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Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge
FEMS Microbiology Letters 205 (2001) 291^297
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Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge Jie Lu, Yuichi Nogi, Hideto Takami * Microbial Genome Research Group, DEEPSTAR, Japan Marine Science and Technology Center, 2-15 Natsushima, Yokosuka 237-0061, Japan Received 6 August 2001; received in revised form 21 October 2001; accepted 21 October 2001 First published online 14 November 2001
Abstract An extremely halotolerant and alkaliphilic bacterium was isolated previously from deep-sea sediment collected at a depth of 1050 m on the Iheya Ridge. The strain, designated HTE831 (JCM 11309, DSM 14371), was Gram-positive, strictly aerobic, rod-shaped, motile by peritrichous flagella, and spore-forming. Strain HTE831 grew at salinities of 0^21% (w/v) NaCl at pH 7.5 and 0^18% at pH 9.5. The optimum concentration of NaCl for growth was 3% at both pH 7.5 and 9.5. The G+C content of its DNA was 35.8%. Low level (12^30%) of DNA^DNA relatedness between strain HTE831 and the species of these genera was found, indicating that HTE831 could not be classified as a member of a new species belonging to known genera. Based on phylogenetic analysis using 16S rDNA sequencing, chemotaxonomy, and the physiology of strain HTE831, it is proposed that this organism is a member of a new species in a new genus, for which the name Oceanobacillus iheyensis is proposed. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Deep-sea isolate ; Halotolerant alkaliphile; 16S rDNA sequence; DNA^DNA hybridization; Oceanobacillus iheyensis
1. Introduction The bottom of the deep-sea is not devoid of organisms, although the deep-sea is an extreme environment with particularly high hydrostatic pressure and low temperature. Numerous microorganisms including nonextremophilic and extremophilic microbes such as piezophiles, psychrophiles, thermophiles, and alkaliphiles have been isolated from deep-sea sediment collected at depths of 10 897 m in the Challenger Deep of the Mariana Trench by the unmanned submersible Kaiko [1]. In addition, benthic organisms such as amphipods and sea cucumbers, which thrive in the Challenger Deep, have been retrieved. Although halophiles were not found in the Challenger Deep, several types of halophilic or halotolerant bacteria similar to the members of genera Bacillus, Halomonas, and Marinobacter on the basis of 16S rDNA comparison
* Corresponding author. Tel. : +81 (468) 67-9643; Fax: +81 (468) 67-9645. E-mail address : [email protected] (H. Takami).
have been isolated from the shallower Izu-Bonin Trench (2759 m deep, 30³07.05PN, 139³58.42PE; and 3400 m deep, 29³04.2PN, 140³43.3PE) and the Iheya Ridge of the Nansei Islands (1050 m deep, 27³47.18PN, 126³54.15PE) [2]. Thus, it was expected that the relative abundance of halophiles and halotolerant microbes among the deep-sea microbial £ora would decrease as the depth of sampling points increased. Halophilic or halotolerant bacteria are often isolated from salterns, hypersaline soils, and lakes [3^5], but little information is available on isolates from the deepsea. We are focusing on halophilic or halotolerant isolates from deep-sea environments. We are especially interested in strain HTE831 from the Iheya Ridge, which can grow on marine agar plates containing 15% NaCl, because 16S rDNA sequencing showed that the phylogenetic placement of this strain is comparatively close to that of Bacillus subtilis, for which the genome sequence has been determined [2]. In this study, we attempted to identify strain HTE831 both on the basis of conventional physiological and biochemical characteristics and through phylogenetic analysis based on 16S rDNA sequences and compari-
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 9 3 - 1
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son of DNA^DNA hybridization patterns. We also attempted to determine the genome size of the HTE831 chromosome by pulsed-¢eld gel electrophoretic (PFGE) analyses. 2. Materials and methods 2.1. Bacterial strain, media, and cultivation A deep-sea bacterium, designated HTE831, isolated from deep-sea mud sample collected at a depth of 1050 m on the Iheya Ridge of the Nansei Islands was studied [2]. The basal medium for strain HTE831 was PY medium, consisting of 1% polypeptone, 0.5% yeast extract, 0.1% K2 HPO4 , and 0.02% MgSO4 W7H2 O supplemented with NaCl (3^21%). The PY medium was adjusted to pH 6.5^ 8.5 with HCl or NaOH solution. Two types of alkaline PY medium supplemented with 1% Na2 CO3 or 2% NaHCO3 were adjusted to pH 10 and pH 9.5 with HCl or NaOH. All media with di¡erent pH values and NaCl concentrations were sterilized with a Falcon ¢lter unit with 0.22-Wm pore size (Becton Dickinson, Franklin Lakes, NJ, USA). Strain HTE831 was grown aerobically at various pH and temperature conditions. The cultivation of this strain under high-pressure conditions was carried out by the methods previously described [2,6].
2.5. Phylogenetic analysis based on 16S rDNA sequence 16S rDNA sequences were aligned using the Clustal multiple-alignment program (Clustal W) [13]. Sites involving gaps were excluded from all analyses. A phylogenetic tree was inferred by the neighbor-joining method [14] using the DNADIST and NEIGHBOR programs in the PHYLIP package, version 3.57 [15]. 2.6. PFGE Chromosomal DNA of HTE831 for PFGE was prepared in agarose plugs as described [16]. Agarose blocks containing chromosomal DNA were washed twice in 50 ml of 0.1UTE bu¡er and then equilibrated with the corresponding restriction bu¡er at 4³C for 1 h. DNA was digested with 100^200 U of ApaI or Sse8387I (Takara Shuzo, Otsu, Japan) at 37³C overnight in 500 Wl of the restriction bu¡er recommended by the manufacturer. PFGE in 1% PFC agarose was performed as previously described [16,17].
2.2. Biolog test and physiological properties Biolog tests were performed according to the manufacturer's instructions (Release 3.50, version DE, Biolog, Hayward, CA, USA). Plates were incubated at 30³C, and color changes were measured at 590 nm. Physiological properties of strain HTE831 were investigated by standard methods, described previously [7]. 2.3. Isoprenoid quinones and fatty acid analysis Isoprenoid quinones were extracted from freeze-dried cells with chloroform:methanol (2:1) and puri¢ed by thin-layer chromatography. The puri¢ed isoprenoid quinones were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) [8] and the absorbance was measured at 270 nm using menaquinone as a standard. Fatty acids were analyzed as methyl ester derivatives prepared from 10 mg of freeze-dried cell materials as previously described [9]. 2.4. DNA studies DNA was extracted by a previously described method [10]. The G+C content was determined by RP-HPLC [11]. For analysis of relatedness, DNA^DNA hybridization was carried out at 40³C for 3 h and measured £uorometrically [12].
Fig. 1. Phase-contrast and transmission electron micrographs of strain HTE831. A: Phase-contrast micrograph. Bar, 10 Wm. B: Transmission electron micrograph. Bar, 1 Wm. The strain was grown aerobically at pH 9.5 in PY medium containing 3% NaCl. For negative staining, one drop of culture was placed on a copper grid coated with 1% potassium phosphotungstic acid adjusted to pH 6.5 with potassium hydroxide.
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38 43 42
+ 5.8^9.6 2^24.8 10^45 MK-7 ND
9 8 7
rods, single or in chain central or subterminal 3 6^9.5 0.5^30 10^44 MK-7 iso-15:0, anteiso-15 :0, anteiso-17 :0
Cells of strain HTE831 were predominantly rod-shaped, 0.6^0.8U2.5^3.5 Wm in size, but ¢lamentous forms were present throughout the growth cycle. The cells were Grampositive, strictly aerobic, and motile by means of peritrichous £agella (Fig. 1 and Table 1). Ellipsoidal endospores were produced terminally or subterminally, causing swelling of the sporangia (Fig. 1). Cell morphology was not a¡ected by the salinity of the growth medium. Colonies of strain HTE831 were circular, creamy white, nontransparent, and approximately 2^3 mm in diameter on PY agar medium after 2 days of growth at 30³C.
rods, single or in chain central or subterminal 3 6^9.5 0.5^25 10^43 MK-7 iso-15:0, anteiso-15:0
3.1. Morphology
rods, single or in small chain NO
3. Results
36.9 40.7 38.0 G+C content (mol%)
35.8
+ ND 0^15 28^50 MK-7 iso-15:0, anteiso-15:0, iso-16:0, anteiso-17:0 40.1 3 5^10 0^20 6^50 MK-7 anteiso-15 :0, iso-16:0, anteiso-17 :0
Spore position
subterminal or terminal Anaerobic growth 3 pH range 6.5^10 NaCl range (%) 0^21 Temperature range (³C) 15^42 Major isoprenoid quinone MK-7 Major cellular fatty acids iso-15:0, anteiso-15:0, iso-14:0
39.5
+ ND 0^10 15^50 MK-7 iso-15:0, anteiso-15:0, anteiso-17:0
rods, single or in chain subterminal or terminal 3 6^9 5^25 15^50 MK-7 iso-15:0, anteiso-15 :0, anteiso-17 :0
6 4
rods, single or in chain central or subterminal 3 6^11 8^16 15^45 MK-7 iso-15:0, anteiso-15:0, iso-16:0 thin rods and ¢laments terminal
3
rods
2 1
Morphology
thin rods and ¢laments terminal
5
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Characteristics
Table 1 Morphological and physiological characteristics of strain HTE831 and some related organisms
Strain HTE831 exhibited extreme halotolerance and was able to grow in PY medium containing 0^21% NaCl at 30³C, pH 7.5. At pH 9.5, this strain could also grow in the same medium containing 0^18% NaCl. The duration of the lag phase and generation time lengthened with increasing NaCl concentration. It is notable that this strain did not require NaCl and that the optimum concentration of NaCl for cell growth was 3% in the pH range 6.5^10.0 (Table 1). Thus, strain HTE831 was shown to be facultatively alkaliphilic, exhibiting extreme halotolerance with broad pH optima for growth ranging from 7.0 to 9.5. Cell growth was observed at temperatures of 15^42³C, and the optimum temperature was 30³C in PY medium. In addition, HTE831 cell growth occurred at pressures of up to 30 MPa, corresponding to the pressure at a depth of 3000 m. When the physiological characteristics of strain HTE831 were investigated by the Biolog test, it assimilated only four substrates (K-D-glucose, maltose, D-mannose, and turanose). This assimilation pattern was characteristic of other related strains listed in Table 2. In contrast to HTE831, the ¢ve other strains, with the exception of Salibacillus salexigens [18], assimilated 15^23 di¡erent substrates under the same conditions. The assimilation patterns of two Gracilibacillus strains [9,19], G. halotolerans and G. dipsosauri, were very similar to each other. Among three Halobacillus strains [20], the substrate assimilation pattern of H. trueperi was very similar to that of H. litoralis, although that of H. halophilus di¡ered from those of the ¢rst two species. S. salexigens, which assimilated nine types of substrate, did not show an assimilation pattern similar to that of any other species (Table 2). Strain HTE831 produced acid from glycerol, fructose, glucose, maltose, and mannose. HTE831 neither produced H2 S nor indol. Production of DNase and urease, VP reaction, and assimilation of citrate by this strain were negative. The major isoprenoid quinone in strain HTE831 was menaquinone-7 (MK-7), which accounted for 99% of the
rods, single or in chain terminal
3.2. Physiological and biochemical properties
1, HTE831; 2, G. halotolerans [9]; 3, G. dipsosauri [9]; 4, S. salexigens [9]; 5, S. marismortui [23]; 6, V. pantothenticus [22]; 7, H. litoralis [20]; 8, H. trueperi [20]; 9, B. halodenitri¢cans [21]. ND: not determined ; NO : not observed.
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Table 2 Speci¢c carbon sources and nutrients showing positive reaction in the Biolog test among halophilic or halotolerant species used in this study Utilization of
1
2
3
4
5
6
7
K-Cyclodextrin L-Cyclodextrin Dextrin Glycogen N-acetyl-D-galactosamine N-acetyl-D-glucosamine Arbutin Cellobiose D-fructose Gentiobiose D-gluconic acid K-D-glucose K-D-lactose Maltose Maltotriose D-mannose D-melezitose D-melibiose 3-Methyl glucose K-Methyl D-glucoside L-Methyl D-glucoside K-Methyl D-mannoside Palatinose D-psicose D-ra¤nose Salicin Sedoheptulosan D-sorbitol Sucrose D-trehalose Turanose Acetic acid L-Hydroxybutyric acid K-Keto valeric acid L-lactic acid Methyl pyruvate Propionic acid Pyruvic acid Alaninamide D-alanine L-alanine L-glutamic acid L-serine Glycerol Adenosine Inosine Uridine
3 3 3 3 3 3 3 3 3 3 3 + 3 + 3 + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
+ + + + + 3 + + + 3 3 + + + + + 3 3 3 3 + 3 + 3 3 + 3 3 + + + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
+ + + 3 3 3 + + + 3 3 + + + + + + + 3 3 + 3 3 + + + 3 + + + 3 3 3 3 3 + 3 + 3 3 3 3 3 + 3 3 3
3 3 + 3 + + 3 3 + 3 + + 3 + + + 3 3 + + 3 + 3 3 3 3 3 3 3 + 3 3 3 + 3 3 3 + 3 + + 3 + + 3 + +
3 3 3 3 3 3 3 3 3 3 3 + 3 3 3 3 3 3 3 + 3 3 3 3 3 3 3 3 3 3 + 3 3 3 + 3 3 3 + + 3 + 3 + + 3 3
3 + 3 3 3 3 3 + + + 3 + 3 + + 3 3 3 3 3 3 3 + + 3 3 3 3 + + + + + + + 3 + + 3 3 3 3 3 3 3 3 3
+ + 3 3 3 + 3 + 3 3 3 3 + + + 3 3 3 3 3 3 3 + + 3 3 3 3 3 + + + 3 + 3 3 + 3 3 3 3 3 3 + 3 3 3
1, HTE831 ; 2, G. halotolerans; 3, G. dipsosauri; 4, S. salexigens; 5, H. halophilus; 6, H. trueperi ; 7, H. litoralis.
total isoprenoid quinones, as in related genera showing halotolerant or halophilic phenotypes (Table 1). The G+C content of the DNA of strain HTE831 was found to be 35.8 mol%, as shown in Table 1. 3.3. Phylogenetic analysis based on 16S rDNA sequence In a previous study, the phylogenetic placement of strain HTE831 was investigated with partial 16S rDNA
Fig. 2. Unrooted phylogenetic tree based on 16S rDNA sequence comparison showing the relationship of strain HTE831 and other related strains. The numbers next to nodes indicate the percentages of bootstrap samples, derived from 1000 samples, which supported the internal branches [15]. Bootstrap probability values less than 50% were omitted from this ¢gure. Five strains (Bacillus halodurans, B. pseudo¢rmus, B. pseudalkaliphilus, B. clarkii, and B. agaradhaerens) are alkaliphile and others are halophilic or halotolerant and alkaliphilic strains. The sequence of Lactobacillus delbrueckii has been included to serve as an outgroup. Bar, 0.01 Knuc unit
sequencing (1419 bp) of this strain [2]. In that analysis, it was found that strain HTE831 was positioned close to Bacillus species such as B. subtilis and B. sporothermodurans. For further characterization of strain HTE831, we determined the complete 16S rDNA sequence and constructed a phylogenetic tree based on a comparison of the 16S rDNA sequence of this strain and those of type strains of Bacillus species and other species belonging to related genera. The 16S rDNA sequence of strain HTE831 (1525 bp) was redeposited in the DDBJ/EMBL/GenBank with the same accession number (AB10863) previously registered. Similarity values in the range of 90.5^94.4% were obtained when comparing the 16S rDNA sequence of strain HTE831 and those of nine other strains belonging to Bacillus halodenitri¢cans [21] and the genera derived from former Bacillus spp., such as Gracilibacillus, Halobacillus, Virgibacillus [22], and Salibacillus. Phylogenetic analysis
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based on 16S rDNA sequence comparison showed that strain HTE831 formed a cluster with the related strains belonging to the genera Virgibacillus and Salibacillus, derived from former Bacillus spp. and B. halodenitri¢cans, but the branch of HTE831 diverged at the bottom of this cluster (Fig. 2). This means that strain HTE831 is phylogenetically distant from these related genera, although their halotolerant phenotypes and other physiological characteristics are similar. 3.4. DNA^DNA hybridization analysis DNA^DNA hybridization analysis was carried out to compare strain HTE831 and eight related strains (Table 3). The DNA^DNA relatedness between HTE831 and two Gracilibacillus strains [9], G. dipsosauri (DSM 11125T ) and G. halotolerans (DSM 11805T ), was 30% and 25%, respectively. Two Salibacillus strains [9], S. salexigens [17] (DSM 11483T ) and S. marismortui [23] (DSM 12325T ), showed low homologies of 12^15% with HTE831. Similarly, three Halobacillus strains [20], H. halophilus (DSM 2266T ), H. trueperi (DSM 10404T ), and H. litoralis (DSM 10405T ), showed only 14^23% homologies with HTE831. The DNA^DNA relatedness of HTE831 and Virgibacillus pantothenticus [22] (DSM 26T ) was low at 15%. Finally, B. halodenitri¢cans [21] (DSM 10037T ) also showed a low homology of 24% with HTE831. Thus, it is clear that
295
Table 3 DNA^DNA hybridization between strain HTE831 and other related strains Source of unlabelled DNA O. iheyensis HTE831 G. dipsosauri DSM 11125T G. halotolerans DSM 11805T H. halophilus DSM 2266T H. trueperi DSM 10404T H. litoralis DSM 10405T S. salexigens DSM 11483T S. marismortui DSM 12325T V. pantothenticus DSM 26T B. halodenitri¢cans DSM 10037T
Homology (%) with H-labelled DNA from strain HTE831 100 30 25 23 21 14 12 15 15 24
strain HTE831 should not be categorized as a member of a new species within a known genus. 3.5. Estimation of genome size of strain HTE831 Restriction endonucleases that recognize an 8-bp sequence were tested for their ability to digest the chromosome of strain HTE831. ApaI (5P-GGGCC/C-3P) and Sse8387I (5P-CCTGCA/GG-3P) generated 37 and 25 resolvable fragments, respectively (Fig. 3). The sizes of these fragments were determined by comparison with size standards on a series of PFGE gels (Fig. 3). The mean total
Fig. 3. Digestion patterns of the chromosomal DNA of strain HTE831 obtained with Sse8387I and ApaI. A: Separation of fragments ranging in size from 200 to 1000 kb. B: Separation of fragments ranging in size from 100 to 400 kb. C: Separation of fragments ranging in size from 5 to 75 kb. Lanes: 1, Molecular size marker; 2, complete ApaI digestion; 3, complete Sse8387I digestion
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size of the genome of HTE831, estimated by totaling the ApaI or Sse8387I fragments, was 3.6 Mb. 4. Discussion In the previous study, halophilic or halotolerant bacteria were recovered from various deep-sea sites at a frequency of 0.9U102 ^3.6U103 g31 of dry sea mud [2]. Strain HTE831 isolated from deep-sea mud collected at a depth of 1050 m on the Iheya Ridge was characterized as an extremely halotolerant and facultatively alkaliphilic bacterium in this study. Halophilic or halotolerant bacteria are often isolated from salterns, hypersaline soils, and lakes [3^5], but little information is available on the isolates from the deep-sea. HTE831 is the ¢rst Gram-positive, spore-forming isolate from the bathypelagic zone, which shows extremely halotolerant and facultatively alkaliphilic phenotypes. Thus, deep-sea is a promising environment to ¢nd new types of halophilic or halotolerant bacteria. However, it is not easy to access such deep-sea sites, not even by using the manned submersible Shinkai 6500, due to climatic problems and economical constraints. Similarity values in the range of 90.5^94.4% were obtained when comparing the 16S rDNA sequence of strain HTE831 and those of nine other strains used in this study. In the phylogenetic tree constructed based on 16S rDNA sequences, HTE831 formed a cluster with the related strains belonging to the genera Virgibacillus and Salibacillus, derived from former Bacillus spp. and B. halodenitri¢cans; the branch of HTE831 diverged at the bottom of this cluster, which is being supported by the bootstrap value of 80% (Fig. 2). It has been suggested that 16S rDNA similarities around 95% would be a practical border zone for genus de¢nition [24]. At 16S rDNA similarity levels of 97% and higher, the DNA^DNA reassociation techniques are the superior method [25], and the genomic DNA similarity of around 70% and higher is shared by strains of a species [26]. The DNA^DNA relatedness between HTE831 and nine other related species in the ¢ve genera, Bacillus, Gracilibacillus, Halobacillus, Virgibacillus, and Salibacillus, was less than 30%, indicating that HTE831 should not be categorized as a new species in any of these genera. On the other hand, there are some signi¢cant di¡erences between HTE831 and other related species in the growth pattern in terms of salinity and temperature. For example, Salibacillus and Halobacillus species show a halophilic phenotype, but no growth occurs in the absence of salt, in contrast to halotolerant HTE831. Although Virgibacillus and Gracilibacillus species also have halotolerant phenotypes like HTE831, Virgibacillus species can only grow in medium containing up to 10% salt, and Gracilibacillus species can grow even at 50³C, which is 8³C higher than the maximum growth temperature for HTE831. Thus, we propose that the new genus Oceanobacillus should be created for the deep-sea isolate HTE831T and
describe a new species within the genus Oceanobacillus called O. iheyensis on the basis of the results presented in this study. We note that it is not recommended to base the description of a genus on a single isolate. However, we did not detect any other strain which could be assigned to the species O. iheyensis among our deep-sea isolates, and access to the deep-sea sampling station is di¤cult. The type strain is O. iheyensis HTE831 (JCM 11309T , DSM 14371T ). 4.1. Description of Oceanobacillus (Lu, Nogi, and Takami) gen. nov. Oceanobacillus (o.ce.a.no.ba.cil'lus. L. n. oceanus, the ocean; L. dim. n. bacillus, a small rod; M.L. masc. Oceanobacillus, the ocean bacillus/rod). Gram-positive, motile by peritrichous £agella, sporeforming rods. Endospores are subterminal or terminal and slightly swell the sporangia. Obligately aerobic, facultatively alkaliphilic, and extremely halotolerant. Growth occurs at temperatures of 15^42³C. Colonies are circular. Chemoorganotrophic. Grows on glucose, mannose, maltose and turanose. Acid production occurred from glucose, mannose, glycerol, fructose, and maltose, but not from xylose, rhamnose, glucitol, trehalose, galactose, lactose, and melibiose. Catalase-positive, oxidase-variable, DNase-negative, and urease-negative. VP reaction, indol and H2 S production, and use of citrate are negative. Aminopeptidase and KOH tests are negative. Nitrate reduction to nitrite is negative. Cells are resistant to erythromycin, but susceptible to ampicillin. On the basis of 16S rRNA gene sequencing analysis, the genus Oceanobacillus does not form a branch with other genera such as Halobacillus, Gracilibacillus, Virgibacillus, and Salibacillus. The G+C content of the type species is 35.8%. The major cellular fatty acids are anteiso-15:0, iso-15:0, and iso-14 :0. The main menaquinone type is MK-7. The type species of the genus is O. iheyensis sp. nov. 4.2. Description of O. iheyensis sp. nov. O. iheyensis (i.he.yen'sis. M.L. masc. adj. iheyensis, pertaining to the Iheya Ridge). In addition to the characteristics listed above for the genus, the following features are characteristic of M. iheyensis. Cells are 0.6^0.8U2.5^3.5 Wm. Endospores are ellipsoid. Colonies are creamy white. Grows at 0^21% (w/v) NaCl, with optimum growth at 3% NaCl. Growth occurs at temperatures of 15^42³C (optimum 30³C). The pH range for growth is 6.5^10 (optimum 7.0^9.5). Hydrolyzes gelatin, casein, Tween 40, and Tween 60. Does not hydrolyze starch. Cells are resistant to nalidixic acid and spectinomycin, but susceptible to gentamycin, kanamycin, tetracycline, bacitracin, carbenicillin, chloramphenicol, novobiocin, penicillin G, and rifampicin. The following four substrates are assimilated in the Biolog test: K-D-glucose,
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maltose, D-mannose, and turanose. The G+C content is 35.8%. The genome size is about 3.6 Mb. 16S rDNA sequence exhibits 90.5^94.4% homology with those from B. halodenitri¢cans and the members of the genera Halobacillus, Gracilibacillus, Virgibacillus, and Salibacillus. The isolate was obtained from a mud sample collected at a depth of 1050 m on the Iheya Ridge. The type strain is O. iheyensis HTE831T , = JCM 11309T , Japan Collection of Microorganisms, RIKEN Institute, Wako-shi, Saitma, Japan, and = DSM 14371T , Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH. Acknowledgements We thank H. Uchiyama and Drs. Y. Takaki and K. Nakasone for their technical assistance. Thanks are also due to K. Uemastu for his assistance in preparing electron micrographs. References [1] Takami, H., Inoue, A., Fuji, F. and Horikoshi, K. (1997) Microbial £ora in the deepest sea mud of the Mariana Trench. FEMS Microbiol. Lett. 152, 279^285. [2] Takami, H., Kobata, K., Nagahama, T., Kobayashi, H., Inoue, A. and Horikoshi, K. (1999) Biodiversity in the deep-sea sites located near the south part of Japan. Extremophiles 3, 97^102. [3] Garabito, M.J., Ma¨rquez, M.C. and Ventosa, A. (1998) Halotolerant Bacillus diversity in hypersaline environments. Can. J. Microbiol. 44, 95^102. [4] Arahal, D.R., Ma¨rquez, M.C., Volcani, B.E., Schleifer, K.H. and Ventosa, A. (1999) Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int. J. Syst. Bacteriol. 49, 521^ 530. [5] Duckworth, A.W., Grant, W.D., Jones, B.E., Meijer, D., Ma¨rquez, M.C. and Ventosa, A. (2000) Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley. Extremophiles 4, 53^60. [6] Abe, F. and Horikoshi, K. (1995) Hydrostatic pressure promotes the acidi¢cation of vacuoles in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 130, 307^312. [7] Sneath, P.H.A., Mair, N.S., Sharp, M.E. and Holt, J.G. (1986) Bergey's Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins, Baltimore, MD. [8] Komogata, K. and Suzuki, K. (1987) Lipid and cell wall analysis in bacterial systematics. Methods Microbiol. 19, 161^207. [9] WainÖ, M., Tindall, B.J., Schumann, P. and Ingvorsen, K. (1999) Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov.. Int. J. Syst. Bacteriol. 49, 821^831. [10] Saito, H. and Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619^629.
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[11] Tamaoka, J. and Komagata, K. (1984) Determination of DNA base composition of by reverse-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25, 125^128. [12] Ezaki, T., Hashimoto, Y. and Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid^deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane ¢lter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39, 224^229. [13] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. [14] Saitou, N. and Nei, M. (1987) The neighbor-joining method : a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406^425. [15] Felsenstein, J. (1995) PHYLIP : Phylogenetic Inference Package, Version 3.57c. University of Washington, Seattle, WA. [16] Takami, H., Nakasone, K., Hirama, C., Takaki, Y., Masui, N., Fuji, F., Nakamura, Y. and Inoue, A. (1999) An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125. Extremophiles 3, 21^28. [17] Nakasone, K., Masui, N., Takaki, Y., Sasaki, R., Maeno, G., Sakiyama, T., Hirama, C., Fuji, F. and Takami, H. (2000) Characterization and comparative study of the rrn operons of alkaliphilic Bacillus halodurans C-125. Extremophiles 4, 209^214. [18] Garabito, M.J., Arahal, D.R., Mellado, E., Ma¨rquez, M.C. and Ventosa, A. (1997) Bacillus salexigens sp. nov., a new moderately halophilic Bacillus species. Int. J. Syst. Bacteriol. 47, 735^741. [19] Lawson, P.A., Deutch, C.E. and Collins, M.D. (1996) Phylogenetic characterization of a novel salt-tolerant Bacillus species : description of Bacillus dipsosauri sp. nov.. J. Appl. Bacteriol. 81, 109^112. [20] Spring, S., Ludwig, W., Ma¨rquez, M.C., Ventosa, A. and Schleifer, K.H. (1996) Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov.. Int. J. Syst. Bacteriol. 46, 492^496. [21] Denariaz, G., Payne, W. and Legall, J. (1989) A halophilic denitri¢er, Bacillus halodenitri¢cans sp. nov.. Int. J. Syst. Bacteriol. 39, 145^ 151. [22] Heyndrickx, M., Lebbe, L., Kersters, K., de Vos, P., Forsyth, G. and Logan, N.A. (1998) Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int. J. Syst. Bacteriol. 48, 99^ 106. [23] Arahal, D.R., Ma¨rquez, M.C., Volcani, B.E., Schleifer, K.H. and Ventosa, A. (2000) Reclassi¢cation of Bacillus marismortui as Salibacillus marismortui comb. nov.. Int. J. Syst. Evol. Microbiol. 50, 1501^ 1503. [24] Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. and Schleifer, K.-H. (1998) Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554^568. [25] Stackebrandt, E. and Goebel, B.M. (1994) Taxonomic note: a place for DNA^DNA reassociation and 16S rRNA sequence analysis in the present species de¢nition in bacteriology. Int. J. Syst. Bacteriol. 44, 846^849. [26] Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D., Kandler, O., Krichevsky, M.I., Moore, L.H., Moore, W.E.C., Murray, R.G.E., Stackebrandt, E., Starr, M.P. and Tru«per, H.G. (1987) Report of the ad hoc committe on reconciliation of approches to bacterial systematics. Int. J. Syst. Bacteriol. 37, 463^464.
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Oceanobacillus iheyensis gen. nov., sp. nov., a deep-sea extremely halotolerant and alkaliphilic species isolated from a depth of 1050 m on the Iheya Ridge Jie Lu, Yuichi Nogi, Hideto Takami * Microbial Genome Research Group, DEEPSTAR, Japan Marine Science and Technology Center, 2-15 Natsushima, Yokosuka 237-0061, Japan Received 6 August 2001; received in revised form 21 October 2001; accepted 21 October 2001 First published online 14 November 2001
Abstract An extremely halotolerant and alkaliphilic bacterium was isolated previously from deep-sea sediment collected at a depth of 1050 m on the Iheya Ridge. The strain, designated HTE831 (JCM 11309, DSM 14371), was Gram-positive, strictly aerobic, rod-shaped, motile by peritrichous flagella, and spore-forming. Strain HTE831 grew at salinities of 0^21% (w/v) NaCl at pH 7.5 and 0^18% at pH 9.5. The optimum concentration of NaCl for growth was 3% at both pH 7.5 and 9.5. The G+C content of its DNA was 35.8%. Low level (12^30%) of DNA^DNA relatedness between strain HTE831 and the species of these genera was found, indicating that HTE831 could not be classified as a member of a new species belonging to known genera. Based on phylogenetic analysis using 16S rDNA sequencing, chemotaxonomy, and the physiology of strain HTE831, it is proposed that this organism is a member of a new species in a new genus, for which the name Oceanobacillus iheyensis is proposed. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Deep-sea isolate ; Halotolerant alkaliphile; 16S rDNA sequence; DNA^DNA hybridization; Oceanobacillus iheyensis
1. Introduction The bottom of the deep-sea is not devoid of organisms, although the deep-sea is an extreme environment with particularly high hydrostatic pressure and low temperature. Numerous microorganisms including nonextremophilic and extremophilic microbes such as piezophiles, psychrophiles, thermophiles, and alkaliphiles have been isolated from deep-sea sediment collected at depths of 10 897 m in the Challenger Deep of the Mariana Trench by the unmanned submersible Kaiko [1]. In addition, benthic organisms such as amphipods and sea cucumbers, which thrive in the Challenger Deep, have been retrieved. Although halophiles were not found in the Challenger Deep, several types of halophilic or halotolerant bacteria similar to the members of genera Bacillus, Halomonas, and Marinobacter on the basis of 16S rDNA comparison
* Corresponding author. Tel. : +81 (468) 67-9643; Fax: +81 (468) 67-9645. E-mail address : [email protected] (H. Takami).
have been isolated from the shallower Izu-Bonin Trench (2759 m deep, 30³07.05PN, 139³58.42PE; and 3400 m deep, 29³04.2PN, 140³43.3PE) and the Iheya Ridge of the Nansei Islands (1050 m deep, 27³47.18PN, 126³54.15PE) [2]. Thus, it was expected that the relative abundance of halophiles and halotolerant microbes among the deep-sea microbial £ora would decrease as the depth of sampling points increased. Halophilic or halotolerant bacteria are often isolated from salterns, hypersaline soils, and lakes [3^5], but little information is available on isolates from the deepsea. We are focusing on halophilic or halotolerant isolates from deep-sea environments. We are especially interested in strain HTE831 from the Iheya Ridge, which can grow on marine agar plates containing 15% NaCl, because 16S rDNA sequencing showed that the phylogenetic placement of this strain is comparatively close to that of Bacillus subtilis, for which the genome sequence has been determined [2]. In this study, we attempted to identify strain HTE831 both on the basis of conventional physiological and biochemical characteristics and through phylogenetic analysis based on 16S rDNA sequences and compari-
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 9 3 - 1
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son of DNA^DNA hybridization patterns. We also attempted to determine the genome size of the HTE831 chromosome by pulsed-¢eld gel electrophoretic (PFGE) analyses. 2. Materials and methods 2.1. Bacterial strain, media, and cultivation A deep-sea bacterium, designated HTE831, isolated from deep-sea mud sample collected at a depth of 1050 m on the Iheya Ridge of the Nansei Islands was studied [2]. The basal medium for strain HTE831 was PY medium, consisting of 1% polypeptone, 0.5% yeast extract, 0.1% K2 HPO4 , and 0.02% MgSO4 W7H2 O supplemented with NaCl (3^21%). The PY medium was adjusted to pH 6.5^ 8.5 with HCl or NaOH solution. Two types of alkaline PY medium supplemented with 1% Na2 CO3 or 2% NaHCO3 were adjusted to pH 10 and pH 9.5 with HCl or NaOH. All media with di¡erent pH values and NaCl concentrations were sterilized with a Falcon ¢lter unit with 0.22-Wm pore size (Becton Dickinson, Franklin Lakes, NJ, USA). Strain HTE831 was grown aerobically at various pH and temperature conditions. The cultivation of this strain under high-pressure conditions was carried out by the methods previously described [2,6].
2.5. Phylogenetic analysis based on 16S rDNA sequence 16S rDNA sequences were aligned using the Clustal multiple-alignment program (Clustal W) [13]. Sites involving gaps were excluded from all analyses. A phylogenetic tree was inferred by the neighbor-joining method [14] using the DNADIST and NEIGHBOR programs in the PHYLIP package, version 3.57 [15]. 2.6. PFGE Chromosomal DNA of HTE831 for PFGE was prepared in agarose plugs as described [16]. Agarose blocks containing chromosomal DNA were washed twice in 50 ml of 0.1UTE bu¡er and then equilibrated with the corresponding restriction bu¡er at 4³C for 1 h. DNA was digested with 100^200 U of ApaI or Sse8387I (Takara Shuzo, Otsu, Japan) at 37³C overnight in 500 Wl of the restriction bu¡er recommended by the manufacturer. PFGE in 1% PFC agarose was performed as previously described [16,17].
2.2. Biolog test and physiological properties Biolog tests were performed according to the manufacturer's instructions (Release 3.50, version DE, Biolog, Hayward, CA, USA). Plates were incubated at 30³C, and color changes were measured at 590 nm. Physiological properties of strain HTE831 were investigated by standard methods, described previously [7]. 2.3. Isoprenoid quinones and fatty acid analysis Isoprenoid quinones were extracted from freeze-dried cells with chloroform:methanol (2:1) and puri¢ed by thin-layer chromatography. The puri¢ed isoprenoid quinones were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) [8] and the absorbance was measured at 270 nm using menaquinone as a standard. Fatty acids were analyzed as methyl ester derivatives prepared from 10 mg of freeze-dried cell materials as previously described [9]. 2.4. DNA studies DNA was extracted by a previously described method [10]. The G+C content was determined by RP-HPLC [11]. For analysis of relatedness, DNA^DNA hybridization was carried out at 40³C for 3 h and measured £uorometrically [12].
Fig. 1. Phase-contrast and transmission electron micrographs of strain HTE831. A: Phase-contrast micrograph. Bar, 10 Wm. B: Transmission electron micrograph. Bar, 1 Wm. The strain was grown aerobically at pH 9.5 in PY medium containing 3% NaCl. For negative staining, one drop of culture was placed on a copper grid coated with 1% potassium phosphotungstic acid adjusted to pH 6.5 with potassium hydroxide.
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38 43 42
+ 5.8^9.6 2^24.8 10^45 MK-7 ND
9 8 7
rods, single or in chain central or subterminal 3 6^9.5 0.5^30 10^44 MK-7 iso-15:0, anteiso-15 :0, anteiso-17 :0
Cells of strain HTE831 were predominantly rod-shaped, 0.6^0.8U2.5^3.5 Wm in size, but ¢lamentous forms were present throughout the growth cycle. The cells were Grampositive, strictly aerobic, and motile by means of peritrichous £agella (Fig. 1 and Table 1). Ellipsoidal endospores were produced terminally or subterminally, causing swelling of the sporangia (Fig. 1). Cell morphology was not a¡ected by the salinity of the growth medium. Colonies of strain HTE831 were circular, creamy white, nontransparent, and approximately 2^3 mm in diameter on PY agar medium after 2 days of growth at 30³C.
rods, single or in chain central or subterminal 3 6^9.5 0.5^25 10^43 MK-7 iso-15:0, anteiso-15:0
3.1. Morphology
rods, single or in small chain NO
3. Results
36.9 40.7 38.0 G+C content (mol%)
35.8
+ ND 0^15 28^50 MK-7 iso-15:0, anteiso-15:0, iso-16:0, anteiso-17:0 40.1 3 5^10 0^20 6^50 MK-7 anteiso-15 :0, iso-16:0, anteiso-17 :0
Spore position
subterminal or terminal Anaerobic growth 3 pH range 6.5^10 NaCl range (%) 0^21 Temperature range (³C) 15^42 Major isoprenoid quinone MK-7 Major cellular fatty acids iso-15:0, anteiso-15:0, iso-14:0
39.5
+ ND 0^10 15^50 MK-7 iso-15:0, anteiso-15:0, anteiso-17:0
rods, single or in chain subterminal or terminal 3 6^9 5^25 15^50 MK-7 iso-15:0, anteiso-15 :0, anteiso-17 :0
6 4
rods, single or in chain central or subterminal 3 6^11 8^16 15^45 MK-7 iso-15:0, anteiso-15:0, iso-16:0 thin rods and ¢laments terminal
3
rods
2 1
Morphology
thin rods and ¢laments terminal
5
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Table 1 Morphological and physiological characteristics of strain HTE831 and some related organisms
Strain HTE831 exhibited extreme halotolerance and was able to grow in PY medium containing 0^21% NaCl at 30³C, pH 7.5. At pH 9.5, this strain could also grow in the same medium containing 0^18% NaCl. The duration of the lag phase and generation time lengthened with increasing NaCl concentration. It is notable that this strain did not require NaCl and that the optimum concentration of NaCl for cell growth was 3% in the pH range 6.5^10.0 (Table 1). Thus, strain HTE831 was shown to be facultatively alkaliphilic, exhibiting extreme halotolerance with broad pH optima for growth ranging from 7.0 to 9.5. Cell growth was observed at temperatures of 15^42³C, and the optimum temperature was 30³C in PY medium. In addition, HTE831 cell growth occurred at pressures of up to 30 MPa, corresponding to the pressure at a depth of 3000 m. When the physiological characteristics of strain HTE831 were investigated by the Biolog test, it assimilated only four substrates (K-D-glucose, maltose, D-mannose, and turanose). This assimilation pattern was characteristic of other related strains listed in Table 2. In contrast to HTE831, the ¢ve other strains, with the exception of Salibacillus salexigens [18], assimilated 15^23 di¡erent substrates under the same conditions. The assimilation patterns of two Gracilibacillus strains [9,19], G. halotolerans and G. dipsosauri, were very similar to each other. Among three Halobacillus strains [20], the substrate assimilation pattern of H. trueperi was very similar to that of H. litoralis, although that of H. halophilus di¡ered from those of the ¢rst two species. S. salexigens, which assimilated nine types of substrate, did not show an assimilation pattern similar to that of any other species (Table 2). Strain HTE831 produced acid from glycerol, fructose, glucose, maltose, and mannose. HTE831 neither produced H2 S nor indol. Production of DNase and urease, VP reaction, and assimilation of citrate by this strain were negative. The major isoprenoid quinone in strain HTE831 was menaquinone-7 (MK-7), which accounted for 99% of the
rods, single or in chain terminal
3.2. Physiological and biochemical properties
1, HTE831; 2, G. halotolerans [9]; 3, G. dipsosauri [9]; 4, S. salexigens [9]; 5, S. marismortui [23]; 6, V. pantothenticus [22]; 7, H. litoralis [20]; 8, H. trueperi [20]; 9, B. halodenitri¢cans [21]. ND: not determined ; NO : not observed.
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Table 2 Speci¢c carbon sources and nutrients showing positive reaction in the Biolog test among halophilic or halotolerant species used in this study Utilization of
1
2
3
4
5
6
7
K-Cyclodextrin L-Cyclodextrin Dextrin Glycogen N-acetyl-D-galactosamine N-acetyl-D-glucosamine Arbutin Cellobiose D-fructose Gentiobiose D-gluconic acid K-D-glucose K-D-lactose Maltose Maltotriose D-mannose D-melezitose D-melibiose 3-Methyl glucose K-Methyl D-glucoside L-Methyl D-glucoside K-Methyl D-mannoside Palatinose D-psicose D-ra¤nose Salicin Sedoheptulosan D-sorbitol Sucrose D-trehalose Turanose Acetic acid L-Hydroxybutyric acid K-Keto valeric acid L-lactic acid Methyl pyruvate Propionic acid Pyruvic acid Alaninamide D-alanine L-alanine L-glutamic acid L-serine Glycerol Adenosine Inosine Uridine
3 3 3 3 3 3 3 3 3 3 3 + 3 + 3 + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
+ + + + + 3 + + + 3 3 + + + + + 3 3 3 3 + 3 + 3 3 + 3 3 + + + 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
+ + + 3 3 3 + + + 3 3 + + + + + + + 3 3 + 3 3 + + + 3 + + + 3 3 3 3 3 + 3 + 3 3 3 3 3 + 3 3 3
3 3 + 3 + + 3 3 + 3 + + 3 + + + 3 3 + + 3 + 3 3 3 3 3 3 3 + 3 3 3 + 3 3 3 + 3 + + 3 + + 3 + +
3 3 3 3 3 3 3 3 3 3 3 + 3 3 3 3 3 3 3 + 3 3 3 3 3 3 3 3 3 3 + 3 3 3 + 3 3 3 + + 3 + 3 + + 3 3
3 + 3 3 3 3 3 + + + 3 + 3 + + 3 3 3 3 3 3 3 + + 3 3 3 3 + + + + + + + 3 + + 3 3 3 3 3 3 3 3 3
+ + 3 3 3 + 3 + 3 3 3 3 + + + 3 3 3 3 3 3 3 + + 3 3 3 3 3 + + + 3 + 3 3 + 3 3 3 3 3 3 + 3 3 3
1, HTE831 ; 2, G. halotolerans; 3, G. dipsosauri; 4, S. salexigens; 5, H. halophilus; 6, H. trueperi ; 7, H. litoralis.
total isoprenoid quinones, as in related genera showing halotolerant or halophilic phenotypes (Table 1). The G+C content of the DNA of strain HTE831 was found to be 35.8 mol%, as shown in Table 1. 3.3. Phylogenetic analysis based on 16S rDNA sequence In a previous study, the phylogenetic placement of strain HTE831 was investigated with partial 16S rDNA
Fig. 2. Unrooted phylogenetic tree based on 16S rDNA sequence comparison showing the relationship of strain HTE831 and other related strains. The numbers next to nodes indicate the percentages of bootstrap samples, derived from 1000 samples, which supported the internal branches [15]. Bootstrap probability values less than 50% were omitted from this ¢gure. Five strains (Bacillus halodurans, B. pseudo¢rmus, B. pseudalkaliphilus, B. clarkii, and B. agaradhaerens) are alkaliphile and others are halophilic or halotolerant and alkaliphilic strains. The sequence of Lactobacillus delbrueckii has been included to serve as an outgroup. Bar, 0.01 Knuc unit
sequencing (1419 bp) of this strain [2]. In that analysis, it was found that strain HTE831 was positioned close to Bacillus species such as B. subtilis and B. sporothermodurans. For further characterization of strain HTE831, we determined the complete 16S rDNA sequence and constructed a phylogenetic tree based on a comparison of the 16S rDNA sequence of this strain and those of type strains of Bacillus species and other species belonging to related genera. The 16S rDNA sequence of strain HTE831 (1525 bp) was redeposited in the DDBJ/EMBL/GenBank with the same accession number (AB10863) previously registered. Similarity values in the range of 90.5^94.4% were obtained when comparing the 16S rDNA sequence of strain HTE831 and those of nine other strains belonging to Bacillus halodenitri¢cans [21] and the genera derived from former Bacillus spp., such as Gracilibacillus, Halobacillus, Virgibacillus [22], and Salibacillus. Phylogenetic analysis
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based on 16S rDNA sequence comparison showed that strain HTE831 formed a cluster with the related strains belonging to the genera Virgibacillus and Salibacillus, derived from former Bacillus spp. and B. halodenitri¢cans, but the branch of HTE831 diverged at the bottom of this cluster (Fig. 2). This means that strain HTE831 is phylogenetically distant from these related genera, although their halotolerant phenotypes and other physiological characteristics are similar. 3.4. DNA^DNA hybridization analysis DNA^DNA hybridization analysis was carried out to compare strain HTE831 and eight related strains (Table 3). The DNA^DNA relatedness between HTE831 and two Gracilibacillus strains [9], G. dipsosauri (DSM 11125T ) and G. halotolerans (DSM 11805T ), was 30% and 25%, respectively. Two Salibacillus strains [9], S. salexigens [17] (DSM 11483T ) and S. marismortui [23] (DSM 12325T ), showed low homologies of 12^15% with HTE831. Similarly, three Halobacillus strains [20], H. halophilus (DSM 2266T ), H. trueperi (DSM 10404T ), and H. litoralis (DSM 10405T ), showed only 14^23% homologies with HTE831. The DNA^DNA relatedness of HTE831 and Virgibacillus pantothenticus [22] (DSM 26T ) was low at 15%. Finally, B. halodenitri¢cans [21] (DSM 10037T ) also showed a low homology of 24% with HTE831. Thus, it is clear that
295
Table 3 DNA^DNA hybridization between strain HTE831 and other related strains Source of unlabelled DNA O. iheyensis HTE831 G. dipsosauri DSM 11125T G. halotolerans DSM 11805T H. halophilus DSM 2266T H. trueperi DSM 10404T H. litoralis DSM 10405T S. salexigens DSM 11483T S. marismortui DSM 12325T V. pantothenticus DSM 26T B. halodenitri¢cans DSM 10037T
Homology (%) with H-labelled DNA from strain HTE831 100 30 25 23 21 14 12 15 15 24
strain HTE831 should not be categorized as a member of a new species within a known genus. 3.5. Estimation of genome size of strain HTE831 Restriction endonucleases that recognize an 8-bp sequence were tested for their ability to digest the chromosome of strain HTE831. ApaI (5P-GGGCC/C-3P) and Sse8387I (5P-CCTGCA/GG-3P) generated 37 and 25 resolvable fragments, respectively (Fig. 3). The sizes of these fragments were determined by comparison with size standards on a series of PFGE gels (Fig. 3). The mean total
Fig. 3. Digestion patterns of the chromosomal DNA of strain HTE831 obtained with Sse8387I and ApaI. A: Separation of fragments ranging in size from 200 to 1000 kb. B: Separation of fragments ranging in size from 100 to 400 kb. C: Separation of fragments ranging in size from 5 to 75 kb. Lanes: 1, Molecular size marker; 2, complete ApaI digestion; 3, complete Sse8387I digestion
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size of the genome of HTE831, estimated by totaling the ApaI or Sse8387I fragments, was 3.6 Mb. 4. Discussion In the previous study, halophilic or halotolerant bacteria were recovered from various deep-sea sites at a frequency of 0.9U102 ^3.6U103 g31 of dry sea mud [2]. Strain HTE831 isolated from deep-sea mud collected at a depth of 1050 m on the Iheya Ridge was characterized as an extremely halotolerant and facultatively alkaliphilic bacterium in this study. Halophilic or halotolerant bacteria are often isolated from salterns, hypersaline soils, and lakes [3^5], but little information is available on the isolates from the deep-sea. HTE831 is the ¢rst Gram-positive, spore-forming isolate from the bathypelagic zone, which shows extremely halotolerant and facultatively alkaliphilic phenotypes. Thus, deep-sea is a promising environment to ¢nd new types of halophilic or halotolerant bacteria. However, it is not easy to access such deep-sea sites, not even by using the manned submersible Shinkai 6500, due to climatic problems and economical constraints. Similarity values in the range of 90.5^94.4% were obtained when comparing the 16S rDNA sequence of strain HTE831 and those of nine other strains used in this study. In the phylogenetic tree constructed based on 16S rDNA sequences, HTE831 formed a cluster with the related strains belonging to the genera Virgibacillus and Salibacillus, derived from former Bacillus spp. and B. halodenitri¢cans; the branch of HTE831 diverged at the bottom of this cluster, which is being supported by the bootstrap value of 80% (Fig. 2). It has been suggested that 16S rDNA similarities around 95% would be a practical border zone for genus de¢nition [24]. At 16S rDNA similarity levels of 97% and higher, the DNA^DNA reassociation techniques are the superior method [25], and the genomic DNA similarity of around 70% and higher is shared by strains of a species [26]. The DNA^DNA relatedness between HTE831 and nine other related species in the ¢ve genera, Bacillus, Gracilibacillus, Halobacillus, Virgibacillus, and Salibacillus, was less than 30%, indicating that HTE831 should not be categorized as a new species in any of these genera. On the other hand, there are some signi¢cant di¡erences between HTE831 and other related species in the growth pattern in terms of salinity and temperature. For example, Salibacillus and Halobacillus species show a halophilic phenotype, but no growth occurs in the absence of salt, in contrast to halotolerant HTE831. Although Virgibacillus and Gracilibacillus species also have halotolerant phenotypes like HTE831, Virgibacillus species can only grow in medium containing up to 10% salt, and Gracilibacillus species can grow even at 50³C, which is 8³C higher than the maximum growth temperature for HTE831. Thus, we propose that the new genus Oceanobacillus should be created for the deep-sea isolate HTE831T and
describe a new species within the genus Oceanobacillus called O. iheyensis on the basis of the results presented in this study. We note that it is not recommended to base the description of a genus on a single isolate. However, we did not detect any other strain which could be assigned to the species O. iheyensis among our deep-sea isolates, and access to the deep-sea sampling station is di¤cult. The type strain is O. iheyensis HTE831 (JCM 11309T , DSM 14371T ). 4.1. Description of Oceanobacillus (Lu, Nogi, and Takami) gen. nov. Oceanobacillus (o.ce.a.no.ba.cil'lus. L. n. oceanus, the ocean; L. dim. n. bacillus, a small rod; M.L. masc. Oceanobacillus, the ocean bacillus/rod). Gram-positive, motile by peritrichous £agella, sporeforming rods. Endospores are subterminal or terminal and slightly swell the sporangia. Obligately aerobic, facultatively alkaliphilic, and extremely halotolerant. Growth occurs at temperatures of 15^42³C. Colonies are circular. Chemoorganotrophic. Grows on glucose, mannose, maltose and turanose. Acid production occurred from glucose, mannose, glycerol, fructose, and maltose, but not from xylose, rhamnose, glucitol, trehalose, galactose, lactose, and melibiose. Catalase-positive, oxidase-variable, DNase-negative, and urease-negative. VP reaction, indol and H2 S production, and use of citrate are negative. Aminopeptidase and KOH tests are negative. Nitrate reduction to nitrite is negative. Cells are resistant to erythromycin, but susceptible to ampicillin. On the basis of 16S rRNA gene sequencing analysis, the genus Oceanobacillus does not form a branch with other genera such as Halobacillus, Gracilibacillus, Virgibacillus, and Salibacillus. The G+C content of the type species is 35.8%. The major cellular fatty acids are anteiso-15:0, iso-15:0, and iso-14 :0. The main menaquinone type is MK-7. The type species of the genus is O. iheyensis sp. nov. 4.2. Description of O. iheyensis sp. nov. O. iheyensis (i.he.yen'sis. M.L. masc. adj. iheyensis, pertaining to the Iheya Ridge). In addition to the characteristics listed above for the genus, the following features are characteristic of M. iheyensis. Cells are 0.6^0.8U2.5^3.5 Wm. Endospores are ellipsoid. Colonies are creamy white. Grows at 0^21% (w/v) NaCl, with optimum growth at 3% NaCl. Growth occurs at temperatures of 15^42³C (optimum 30³C). The pH range for growth is 6.5^10 (optimum 7.0^9.5). Hydrolyzes gelatin, casein, Tween 40, and Tween 60. Does not hydrolyze starch. Cells are resistant to nalidixic acid and spectinomycin, but susceptible to gentamycin, kanamycin, tetracycline, bacitracin, carbenicillin, chloramphenicol, novobiocin, penicillin G, and rifampicin. The following four substrates are assimilated in the Biolog test: K-D-glucose,
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maltose, D-mannose, and turanose. The G+C content is 35.8%. The genome size is about 3.6 Mb. 16S rDNA sequence exhibits 90.5^94.4% homology with those from B. halodenitri¢cans and the members of the genera Halobacillus, Gracilibacillus, Virgibacillus, and Salibacillus. The isolate was obtained from a mud sample collected at a depth of 1050 m on the Iheya Ridge. The type strain is O. iheyensis HTE831T , = JCM 11309T , Japan Collection of Microorganisms, RIKEN Institute, Wako-shi, Saitma, Japan, and = DSM 14371T , Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH. Acknowledgements We thank H. Uchiyama and Drs. Y. Takaki and K. Nakasone for their technical assistance. Thanks are also due to K. Uemastu for his assistance in preparing electron micrographs. References [1] Takami, H., Inoue, A., Fuji, F. and Horikoshi, K. (1997) Microbial £ora in the deepest sea mud of the Mariana Trench. FEMS Microbiol. Lett. 152, 279^285. [2] Takami, H., Kobata, K., Nagahama, T., Kobayashi, H., Inoue, A. and Horikoshi, K. (1999) Biodiversity in the deep-sea sites located near the south part of Japan. Extremophiles 3, 97^102. [3] Garabito, M.J., Ma¨rquez, M.C. and Ventosa, A. (1998) Halotolerant Bacillus diversity in hypersaline environments. Can. J. Microbiol. 44, 95^102. [4] Arahal, D.R., Ma¨rquez, M.C., Volcani, B.E., Schleifer, K.H. and Ventosa, A. (1999) Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int. J. Syst. Bacteriol. 49, 521^ 530. [5] Duckworth, A.W., Grant, W.D., Jones, B.E., Meijer, D., Ma¨rquez, M.C. and Ventosa, A. (2000) Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley. Extremophiles 4, 53^60. [6] Abe, F. and Horikoshi, K. (1995) Hydrostatic pressure promotes the acidi¢cation of vacuoles in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 130, 307^312. [7] Sneath, P.H.A., Mair, N.S., Sharp, M.E. and Holt, J.G. (1986) Bergey's Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins, Baltimore, MD. [8] Komogata, K. and Suzuki, K. (1987) Lipid and cell wall analysis in bacterial systematics. Methods Microbiol. 19, 161^207. [9] WainÖ, M., Tindall, B.J., Schumann, P. and Ingvorsen, K. (1999) Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov.. Int. J. Syst. Bacteriol. 49, 821^831. [10] Saito, H. and Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619^629.
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