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1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
2 Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken 278-0037, Japan
3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, China
4 Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa
5 Genencor International BV, Archimedesweg 30, 2333 CN Leiden, The Netherlands
6 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, UK
Correspondence
A. Ventosa
ventosa{at}us.es
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A phase-contrast micrograph and TLC of polar lipids of strain EJ-52T are available as supplementary figures in IJSEM Online.
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; Oren, 2000; Grant et al., 2001). They are able to grow over a wide range of salt concentrations, i.e. between 8 and 30 % NaCl (Grant et al., 2001). Currently, they are included in the order Halobacteriales, family Halobacteriaceae, with a large number of species being grouped in 20 genera (Grant et al., 2001; Ventosa, 2006). The genus Halorubrum was formally proposed by McGenity & Grant (1995) to accommodate several species previously included in the genus Halobacterium: Halorubrum saccharovorum (Tomlinson & Hochstein, 1976), Halorubrum sodomense (Oren, 1983), Halorubrum trapanicum (Petter, 1931) and Halorubrum lacusprofundi (Franzmann et al., 1988). In addition, Halorubrobacterium distributum (Zvyagintseva & Tarasov, 1987) and Halorubrobacterium coriense (Kamekura & Dyall-Smith, 1995) were transferred to the genus Halorubrum by Oren & Ventosa (1996). 16S rRNA gene sequence studies showed that Natronobacterium vacuolatum (Mwatha & Grant, 1993) was phylogenetically related to the genus Halorubrum, and it was designated Halorubrum vacuolatum (Kamekura et al., 1997). Lizama et al. (2002) described the species Halorubrum tebenquichense, isolated from Lake Tebenquiche, located in the Salar de Atacama, Chile. More recently, four novel species have been described: Halorubrum terrestre (Ventosa et al., 2004), Halorubrum tibetense (Fan et al., 2004), Halorubrum xinjiangense (Feng et al., 2004) and Halorubrum alkaliphilum (Feng et al., 2005). Strain EJ-52T was isolated from water from a saline lake in Inner Mongolia, China, and was subjected to 16S rRNA gene sequence analysis, polar lipid determination and phenotypic study. The results show that strain EJ-52T is closely related to members of the genus Halorubrum.
Strain EJ-52T was isolated from a sample of water from Lake Ejinor (45° 14' N 116° 31' E) by enrichment in liquid medium and subsequent plating on the same medium with added agar until clonal purity was obtained. The medium contained the following (l–1): NaCl, 195 g; MgCl2.6H2O, 32.5 g; MgSO4.7H2O, 50.8 g; CaCl2, 0.8 g; KCl, 5 g; NaHCO3, 0.16 g; NaBr, 0.6 g; and yeast extract, 5 g. The pH was adjusted to 8 with 1 M NaOH. The water of the lake had a conductivity of 166 mS cm–1, a pH of 8.5 and a temperature of 32 °C at the time of sampling in September 2003. Strain EJ-52T grew at temperatures in the range 25–50 °C (optimum, 35–45 °C) and at pH values in the range 6.0–10.0 (optimum, pH 8.0). Routine cultivation was conducted at 37 °C and pH 8. The NaCl and MgCl2 requirements for growth were determined in media containing 0.9–5.2 M NaCl or 0–0.5 M MgCl2. Strain EJ-52T was capable of growing in a wide range of NaCl concentrations, from 15 % (2.5 M) to 30 % (5 M); it grew optimally in the presence of 20 % (3.4 M) NaCl. The salt range and optimum for growth were found to be similar to those of most extremely halophilic archaea belonging to the Halobacteriales (Oren, 2000; Grant et al., 2001). MgCl2 was not required for growth.
Phenotypic tests were performed according to the proposed minimal standards for the description of novel taxa of the order Halobacteriales (Oren et al., 1997). Cell motility and morphology of exponentially growing liquid cultures were examined using an Olympus BX41 microscope equipped with phase-contrast optics. The cells were found to be motile and pleomorphic (see Supplementary Fig. S1 available in IJSEM Online). Colony morphology was observed under optimal growth conditions on agar medium after incubation at 37 °C for 10 days. On agar plates, the colonies of strain EJ-52T were circular, smooth, entire and red-pigmented. Anaerobic growth was tested in the presence of nitrate (5 g l–1) or L-arginine (5 g l–1) in filled stoppered tubes. The following characteristics were tested as described by Oren et al. (1997): hydrolysis of starch, gelatin, urea, DNA, aesculin, casein and Tween 80; nitrate reduction, production of indole and H2S, catalase and oxidase activities and utilization of sugars, alcohols, amino acids and organic acids as carbon and energy sources or carbon, nitrogen and energy sources. Strain EJ-52T was found to be oxidase- and catalase-positive. Negative results were obtained for indole production from tryptophan and in methyl red, Voges–Proskauer and Simmons' citrate tests. Nitrate was reduced without the production of gas. Tween 80 and urea were hydrolysed, but starch, gelatin, casein, DNA and aesculin were not.
Susceptibility to antibiotics was determined on agar plates with antibiotic discs containing the following: ampicillin (10 µg), bacitracin (10 U), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), nalidixic acid (30 µg), neomycin (10 µg), novobiocin (30 µg), penicillin G (10 U), rifampicin (30 µg), streptomycin (10 µg) and tetracycline (30 µg). The results for antibiotic susceptibility and for the utilization of various substrates are included in the species description.
Polar lipids were extracted with chloroform/methanol as described previously (Kamekura, 1993). TLC was performed using Merck HPTLC plates (silica gel 60; art. no. 5641) in the solvent system chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.). Glycolipids were detected as purple spots by spraying 0.5 % with 
-naphthol in methanol/water (1 : 1) and then with sulfuric acid/ethanol (1 : 1), followed by heating at 160 °C. The polar lipids of strain EJ-52T (see Supplementary Fig. S2 available in IJSEM Online) are C20C20 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and the characteristic glycolipid of the neutrophilic species of Halorubrum, a sulfated mannosyl-glucosyl-glycerol diether (McGenity & Grant, 2001).
Chromosomal DNA of strain EJ-52T was isolated and purified according to the method described by Marmur (1961). The G+C content of the genomic DNA was determined from the mid-point (Tm) of the thermal denaturation profile (Marmur & Doty, 1962) using the equation of Owen & Hill (1979), as described previously (Ventosa et al., 2004), and was found to be 64.2 mol%. The 16S rRNA gene of strain EJ-52T was amplified by means of a PCR with three universal primers, as described previously (López-Garcia et al., 2001; Arahal et al., 1996), and an almost-complete nucleotide sequence (1409 bp) was determined. The ARB software package (Ludwig et al., 2004) was used for analysis of the 16S rRNA gene sequence. Base-frequency filters were applied in the sequence comparison analysis and the effects on the results were evaluated. Alignment of the 16S rRNA gene sequence with all published sequences of haloarchaea clearly showed that strain EJ-52T belonged to the genus Halorubrum, since it possessed 18 of the 19 signature bases of the genus Halorubrum (Kamekura et al., 2004). The phylogenetic tree (Fig. 1) constructed by maximum parsimony (Saitou & Nei, 1987) indicated that strain EJ-52T was related to Hrr. saccharovorum ATCC 29252T (96.1 %), Hrr. lacusprofundi JCM 8891T (95.9 %), Hrr. tibetense AS 1.3239T (95.2 %), Hrr. alcaliphilum AS 1.3528T (95.2 %) and Hrr. vacuolatum JCM 9060T (95.1 %). Similar topologies were obtained when other treeing methods (neighbour joining and maximum likelihood) were used. The low levels of similarity (<97 % in all cases) between the 16S rRNA gene sequence of strain EJ-52T and those of the species of Halorubrum suggest that the novel isolate could represent a novel species (Stackebrandt & Goebel, 1994; Vandamme et al., 1996). Additionally, there are phenotypic differences between strain EJ-52T and the species of Halorubrum (Table 1).
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Description of Halorubrum orientale sp. nov.
Halorubrum orientale (o.ri.en.ta'le. L. neut. adj. orientale of, or belonging to, the East, eastern, pertaining to the isolation of the type strain from China).
Cells are pleomorphic, 1.5–2x1–2 µm in size and motile. Colonies are small, entire, smooth and red-pigmented. Growth occurs in the presence of 2.5–5.0 M NaCl, with optimum growth occurring at 3.4 M NaCl. MgCl2 is not required. Growth occurs between 25 and 50 °C (optimum, 35–45 °C) and at pH 6.0–10.0 (optimum, pH 8.0). Chemo-organotrophic. Oxidase- and catalase-positive. Nitrate is reduced to nitrite. Tween 80 and urea are hydrolysed, whereas starch, casein and gelatin are not. H2S is not produced. Indole is not produced from tryptophan. The Voges–Proskauer test is negative. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are not produced. Anaerobic growth with nitrate or arginine does not occur. Acid is not produced from lactose, glycerol, D-glucose, sucrose, D-fructose, D-arabinose, maltose, D-xylose, D-galactose, D-trehalose and D-mannose. Growth occurs on glycerol, D-fructose, maltose, D-glucose, starch, L-glutamate and fumarate as single carbon and energy sources. The following compounds are not used as sole carbon and energy sources: D-mannitol, D-sorbitol, lactose, D-arabinose, D-raffinose, D-ribose, D-xylose, succinate, propionate, malate and acetate. The following compounds are used as sole carbon, nitrogen or energy sources: L-asparagine, glycine, L-lysine, L-serine and L-threonine. Susceptible to novobiocin, bacitracin, gentamicin, ampicillin, neomycin, erythromycin, chloramphenicol, nalidixic acid and streptomycin; resistant to penicillin G and rifampicin. The polar lipids are C20C20 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and a sulfated mannosyl-glucosyl-glycerol diether. The DNA G+C content is 64.2 mol% (Tm method).
The type strain, EJ-52T (=CECT 7145T=JCM 13889T=CGMCC 1.6295T), was isolated from Lake Ejinor, a saline lake in Inner Mongolia, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Fan, H., Xue, Y., Ma, Y., Ventosa, A. & Grant, W. D. (2004). Halorubrum tibetense sp. nov., a novel haloalkaliphilic archaeon from Lake Zabuye in Tibet, China. Int J Syst Evol Microbiol 54, 1213–1216.
Feng, J., Zhou, P. & Liu, S. (2004). Halorubrum xinjiangense sp. nov., a novel halophile isolated from saline lakes in China. Int J Syst Evol Microbiol 54, 1789–1791.
Feng, J., Zhou, P., Zhou, Y., Liu, S. & Warren-Rhodes, K. (2005). Halorubrum alkaliphilum sp. nov., a novel haloalkaliphile isolated from a soda lake in Xinjiang, China. Int J Syst Evol Microbiol 55, 149–152.
Franzmann, P. D., Stackebrandt, E., Sanderson, K., Volkman, J. K., Cameron, D. E., Stevenson, P. L., McMeekin, T. A. & Burton, H. R. (1988). Halobacterium lacusprofundi sp. nov., a halophilic bacterium isolated from Deep Lake, Antarctica. Syst Appl Microbiol 11, 20–27.
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Kamekura, M., Mizuki, T., Usami, R., Yoshida, Y., Horikoshi, K. & Vreeland, R. H. (2004). The potential use of signature bases from 16S rRNA gene sequences to aid the assignment of microbial strains to genera of halobacteria. In Halophilic Microorganisms, pp. 77–87. Edited by A. Ventosa. Heidelberg: Springer.
Lizama, C., Monteoliva-Sánchez, M., Suárez-García, A., Rosselló-Mora, R., Aguilera, M., Campos, V. & Ramos-Cormenzana, A. (2002). Halorubrum tebenquichense sp. nov., a novel halophilic archaeon isolated from the Atacama Saltern, Chile. Int J Syst Evol Microbiol 52, 149–155.[Abstract]
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