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Taxonomic study of Halorubrum distributum and proposal of Halorubrum terrestre sp. nov.

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 Institute of Microbiology of the Russian Academy of Sciences, 117811 Moscow, Russia
⁴ Division of Microbial and Molecular Ecology, Institute of Life Sciences and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Correspondence
Antonio Ventosa
ventosa{at}us.es

	ABSTRACT

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Halorubrum distributum (basonym, Halobacterium distributum) is an extremely halophilic, aerobic archaeon isolated from saline soils, which was described on the basis of phenotypic features of several strains. The designated type strain of the species (1m^T=VKM B-1733^T=JCM 9100^T) was shown recently to differ from the other strains. In this study, Halorubrum distributum isolates have been characterized with regard to phenotypic features, polar lipid content, comparison of 16S rRNA gene sequences and DNA–DNA hybridization. On the basis of these data, a novel species that includes the other isolates is proposed, with the name Halorubrum terrestre sp. nov. The type strain of this novel species is 4p^T (=VKM B-1739^T=JCM 10247^T). The DNA G+C content of this novel species is 64·2–64·9 mol% (64·4 mol% for the type strain).

	MAIN TEXT

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In the course of a study of extremely halophilic micro-organisms from saline soils, Zvyagintseva & Tarasov (1987) proposed the species Halobacterium distributus, based on the features of four strains (designated 1m^T, 4p, 13 and 32). This name was validly published and corrected to Halobacterium distributum (Zvyagintseva & Tarasov, 1989). The strain designated as the type strain of this species is strain 1m^T, deposited as VKM B-1733^T (Zvyagintseva & Tarasov, 1989). Two almost-simultaneous studies (Kamekura & Dyall-Smith, 1995; McGenity & Grant, 1995) showed that most species that were previously assigned to the genus Halobacterium were not related to Halobacterium salinarum, the type species of this genus, and they were placed in the novel genus Halorubrum (McGenity & Grant, 1995). On the basis of 16S rRNA gene sequence comparison, Halobacterium distributum was also included in the genus Halorubrum, as Halorubrum distributum (Oren & Ventosa, 1996).

Zvyagintseva et al. (1996) showed that the features of strain 1m^T were different from those of the other three strains that were assigned to the species Halorubrum distributum and they proposed strain 4p (=VKM B-1739) as the new type strain of Halorubrum distributum. As this proposal was not in accordance with the rules of the International Code of Nomenclature of Bacteria (Lapage et al., 1992), Oren et al. (1997a) confirmed strain 1m^T (=VKM B-1733^T=JCM 9100^T) as the type strain of Halorubrum distributum and suggested that an exhaustive study should be carried out in order to ‘describe and validate a new species of Halorubrum and include a full phenotypic characterization’, and to propose (preferably) strain 4p (=VKM B-1739) as its type strain.

In the present paper, we study in detail the type strain of Halorubrum distributum, a species that has not been well characterized phenotypically, as well as the other strains that were previously assigned to this species; we describe these as a novel species, for which we propose the name Halorubrum terrestre sp. nov.

Strains used in this study are shown in Table 1. Halorubrum distributum VKM B-1733^T and the other four strains (VKM B-1739, VKM B-1916, VKM B-1954 and VKM B-2151) were isolated from saline soils (Zvyagintseva & Tarasov, 1987). Reference strains are type strains of species that belong to different halobacterial genera. They were cultured in a medium with a final concentration of approximately 25 % salts and 0·5 % yeast extract (Difco). The pH was adjusted to 7·5 with 1 M KOH (Torreblanca et al., 1986). Incubation was carried out at 37 °C in an orbital shaker at 200 r.p.m. For phenotypic characterization, we followed the Minimal Standards for Description of New Taxa in the Order Halobacteriales (Oren et al., 1997b). The tests performed are included in the species description. The methodology used has been described previously (Torreblanca et al., 1986). Unless otherwise indicated, tests were carried out in media that contained 25 % salts, at pH 7·5 and incubated at 37 °C.

For extraction of genomic DNA and determination of DNA G+C content, cells were harvested, washed and suspended in 0·15 M NaCl/0·1 M EDTA buffer (pH 8·0). Lysis was accomplished at 60 °C for 10 min by adding SDS at a final concentration of 2 % (w/v). DNA was extracted and purified by the method of Marmur (1961). Purity was assessed from A₂₆₀/A₂₈₀ and A₂₃₀/A₂₆₀ absorbance ratios (Johnson, 1994). DNA G+C content was determined from the mid-point value (T_m) of the thermal denaturation profile (Marmur & Doty, 1962), which was obtained by using a Perkin-Elmer UV-Vis 551S spectrophotometer at 260 nm. This instrument was programmed for temperature increases of 1·0 °C min^-1. The T_m was determined by a graphic method described by Ferragut & LeClerc (1976), and the G+C content was calculated from this temperature by using the equation of Owen & Hill (1979), in 0·1x SSC buffer (0·15 M NaCl buffered with 0·015 M trisodium citrate, pH 7·0). The T_m value of reference DNA from Escherichia coli NCTC 9001^T was taken as 74·6 °C in 0·1x SSC (Owen & Pitcher, 1985).

DNA was labelled by the multiprime system with a commercial kit with deoxy(1',2',5'-³H)cytidine 5'-triphosphate (Amersham Biosciences). The mean specific activity obtained with this procedure was 8·4x10⁶ c.p.m. (mg DNA)^-1. Labelled DNA was denatured before hybridization by heating at 100 °C for 5 min and then placed on ice. DNA–DNA hybridization studies were performed by the competition procedure of the membrane method, as described by Johnson (1994). Competitor DNA was sonicated (B. Braun Melsungen) at 50 W for two intervals of 15 s. Membrane filters (HAHY; Millipore) that contained reference DNA (25 µg cm^-2) were placed in 5 ml screw cap vials that contained labelled, sheared, denatured DNA and denatured, sheared competitor DNA. The ratio of the concentrations of competitor to labelled DNA was at least 150 : 1. The final volume and concentration were adjusted to 140 ml, 2x SSC and 30 % formamide. The hybridization temperature was 57 °C, which is within the limits of validity for the filter method (De Ley & Tijtgat, 1970). Vials were shaken slightly for 18 h in a water bath; these procedures were done in triplicate. After hybridization, filters were washed in 2x SSC at the optimal renaturation temperature (58 °C). Radioactivity bound to the filters was measured in a liquid scintillation counter (Beckman) and relatedness (%) was calculated according to Johnson (1994). At least two independent determinations were carried out for each experiment; reported results are mean values.

16S rRNA genes were amplified by PCR with a primer set [5'-ATTCCGGTTGATCCTGCCGG-3' (positions 6–25, according to E. coli numbering) and 5'-AGGAGGTGATCCAGCCGCAG-3' (positions 1540–1521)] by using Platinum Taq DNA Polymerase High Fidelity (Invitrogen). PCR products were cloned into the SmaI site of vector pUC119 and sequencing was carried out with a Beckman Coulter capillary DNA sequencer SEQ2000XL. 16S rDNA sequences were obtained from databases and aligned according to the Ribosome Database Project II. Tree reconstructions were performed by using the neighbour-joining and Kimura's two-parameter calculation methods.

Strains VKM B-1739, VKM B-1916, VKM B-1954 and VKM B-2151 are pleomorphic, motile, Gram-negative and able to grow in a wide range of salinities (15–30 % salt; optimal growth at 25 %). They lyse in distilled water. Colonies on solid media are orange–red-pigmented. Results of phenotypic tests and nutritional features of these strains are included in the species description. The polar lipid composition of these four strains are C₂₀C₂₀ derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a sulfated diglycosyl diether, which has been shown to be a distinctive feature of species of the genus Halorubrum (McGenity & Grant, 2001).

The DNA G+C content of the four strains studied ranged from 64·2 to 64·9 mol% (Table 1). This result is within the range of G+C contents reported for the genus Halorubrum (McGenity & Grant, 2001). The DNA G+C content of Halorubrum distributum VKM B-1733^T was 63·9 mol%, a very similar value to that reported previously (63·6 mol%) (McGenity & Grant, 2001). Results of DNA–DNA hybridization experiments are shown in Table 1. High levels of relatedness were found between strain VKM B-1739 and the other three related strains (95–100 %); however, levels of DNA relatedness with the type strains of species of the genus Halorubrum, as well as other genera of the Halobacteriaceae, were <70 %, which is the value that is currently accepted for the delineation of prokaryotic species (Stackebrandt & Goebel, 1994).

The 16S rRNA gene sequence of strain VKM B-1739 was most closely related to those of Halorubrum coriense (97·1 % similarity) and Halorubrum distributum (97·0 % similarity). Fig. 1 shows a phylogenetic tree, in which the relationships of strain VKM B-1739 and other related halobacteria are seen. The 19 signature bases that are specific for the genus Halorubrum or are shared by only one other genus (Grant et al., 2001; Kamekura et al., 2004) were completely conserved.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree of strain VKM B-1739 and other related halobacterial species based on their 16S rRNA gene sequences (GenBank accession numbers are shown in parentheses). Numbers on branches represent confidence levels from 1000 replicate bootstrap resamplings; values >70 % are shown. Bar, 0·02 expected changes per site.

Description of Halorubrum terrestre sp. nov.
Halorubrum terrestre (ter.res'tre. L. neut. adj. terrestre of the soil, from which the strains were isolated).

Cells are pleomorphic, flat and disc-shaped, 1·0–1·5x1·5–2·5 µm in size. Motile. Gas vacuoles are not produced. Colonies are orange–red. Growth occurs in media that contain 15–30 % NaCl, with optimum growth at 25 % NaCl. Growth occurs between 28 and 50 °C (optimum, 37–45 °C) and pH 5–9 (optimum, 7·5). Chemo-organotrophic. Aerobic. Oxidase- and catalase-positive. Acid is produced from glycerol, but not from arabinose, fructose, galactose, glucose, lactose, maltose, sucrose or trehalose. Nitrate is not reduced to nitrite. Indole is not produced from tryptophan. Voges–Proskauer test is negative. Starch, gelatin and casein are not hydrolysed. H₂S is not produced. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are not produced. The following compounds are not used as sole carbon and energy sources: arabinose, cellobiose, aesculin, fructose, fucose, gluconolactone, glucose, glucosamine, inulin, mannose, melibiose, raffinose, rhamnose, ribose, sucrose, trehalose, xylose, adonitol, dulcitol, erythritol, ethanol, glycerol, mannitol, meso-inositol, propanol, sorbitol, -aminovalerate, butyrate, caprylate, citrate, fumarate, glutamate, glycerate, 2-oxoglutarate, malate, malonate, oxalate, propionate, saccharate and tartrate. The following compounds are not used as sole carbon, nitrogen or energy sources: L-alanine, L-arginine, L-asparagine, betaine, creatine, L-glutamine, glycine, L-histidine, L-lysine, L-methionine, L-ornithine, L-proline, putrescine, sarcosine, L-serine, L-threonine and L-valine. Susceptible to anisomycin, bacitracin and novobiocin; resistant to ampicillin, chloramphenicol, kanamycin, nalidixic acid, penicillin G, polymyxin, streptomycin and tetracycline. Polar lipids are C₂₀C₂₀ derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a sulfated diglycosyl diether. DNA G+C content is 64·2–64·9 mol% (T_m method).

Type strain is 4p^T (=VKM B-1739^T=JCM 10247^T). DNA G+C content of this strain is 64·4 mol% (T_m method). Isolated from saline soils.

	ACKNOWLEDGEMENTS

 
We thank H. G. Trüper for his advice on nomenclature of the novel species. This work was supported by grants from the Quality of Life and Management of Living Resources Programme of the European Commission (QLK3-CT-2002-01972), Spanish Ministerio de Ciencia y Tecnología (PB98-1150 and BMC2003-01344) and from Junta de Andalucía (to A. V.).

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