, v/v), S0 Final product(s) of nitrate reduction N2 NHz 4 Major fatty acids (% of total) C18 : 1 (43?6), C16 : 0 C16 : 0 (37?4), ND C16 : 1 (28?8) (31?6), 3-OH C14 : 0 and C18 : 1 (20?0) (9?9) and C12 : 0 (8?1) DNA G+C content (mol%) 29?6 34?6 34?7 Phylogenetic group|| Group A Group A Group D Electron acceptor(s)

*Under anaerobic conditions, motility declines in stationary growth phase. DSea salt concentrations. dPoor growth. §S0 could not serve as a sole electron acceptor. ||Based on the classification of Corre et al. (2001) and Takai et al. (2003a).

ND

29±1 Group D

0 NO{ NO{ NO{ 3 , O2 3 , O2 3 , S (up to 0?6 %, v/v) (up to 7?5 %, v/v) N2 N2 NHz 4 C16 : 1 (53?7), C18 : 1 (65?6), C18 : 1 (42?3), C16 : 1 (30?7) and C16 : 0 (31?3) C16 : 0 (22?4) C16 : 0 (24?3) and C18 : 0 (15?0) and C16 : 1 (5?1) 35?5 48?0 37?2 Group F Group F Group G

O2 (up to 15 %, v/v) – C16 : 1 (45?2), C16 : 0 (37?1) and C18 : 1 (9?4) 35?2 Group B


928

Table 1. Comparison of physiological characteristics of strains MI55-1T and E9I37-1T with related genera of deep-sea hydrothermal vent e-proteobacteria

Two novel genera of the e-Proteobacteria

relatively narrow temperature, pH and NaCl ranges for growth (Table 1). Strain MI55-1T grew at temperatures in the range 40–57 uC, showing optimum growth at 55 uC. The generation time and maximum cell yield at 55 uC were about 2?5 h and 5?26109 cells ml21, respectively. No growth was observed below 37 uC or above 57 uC (see Supplementary Fig. A available in IJSEM Online). Growth occurred between pH 5?4 and 6?9, with optimum growth at about pH 6?4. No growth was detected below pH 4?8 or above pH 7?6 (Supplementary Fig. B). The isolate had an absolute requirement for NaCl for growth, and grew at concentrations in the range 1?5–4?0 % (w/v) NaCl; optimum growth occurred at around 2?5 % NaCl. No growth was observed below 1?0 % NaCl or above 5?0 % NaCl (Supplementary Fig. C). Strain MI55-1T grew with H2 as electron donor and NO{ 3 or O2 as electron acceptor. Final O2 concentrations from 0?21 to 0?50 % (v/v) supported growth (optimum, 0?40 %, v/v) [see graph (d) available as a supplementary figure in IJSEM Online]. Growth with optimal O2 concentrations and nitrate produced lower cell yields (8?66107 and 4?56108 cells ml21, respectively) than that in MMJHS medium, suggesting that the isolate uses S0 as a sulfur source. The isolate was unable to use any organic compounds as energy or carbon sources. Strain MI55-1T utilized ammonium or nitrate as a nitrogen source but could not utilize molecular nitrogen or nitrite. During growth of strain MI55-1T, consumption of nitrate and production of N2 were observed (Fig. 2a). The con2{ 2{ sumption or production of S2 O2{ 3 , SO4 or SO3 was not detected (data not shown). Although the consumption of H2 could not be measured, these results indicated that the isolate utilized H2 as an electron donor and nitrate as an electron acceptor. The accumulation of potential endproducts and intermediate products of nitrate reduction, such as nitrite, ammonium, NO and N2O, was not detected. None of the thermophilic members of the e-Proteobacteria isolated so far produce N2 as a final product of nitrate reduction (Table 1). After the exhaustion of nitrate, production of H2S was detected (Fig. 2a). Throughout 2{ growth, the consumption or production of S2 O2{ 3 , SO4 2{ or SO3 was not detected (data not shown), suggesting that the isolate reduced S0 with H2. These results suggest that the isolate potentially utilizes S0 as an electron acceptor, although S0 could not serve as a sole electron acceptor to support growth. Strain MI55-1T was sensitive to ampicillin, kanamycin, streptomycin and chloramphenicol (each at 50 mg ml21), but was insensitive to 50 mg rifampicin ml21 (though sensitive at 100 mg ml21). Growth characteristics of strain E9I37-1T Strain E9I37-1T grew at temperatures in the range 28– 40 uC, showing optimum growth at 37 uC. The optimum growth temperature of the isolate was higher than those of http://ijs.sgmjournals.org

Fig. 2. Growth and production of N2 from nitrate during growth of strain MI55-1T (a) and strain E9I37-1T (b). MMJHS medium with a gas phase of H2/CO2 (80 : 20; 300 kPa) was used. Symbols: $, growth; %, H2S production; m, N2 production; &, nitrate concentration.

other mesophilic e-proteobacterial species (Table 1). The generation time and maximum cell yield at 37 uC were about 2?5 h and 1?56109, respectively. No growth was observed at 25 or 45 uC (Supplementary Fig. A). Growth occurred between pH 5?6 and 7?6, with optimum growth at about pH 7?0. No growth was detected below pH 5?2 or above pH 8?1 (Supplementary Fig. B). The isolate had an absolute requirement for NaCl for growth, and grew at concentrations in the range 1?5–3?5 % (w/v) NaCl, with optimum growth at around 3?0 % NaCl. No growth was observed below 1?0 % NaCl or above 4?0 % NaCl (Supplementary Fig. C). Strain E9I37-1T represents the first mesophilic and facultatively anaerobic member of the e-Proteobacteria reported to grow on molecular hydrogen (Table 1). Strain E9I37-1T utilized H2 as an electron donor and NO{ 3 or O2 as an electron acceptor. Final O2 concentrations from 0?09 to 0?55 % (v/v) supported growth (optimum around 0?2 %, v/v) [supplementary graph (d) in IJSEM Online]. Growth at optimal O2 concentrations and with nitrate produced lower cell yields (9?86107 and 1?26108 cells ml21, respectively) than that in MMJHS medium, suggesting that the isolate uses S0 as a sulfur source. The isolate was unable to use any organic compounds as energy or carbon sources. 929


0.02

Desulfurella acetivorans DSM 5264T (X72768) Caminibacter hydrogenophilus AM1116T (AJ309655) 100 Lebetimonas acidiphila Pd55T (AB167820) Group D 79 Nautilia lithotrophica 525T (AJ404370) T (AB175499) Nitratiruptor tergarcus MI55-1 63 Env. clone Paralvinella palmiformis C/A26 (AJ441213) 92 89 Group A Strain BKB55-1 (AB091294) 98 100 Hydrogenimonas thermophila EP1-55-1%T (AB105048) Env. clone VC1.2-Cl06 (AF367484) Thioreductor micantisoli BKB25Ts-YT (AB175498) 78 100 100 Group G Env. clone S17sBac16 (AF299121) Env. clone S17sBac17 (AF299122) Env. clone Alvinella pompejana epibiont APG56B (L35522) 92 71 Nitratifractor salsuginis E9137-1T (AB175500) 74 97 Env. clone Paralvinella palmiformis C140 (AJ441202) 95 Sulfurovum lithotrophicum 42BKTT (AB091292) 100 Env. clone A1 B001 (AF420344) Env. clone Alvinella pompejana APB13b (L35520) 86 Env. clone Rimicaris exoculata ectosymbiont (U29081) 98 Helicobacter pylori ATCC 43504T (U01330) 100 Wolinella succinogenes ATCC 29453T (M26636) 17 Campylobacter jejuni CCUG 24567T (L14630) 28 Arcobacter cryaerophilus CCUG 17801T (L14624) 100 Sulfurospirillum barnesii SES3T (U41564) 26 32 Strain EX18.1 (AF357199) Thiomicrospira denitrificans DSM 1251T (L40808) 100 Strain 42GO-1 (AB091295) 100 Env. clone PVB_OTU10 (U15103) 53 Sulfurimonas autotrophica OK10T (AB088431)

Group F

Arcobacter group

Group B

Fig. 3. Phylogenetic tree of representative members and environmental clones within the e-Proteobacteria, inferred from 16S rRNA gene sequences by the neighbour-joining method using 1105 homologous sequence positions for each organism. Numbers at branches are bootstrap values based on 100 replicates. EMBL/GenBank/DDBJ database accession numbers are shown in parentheses. Bar, 2 substitutions per 100 nt.

Strain E9I37-1T utilized ammonium or nitrate as a nitrogen source. During growth of strain E9I37-1T in MMJHS medium, nitrate consumption and N2 production were observed 2{ (Fig. 2b). Consumption or production of S2 O2{ 3 , SO4 , or H S was not detected (data not shown). These SO2{ 2 3 results indicated that the isolate utilized H2 as an electron donor and nitrate as an electron acceptor. The accumulation of potential end-products and intermediate products of nitrate reduction was not detected. Strain E9I37-1T was sensitive to ampicillin, rifampicin, streptomycin and chloramphenicol (each at 50 mg ml21), but was insensitive to ~150 mg ml21 kanamycin (though sensitive at 200 mg ml21). Fatty acid and DNA base compositions Each of the two isolates had a distinctive fatty acid composition (Table 1). The major cellular fatty acids of strain MI55-1T were C18 : 1 (43?6 %), C16 : 0 (31?6 %), 3-OH C14 : 0 (9?9 %), C12 : 0 (8?1 %), C16 : 1 (3?6 %), C18 : 0 (1?6 %) and C14 : 0 (1?6 %). The major cellular fatty acids of strain E9I37-1T were C18 : 1 (42?3 %), C16 : 1 (30?7 %), C16 : 0 (24?3 %), 3-OH C14 : 0 (1?1 %), C14 : 0 (0?9 %) and C18 : 0 (0?7 %). The G+C contents of the genomic DNA of strains MI55-1T and E9I37-1T were found to be 29?6 and 35?5 mol%, respectively, both being lower than those of the closest relatives (described below) (Table 1). Phylogenetic analyses Almost-complete 16S rRNA gene sequences from strains MI55-1T and E9I37-1T were determined (1409 and 1439 bp). According to neighbour-joining analysis, strains MI55-1T 930

and E9I37-1T were members of groups A and F, respectively, of the e-Proteobacteria (Corre et al., 2001) (Fig. 3). H. thermophila EP1-55-1%T and Sulfurovum lithotrophicum 42BKTT are the only species described to date within each of these phylogroups. The sequences of strains MI55-1T and E9I37-1T were distantly related to those of H. thermophila EP1-55-1%T (90?2 % 16S rRNA gene sequence similarity) and Sulfurovum lithotrophicum 42BKTT (88?5 % similarity), respectively. This low phylogenetic relatedness is below the common index of 16S rRNA gene sequence similarity for differentiation of micro-organisms at the genus level (Gillis et al., 2001). Conclusions Strains MI55-1T and E9I37-1T are hydrogen-oxidizing, facultatively anaerobic, strict chemolithoautotrophs. On the basis of their physiological and phylogenetic characteristics, strains MI55-1T and E9I37-1T belong to groups A and F, respectively, of the e-Proteobacteria. Although all of the deep-sea members of the e-Proteobacteria isolated so far share the ability to utilize molecular hydrogen and/or sulfur-bearing compounds as energy sources (Table 1), members of each subgroup appear to have consistent physiological characteristics (i.e. group A, facultatively anaerobic thermophiles; group F, facultatively anaerobic mesophiles). Compared with other members of the e-Proteobacteria, strains MI55-1T and E9I37-1T have distinctive physiological, chemotaxonomic and molecular characteristics (Table 1). In addition, 16S rRNA gene sequence comparisons demonstrate that each of the two strains represents a novel genus within the e-Proteobacteria. Therefore we propose the names Nitratiruptor tergarcus gen. nov., sp. nov. International Journal of Systematic and Evolutionary Microbiology 55


for strain MI55-1T and Nitratifractor salsuginis gen. nov., sp. nov. for strain E9I37-1T. Previous reports (Wirsen et al., 1993; Taylor et al., 1999) have classified the e-Proteobacteria as microaerobic sulfuroxidizers. However, new insights (including those in this report) point to their metabolic versatility (Table 1) and therefore to their importance in the cycling of other elements in addition to sulfur. Strains MI55-1T and E9I371T were able to utilize hydrogen and nitrate as electron donor and electron acceptor, respectively. Thus, members of the e-Proteobacteria probably play a significant role not only in sulfur-cycling, but in hydrogen- and nitrogencycling in deep-sea hydrothermal environments. Description of Nitratiruptor gen. nov. Nitratiruptor [Ni.tra.ti.rup9tor. N.L. masc. n. nitras, -atis nitrate; L. masc. n. ruptor breaker; N.L. masc. n. Nitratiruptor nitrate-breaker (-reducer)]. Non-motile short rods that stain Gram-negative. Anaerobic to microaerobic. Thermophilic. Strictly chemolithoautotrophic. Able to utilize molecular hydrogen as an electron donor and oxygen or nitrate as electron acceptors. NaCl absolutely required for growth. The G+C content of genomic DNA is about 30 mol%. Major cellular fatty acids are C18 : 1, C16 : 0 and 3-OH C14 : 0. On the basis of the 16S rRNA gene sequence, the genus Nitratiruptor is distantly related to the genus Hydrogenimonas. Members of the genus Nitratiruptor occur in deep-sea hydrothermal fields. The type species is Nitratiruptor tergarcus. Description of Nitratiruptor tergarcus sp. nov. Nitratiruptor tergarcus [terg.ar9cus. L. neut. n. tergum back; L. gen. masc. n. arcus (pronounced with long u) of an arc; N.L. gen. n. tergarcus from a black arc (geological term)]. Cells have a mean length of 2?5 mm and a width of approximately 0?8 mm. The temperature range for growth is 40–57 uC (optimum 55 uC). The pH range for growth is 5?4–6?9 (optimum, pH 6?4). NaCl in the concentration range 15–40 g l2l is an absolute growth requirement; optimum growth occurs at 25 g NaCl l2l. Strictly chemolithoautotrophic growth occurs with molecular hydrogen as an electron donor and with oxygen or nitrate as electron acceptor. Nitrate is reduced to N2. Major cellular fatty acids are C18 : 1 (43?6 %), C16 : 0 (31?6 %), 3-OH C14 : 0 (9?9 %) and C12 : 0 (8?1 %). The G+C content of the genomic DNA is 29?6 mol% (HPLC). The type strain, MI55-1T (=JCM 12459T=DSM 16512T), was isolated from the Iheya North hydrothermal field in the Mid-Okinawa Trough, Japan. Description of Nitratifractor gen. nov. Nitratifractor [Ni.tra.ti.frac9tor. N.L. masc. n. nitras, -atis nitrate; L. masc. n. fractor breaker; N.L. masc. n. Nitratifractor nitrate-breaker (-reducer)]. http://ijs.sgmjournals.org

Non-motile short rods that stain Gram-negative. Anaerobic to microaerobic. Mesophilic. Strictly chemolithoautotrophic. Able to utilize molecular hydrogen as an electron donor and oxygen or nitrate as an electron acceptor. NaCl absolutely required for growth. The G+C content of genomic DNA is about 35 mol%. Major cellular fatty acids are C18 : 1, C16 : 1 and C16 : 0. On the basis of the 16S rRNA gene sequence, the genus Nitratifractor is distantly related to the genus Sulfurovum. Members of the genus Nitratifractor occur in deep-sea hydrothermal fields. The type species is Nitratifractor salsuginis. Description of Nitratifractor salsuginis sp. nov. Nitratifractor salsuginis (sal.su9gi.nis. L. gen. fem. n. salsuginis from brine). Cells have a mean length of 2?5 mm and a width of approximately 0?6 mm. The temperature range for growth is 28–40 uC (optimum, 37 uC). The pH range for growth is 5?6–7?6 (optimum, pH 7?0). NaCl in the concentration range 15–35 g l2l is an absolute growth requirement; optimum growth occurs at 30 g NaCl l2l. Strictly chemolithoautotrophic growth occurs with molecular hydrogen as an electron donor and with oxygen or nitrate as an electron acceptor. Nitrate is reduced to N2. Major cellular fatty acids are C18 : 1 (42?3 %), C16 : 1 (30?7 %) and C16 : 0 (24?3 %). The G+C content of genomic DNA is 35?5 mol% (HPLC). The type strain, E9I37-1T (=JCM 12458T=DSM 16511T), was isolated from the Iheya North hydrothermal field in the Mid-Okinawa Trough, Japan.

ACKNOWLEDGEMENTS We would like to thank the captain and the crew of R/V Natsushima and R/V Shinkai 2000 for helping us to obtain deep-sea hydrothermal vent samples. We are grateful to Dr Katsuyuki Uematsu for assistance with the preparation of electron micrographs, and to Professor Dr Hans G. Tru¨per for help with nomenclature. This work was partially supported by a Grant-in-Aid for Science Research (no. 12460093) and a Center of Excellence for Microbial-Process Development Pioneering Future Production Systems from the Ministry of Education, Culture, Sports, Science and Technology of Japan. S. N. was supported by the Research Fellowship of the Japan Society for the Promotion of Science.

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