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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, P. R. China
2 Graduate University of Chinese Academy of Sciences, Beijing 100049, P. R. China
Correspondence
Shuang-Jiang Liu
liusj{at}sun.im.ac.cn
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ABSTRACT |
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A thin-layer chromatogram of total polar lipids from strain BD-3T and members of the genus Haloarcula is available as supplementary material in IJSEM Online.
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, adopting the generic name first suggested by Javor et al. (1982). At the time of writing, the genus Haloarcula comprises six recognized species (Fig. 1): H. vallismortis (Gonzalez et al., 1978; Torreblanca et al., 1986), H. argentinensis (Ihara et al., 1997), H. hispanica (Juez et al., 1986), H. japonica (Takashina et al., 1990), H. marismortui (Elazari-Volcani, 1957; Oren et al., 1990) and H. quadrata (Oren et al., 1999). A common feature of representative strains of the genus Haloarcula is the presence of at least two heterogeneous 16S rRNA genes. During our surveys on halophilic archaeal diversity of the Aibi salt lake (82° 35'–83° 16' E 44° 05'–45° 08' N) in the Xin-Jiang region of China (Cui et al., 2006), a strain that harboured three heterogeneous 16S rRNA genes and that clustered tightly with members of the genus Haloarcula was obtained.
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Phenotypic tests were performed according to the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Cell motility and morphology were examined by phase-contrast microscopy of exponentially growing liquid cultures. Gram staining was carried out as described by Dussault (1955). Colony morphology was observed on salt-milk and the standard growth agar medium after incubation at 40 °C for 6–10 days. Anaerobic growth was tested in the presence of 5 g sodium nitrate, L-arginine or DMSO per litre in filled, stoppered tubes. To determine growth pH range (using increments of 0.5 pH units, from pH 5.0 to 10.0), 25 mM MES (pH 5.0–6.7), 25 mM PIPES (pH 6.1–7.5), 25 mM MOPS (pH 6.5–7.9), 25 mM HEPES (pH 6.8–8.2), 25 mM Tricine (pH 7.4–8.8) and 25 mM CHES (pH 8.6–10.0) were used. Tests for catalase and oxidase activities and hydrolysis of starch and of Tweens 20, 40, 60 and 80 were performed as described by Gonzalez et al. (1978). Nitrate reduction, H2S formation, indole formation, and utilization of carbohydrates, sugar alcohols, amino acids and organic acids were examined as described by Oren et al. (1997).
Polar lipids were extracted with chloroform/methanol as described by Kamekura (1993). One- and two-dimensional TLC was performed by using silica gel plates (Kieselgel 60 F; Merck) (Kates, 1986).
DNA was extracted by using phenol/chloroform extraction followed by ethanol precipitation, as modified according to Ng et al. (1995). The gene encoding 16S rRNA was amplified by PCR with the forward primer 5'-TTCCGGTTGATCCTGCC-3' (positions 7–23, according to the Escherichia coli numbering scheme) and the reverse primer 5'-AAGGAGGTGATCCAGCC-3' (positions 1541–1525), modified according to Achenbach & Woese (1995). PCR was performed for 30 cycles with denaturation for 1 min at 95 °C, annealing for 1 min at 49 °C and polymerization for 90 s at 72 °C. The PCR product was cloned into T-vector, and sequenced by using an ABI BigDye3.1 sequencing kit (Applied Biosystems) and an automated DNA sequencer (model ABI3730; Applied Biosystems). Phylogenetic analyses were conducted using the neighbour-joining method (MEGA version 3.1; Kumar et al., 2004).
The G+C content of the DNA was determined by using the thermal denaturation method (Marmur & Doty, 1962), with a Beckman UV-Vis DU800 spectrophotometer at 260 nm. DNA–DNA hybridization was carried out by the thermal denaturation and renaturation method (De Ley et al., 1970) as modified by Huß et al. (1983).
Cells of strain BD-3T were Gram-negative, motile rods, and were able to grow over a wide range of salinities (2.0–5.1 M NaCl; optimal growth at 2.9–3.2 M). Cells lysed in distilled water and 5 % brine. Colonies on salt-milk agar medium were red-pigmented. Phenotypic characteristics of strain BD-3T are given in the species description below and Table 1. As detailed in Table 1, strain BD-3T could be phenotypically differentiated from recognized Haloarcula species but had very similar phenotypic properties to H. hispanica ATCC 33960T.
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Polymorphism of 16S rRNA genes of strain BD-3T was observed. Three 16S rRNA genes were detected, designated rrnA (1472 bp), rrnB (1472 bp) and rrnC (1472 bp) (Fig. 1
). The rrnA gene sequence of BD-3T showed high similarities to the rrnA gene sequence of H. hispanica (98.7 %), the 16S rRNA gene sequence of H. argentinensis (98.6 %), and the rrnB (98.6 %) and rrnC (98.6 %) gene sequences of H. marismortui. The rrnB gene sequence of BD-3T showed highest similarity (99.5 %) to the H. marismortui rrnA gene sequence. The rrnC gene sequence of BD-3T showed highest similarity (98.8 %) to the H. marismortui rrnA gene sequence and H. quadrata rrnB gene sequence. Levels of similarity between the three 16S rRNA genes of strain BD-3T were 95.2 % (rrnA and rrnB), 94.6 % (rrnA and rrnC) and 99.0 % (rrnB and rrnC). DNA–DNA hybridization values between strain BD-3T and H. japonica JCM 7785T, H. vallismortis ATCC 29715T, H. hispanica ATCC 33960T, H. argentinensis JCM 9737T, H. quadrata JCM 11048T and H. marismortui ATCC 43049T were 30, 30, 34, 38, 39 and 39 %, respectively.
Based on these results, it is concluded that strain BD-3T represents a novel species of the genus Haloarcula, for which the name Haloarcula amylolytica sp. nov. is proposed.
Description of Haloarcula amylolytica sp. nov.
Haloarcula amylolytica (a.my.lo.ly'ti.ca. Gr. n. amylos starch; Gr. adj. lytikos dissolving; N.L. f. adj. amylolytica producing lysis of starch).
Cells are rod-shaped (0.6–0.8x2.0–2.5 µm), motile and Gram-negative. Colonies on salt-milk and standard growth agar plates containing 18 % (w/v) NaCl are red, elevated and round. Chemo-organotrophic and aerobic. Growth occurs at NaCl concentrations of 2.0–5.1 M, Mg2+ concentrations of 0.005–0.70 M, pH 6.5–9.0 and temperature of 20–52 °C. Optimal NaCl concentration, Mg2+ concentration, pH and temperature for growth are 2.9–3.2 M, 0.10–0.30 M, pH 7.0–7.5 and 41 °C, respectively. Catalase- and oxidase-positive. Anaerobic growth occurs in the presence of nitrate with formation of nitrite and gas. No anaerobic growth in the presence of arginine or DMSO. H2S is produced from Na2S2O3. Indole formation is positive. Tweens 20, 40, 60, 80 and starch are hydrolysed. Casein is not hydrolysed. Gelatin is liquefied. Glucose, sucrose, galactose, sorbitol, mannose, mannitol and maltose are utilized and acids are produced. No growth is observed on fructose, sorbose, xylose, lactose or D-ribose. Sensitive to the following antibiotics: novobiocin (30 µg per disc) and bacitracin (0.04 IU per disc). Resistant to the following antibiotics (µg per disc, unless otherwise indicated): ampicillin (10), rifampicin (5), erythromycin (15), tetracycline (30), ciprofloxacin (5), chloramphenicol (30), kanamycin (30), neomycin (30), vancomycin (30), norfloxacin (10), streptomycin (10) and penicillin G (10 IU per disc). The molar G+C content of the DNA is 62.4 % (Tm). The major polar lipids are phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerosulfate, triglycosyl diether and an unknown diglycosyl diether.
The type strain, BD-3T (=CGMCC 1.5335T=JCM 13557T), was isolated from Aibi salt lake in Xin-Jiang, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Cui, H.-L., Yang, Y., Tohty, D., Zhou, P.-J. & Liu, S.-J. (2006). Biodiversity of halophilic archaea isolated from two salt lakes in Xin-Jiang region of China. Acta Microbiol Sinica 46, 171–176.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
Elazari-Volcani, B. (1957). Genus XII. Halobacterium Elazari-Volcani, pp. 207–212. In Bergey's Manual of Determinative Bacteriology, 7th edn. Edited by R. S. Breed, R. G. D. Murray & N. R. Smith. Baltimore: Williams & Wilkins.
Gonzalez, C., Gutierrez, C. & Ramirez, C. (1978). Halobacterium vallismortis sp. nov. An amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium. Can J Microbiol 24, 710–715.[Medline]
Huß, V. A. R., Festl, H. & Schleifer, K.-H. (1983). Studies on the spectrometric determination of DNA hybridisation from renaturation rates. Syst Appl Microbiol 4, 184–192.
Ihara, K., Watanabe, S. & Tamura, T. (1997). Haloarcula argentinensis sp. nov. and Haloarcula mukohataei sp. nov., two new extremely halophilic archaea collected in Argentina. Int J Syst Bacteriol 47, 73–77.
Javor, B., Requadt, C. & Stoeckenius, W. (1982). Box-shaped halophilic bacteria. J Bacteriol 151, 1532–1542.
Juez, G., Rodriguez-Valera, F., Ventosa, A. & Kushner, D. J. (1986). Haloarcula hispanica spec. nov. and Haloferax gibbonsii spec. nov., two new species of extremely halophilic archaebacteria. Syst Appl Microbiol 8, 75–79.
Kamekura, M. (1993). Lipids of extreme halophiles. In The Biology of Halophilic Bacteria, pp. 135–161. Edited by R. H. Vreeland & L. I. Hochstein. Boca Raton, FL: CRC Press.
Kates, M. (1986). Techniques of Lipidology, 2nd edn. Amsterdam: Elsevier.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Ng, W. L., Yang, C. F., Halladay, J. T., Arora, A. & DasSarma, S. (1995). Protocol 25: isolation of genomic and plasmid DNAs from Halobacterium halobium. In Archaea: a Laboratory Manual: Halophiles, pp. 179–180. Edited by S. DasSarma & E. M. Fleischmann. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Oren, A., Ginzburg, M., Ginzburg, B. Z., Hochstein, L. I. & Volcani, B. E. (1990). Haloarcula marismortui (Volcani) sp. nov., nom. rev., an extremely halophilic bacterium from the Dead Sea. Int J Syst Bacteriol 40, 209–210.[Medline]
Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233–238.
Oren, A., Ventosa, A., Gutiérrez, M. C. & Kamekura, M. (1999). Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt). Int J Syst Bacteriol 49, 1149–1155.
Takashina, T., Hamamoto, T., Otozai, K., Grant, W. D. & Horikoshi, K. (1990). Haloarcula japonica sp. nov., a new triangular halophilic archaebacterium. Syst Appl Microbiol 13, 177–181.
Torreblanca, M., Rodriguez-Valera, F., Juez, G., Ventosa, A., Kamekura, M. & Kates, M. (1986). Classification of non-alkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst Appl Microbiol 8, 89–99.
Xin, H., Itoh, T., Zhou, P., Suzuki, K., Kamekura, M. & Nakase, T. (2000). Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China. Int J Syst Evol Microbiol 50, 1297–1303.[Abstract]
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