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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, People's Republic of 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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49 %, indicating that EJ-32T constitutes a different genospecies. The data show that strain EJ-32T represents a novel species of the genus Halorubrum, for which the name Halorubrum ejinorense sp. nov. is proposed. The type strain is EJ-32T (=CECT 7194T=CGMCC 1.6782T=JCM 14265T).
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; Oren, 2002). Members of the family Halobacteriaceae are ubiquitous in these hypersaline environments. They are aerobic or facultatively anaerobic, red-pigmented due to the presence of carotenoid pigments (except for a few species), chemo-organotrophic archaea requiring at least 1.5 M NaCl for growth (Grant et al., 2001; Ventosa, 2006). The halophilic archaea have been classified into 23 genera on the basis of 16S rRNA gene sequence comparisons and chemotaxonomic criteria, particularly polar lipid composition, but there is no strict correlation between the two (Ventosa, 2006).
At the time of writing the genus Halorubrum (McGenity & Grant, 1995, 2001) contained 16 recognized species: Halorubrum aidingense (Cui et al., 2006), Halorubrum alkaliphilum (Feng et al., 2005), Halorubrum coriense (Kamekura & Dyall-Smith, 1995; Oren & Ventosa, 1996), Halorubrum distributum (Zvyagintseva & Tarasov, 1987; Oren & Ventosa, 1996), Halorubrum ezzemoulense (Kharroub et al., 2006), Halorubrum lacusprofundi (Franzmann et al., 1988), Halorubrum lipolyticum (Cui et al., 2006), Halorubrum orientale (Castillo et al., 2006), Halorubrum sodomense (Oren, 1983), Halorubrum tebenquichense (Lizama et al., 2002), Halorubrum terrestre (Ventosa et al., 2004), Halorubrum tibetense (Fan et al., 2004), Halorubrum trapanicum (Petter, 1931; McGenity & Grant, 1995), Halorubrum vacuolatum (Mwatha & Grant, 1993; Kamekura et al., 1997), Halorubrum xinjiangense (Feng et al., 2004) and Halorubrum saccharovorum (type species) (Tomlinson & Hochstein, 1976). These species can be classified into two groups according to their growth at various pH values and their origins. Group 1 contains neutrophilic species, such as Hrr. aidingense, Hrr. coriense, Hrr. distributum, Hrr. ezzemoulense, Hrr. lacusprofundi, Hrr. lipolyticum, Hrr. orientale, Hrr. saccharovorum, Hrr. sodomense, Hrr. tebenquichense, Hrr. terrestre, Hrr. trapanicum and Hrr. xinjiangense. Group 2 contains three alkaliphilic species, Hrr. alkaliphilum, Hrr. tibetense and Hrr. vacuolatum (Grant et al., 2001).
Here, we report the isolation and description of a novel neutrophilic strain (EJ-32T) isolated from Lake Ejinor in Inner Mongolia, China, and its assignment as a novel species of the genus Halorubrum.
Strain EJ-32T was isolated from a water sample collected from Lake Ejinor (4 ° 14' N, 11 ° 32' E) in Inner Mongolia, China. At the time of sampling (September 2003) the water of the lake had a temperature of 32 °C, a pH of 8.5 and a salinity of 18 % (w/v). The isolation procedure was as described previously (Castillo et al., 2006; Gutiérrez et al., 2007). Characterization of strain EJ-32T was performed according to the proposed minimal standards for the description of novel taxa in the order Halobacteriales (Oren et al., 1997). Cell motility and morphology were examined by phase-contrast microscopy of exponentially growing liquid cultures by using an Olympus BX41 microscope equipped with phase-contrast optics. The cells of strain EJ-32T were rod-shaped and non-motile, with a tendency to form clumps. Colony morphology was observed under optimal growth conditions on agar medium with 25 % (w/v) salts, after incubation at 37 °C for 10 days. Anaerobic growth was tested in filled, stoppered tubes in the presence of 5 g nitrate l–1 or 5 g arginine l–1. Tests for the following features were carried out as described by Oren et al. (1997): catalase and oxidase activities; hydrolysis of starch, Tween 80, gelatin, casein, DNA and aesculin; reduction of nitrate and nitrite; formation of H2S and indole; utilization of sugars, alcohols, amino acids and organic acids. Antibiotic-sensitivity tests were performed by spreading bacterial suspensions on culture plates and applying discs impregnated with the following concentrations (µg unless indicated otherwise): ampicillin (10), bacitracin (10 U), cephalothin (30), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic acid (30), neomycin (10), novobiocin (30), penicillin G (10 U), rifampicin (30), polymyxin (300 U), streptomycin (10), sulfamethoxazole (25), tetracycline (30) and vancomycin (30). The physiological and biochemical characteristics, as well as the antibiotic susceptibilities, of strain EJ-32T are provided in the species description below.
Polar lipids were extracted with chloroform/methanol as described by Kamekura (1993). TLC was performed using Merck HPTLC silica gel 60 plates (Art. 5641) in a solvent system comprising chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.). The polar lipid composition of strain EJ-32T comprised phosphatidylglycerol and phosphatidylglycerophosphate methyl ester, but no glycolipids were detected.
The 16S rRNA gene sequence was amplified by PCR using three universal primer sets as described by Lopez-Garcia et al. (2001) and Arahal et al. (1996), and the almost-complete nucleotide sequence was determined by NBT-Newbiotechnic (Sevilla, Spain) using an automated DNA sequencer (model 3100; Applied Biosystems). A subsequent sequence analysis was conducted using the ARB software package (Ludwig et al., 2004). Alternative treeing methods (maximum parsimony, neighbour joining and maximum likelihood) were used according to the recommendations of Ludwig et al. (1998). A comparison of 16S rRNA gene sequences revealed that the sequence of strain EJ-32T (1404 bp) displayed the highest level of similarity with those of Halorubrum species. Fig. 1 shows the phylogenetic tree constructed with the maximum-parsimony method (Fitch, 1971), in which strain EJ-32T clustered with the type strains of species of the genus Halorubrum. Sequence similarity calculations indicated that strain EJ-32T was related to Hrr. coriense (97.9 % sequence similarity), Hrr. trapanicum (97.9 %), Hrr. sodomense (97.8 %), Hrr. tebenquichense (97.8 %), Hrr. xinjiangense (97.6 %), Hrr. terrestre (97.4 %), Hrr. distributum (97.1 %) and Hrr. saccharovorum (96.4 %). Similar tree topologies were obtained when other tree-construction methods were used.
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), using the equation of Owen & Hill (1979). The DNA G+C content of strain EJ-32T was 64.0 mol%, which is within the G+C range reported for the species of the genus Halorubrum (62.7–71.2 mol%) (Grant et al., 2001).
DNA–DNA hybridizations between strain EJ-32T and the type strains of the phylogenetically most closely related Halorubrum species were performed using the competition procedure of Johnson (1994), as described in detail by Gutierrez et al. (2002). The levels of DNA–DNA relatedness between strain EJ-32T and Hrr. coriense JCM 9275T, Hrr. trapanicum NRC 34021T, Hrr. sodomense ATCC 33755T, Hrr. tebenquichense CECT 5317T, Hrr. xinjiangense JCM 12388T, Hrr. terrestre VKM B-1739T, Hrr. distributum JCM 9100T and Hrr. saccharovorum NCIMB 2081T were 40, 46, 31, 44, 49, 17, 36 and 3 %, respectively. These levels of DNA–DNA hybridization are low enough to justify the classification of strain EJ-32T as representing a genotypically distinct species within the genus Halorubrum (Wayne et al., 1987; Stackebrandt & Goebel, 1994).
The differentiating characteristics of strain EJ-32T compared with those of the other Halorubrum species are listed in Table 1. Overall, our data show that strain EJ-52T represents a novel species of the genus Halorubrum, for which the name Halorubrum ejinorense sp. nov. is proposed.
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Cells are Gram-negative rods 1.0–1.5x5.0–8.0 µm in size. Colonies on agar plates containing 25 % (w/v) total salt are red, elevated and round. Growth occurs with 2.5–5.0 M NaCl at pH 6–10 and 25–50 °C. The optimal NaCl concentration, pH and temperature for growth are 3.4 M, pH 7.5 and 37 °C, respectively. Magnesium is not required for growth. Chemo-organotrophic and aerobic. Catalase- and oxidase-positive. Anaerobic growth with nitrate or L-arginine does not occur and nitrate reduction to nitrite is observed. H2S is not produced from cysteine. Indole is not produced from tryptophan. Methyl red, Voges–Proskauer and Simmons citrate test results are negative. Tween 80 and DNA are hydrolysed. Casein, gelatin and starch are not hydrolysed. Acid is not produced from D-arabinose, D-fructose, D-galactose, D-glucose, glycerol, lactose, maltose, D-mannitol, sucrose, trehalose or D-xylose. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are not produced. Growth occurs on trehalose as a single carbon and energy source. The following compounds are not used as sole carbon and energy sources: acetate, D-arabinose, fumarate, D-fructose, D-glucose, D-glutamate, glycerol, lactose, malate, maltose, D-mannitol, propionate, D-raffinose, D-ribose, D-sorbitol, succinate and D-xylose. The following compounds are used as sole carbon, nitrogen or energy sources: L-asparagine, isoleucine, L-lysine and L-threonine. Serine and glycine are not used as sole carbon, nitrogen or energy sources. Susceptible to bacitracin (10 U) and novobiocin (30 µg). Resistant to the following antibiotics (µg unless indicated otherwise): ampicillin (10), cephalothin (30), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), nalidixic acid (30), neomycin (10), penicillin G (10 U), rifampicin (30), polymyxin (300 U), streptomycin (10), sulfamethoxazole (25), tetracycline (30) and vancomycin (30). The polar lipids are phosphatidylglycerol and phosphatidylglycerophosphate methyl ester. Glycolipids not detected. The DNA G+C content is 64.0 mol%.
The type strain, EJ-32T (=CECT 7194T=CGMCC 1.6782T=JCM 14265T), was isolated from Lake Ejinor, a salt lake in Inner Mongolia, China.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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