APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1996, p. 3779–3786 Vol. 62, No. 10 0099-2240/96/$04.0010 Copyright q 1996, American Society for Microbiology Phylogenetic Analyses of Some Extremely Halophilic Archaea Isolated from Dead Sea Water, Determined on the Basis of Their 16S rRNA Sequences DAVID R. ARAHAL,1,2 FLOYD E. DEWHIRST,2 BRUCE J. PASTER,2 BENJAMIN E. VOLCANI,3 1 AND ANTONIO VENTOSA * Departamento de Microbiologı´a y Parasitologı´a, Facultad de Farmacia, Universidad de Sevilla, Seville 41012, Spain1; Department of Molecular Genetics, Forsyth Dental Center, Boston, Massachusetts 021152; and Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-02023 Received 23 April 1996/Accepted 13 August 1996 Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera—Haloferax, Halobacterium, and Halo- arcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized. The Dead Sea is a hypersaline lake with a neutral pH from Dead Sea water plus 1.0% (wt/vol) peptone, were kept in closed 500-ml bottles which a large number of extremely halophilic archaea have under sterile conditions and stored in the dark in a dry place at 18 to 208C. These enrichments remained unopened over the entire storage period. Inocula were been isolated. The first microbiological studies were carried obtained from the bottles under sterile conditions. Several isolation media were out in 1940 by B. E. Volcani (44), who described several new used, varying the total salt concentration and its composition—especially the halophilic species distinct from other species in other catego- amount of Mg21 ion. SWYE medium contained a final total salt concentration ries of halophilic microorganisms. Years later, most of the of ca. 25% (wt/vol) and 0.5% (wt/vol) yeast extract (Difco Laboratories, Detroit, cultures were lost. Since then, many other isolates from the Mich.) (28). HM medium (40) contained 1% (wt/vol) yeast extract (Difco), 0.5% (wt/vol) Proteose Peptone no. 3 (Difco), and 0.1% (wt/vol) glucose. This medium Dead Sea have provided some novel members of the family was supplemented with a balanced mixture of salts, giving a final concentration Halobacteriaceae, such as Haloferax volcanii (26), Haloarcula of 25% (wt/vol) (36), and with 500 IU of sodium penicillin G ml21 to inhibit the marismortui (32), Halorubrum (formerly Halobacterium) sodo- growth of moderately halophilic and halotolerant bacteria (24). The last medium mense (23, 31), and, very recently, Halobaculum gomorrense used, designated HS, was described by Oren for the isolation of Halorubrum (33). The purpose of our study was to isolate and characterize (Halobacterium) sodomense from the Dead Sea. This medium contained (wt/vol) 12.5% NaCl, 16.0% MgCl2 z 6H2O, 0.5% K2SO4, 0.01% CaCl2 z 2H2O, 0.1% pure cultures of extreme halophiles from enrichments ob- yeast extract (Difco), 0.1% Proteose Peptone no. 3 (Difco), and 0.2% soluble tained from Dead Sea water samples used by Volcani in 1936 starch (31). The pH was always adjusted to 7.2. Incubation was done at 378Cin and kept by him for more than 50 years in closed bottles. an orbital shaker (New Brunswick Scientific Co. Inc., Edison, N.J.) at 200 strokes Inocula were taken from these old enrichments, and by using per min. Twenty-two strains, designated E1 to E22, were isolated between March and September 1993. For comparison, the following culture collection strains appropriate media and growth conditions, we isolated 22 ex- were used in this study: Haloarcula hispanica ATCC 33960T (T 5 type strain), H. tremely halophilic microorganisms. Among these, five strains japonica JCM 7785T, H. marismortui ATCC 43049T, H. vallismortis ATCC 29715T, Halobacterium distributum VKM B-1733T, H. salinarum CCM 2148, were selected for phylogenetic analysis of their 16S rRNA gene T T sequences. The 16S rRNA gene sequence of Haloarcula his- Halococcus morrhuae CCM 537 , H. saccharolyticus ATCC 49257 , Haloferax denitrificans DSM 4425T, H. gibbonsii ATCC 33959T, H. mediterranei CCM panica ATCC 33960 was also determined since it has not been 3361T, H. volcanii NCIMB 2012T, Halorubrum lacusprofundi DSM 5036T, and H. previously reported. saccharovorum NCIMB 2081T. SWYE medium was used for cultivation and maintenance of the strains. When necessary, plates and agar slants were pre- pared by adding 2% (wt/vol) Bacto Agar (Difco) to this medium. MATERIALS AND METHODS Phenotypic characterization. The tests conducted for this study are listed in Strain isolation and culture conditions. The source of our isolates was seven Table 1. Cell morphology and motility were examined on freshly prepared wet enrichments obtained from water samples taken in 1936 by B. E. Volcani at mounts by phase-contrast microscopy of exponentially growing liquid cultures. surface level in the north basin of the Dead Sea by sampling at different spots Gram staining was performed by using acetic acid-fixed samples as described by close to the mouth of the Jordan river (42–48). The enrichments, prepared with Dussault (2). Growth rates at different salt concentrations were determined by using maintenance medium prepared with salt concentrations of 0.5, 3, 5, 10, 15, 20, 25, and 30% (wt/vol) (35). Catalase and oxidase activities and starch hydro- lysis were tested by using standard procedures (3, 15, 41). Gelatin hydrolysis * Corresponding author. Phone: 34 5455 6765. Fax: 34 5462 8162. experiments were performed as recommended by Gonza´lez et al (4). Procedures Electronic mail address: [email protected]. for determination of caseinase, phosphatase, and DNase activities have been 3779 3780 ARAHAL ET AL. APPL.ENVIRON.MICROBIOL. TABLE 1. Phenotypic features of 22 isolates studied Strain Characteristic E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 Cell shape Flattened Irregular Pleomorphic Irregular Pleomorphic Irregular Irregular Pleomorphic Irregular Rods, disks, rods rods rods, rods rods rods, rods triangles, triangles, cups triangles disks rectangles rectangles Motility 1111111111 Salt requirementsa: Range for growth 15–25 20–30 20–30 20–30 20–25 20–30 15–20 15–25 15–30 20–30 Optimal growth 20 25 25 25 20 25 20 20 20 25 Pigmentation Red Red Red Red Red Red Red Pink Red Red Oxidase 1111111111 Catalase 1111111111 Gelatin hydrolysis 2221222111 Starch hydrolysis 2221222111 Casein hydrolysis 2221222211 Phosphatase 2222222222 DNase 2222222222 Acid productionb from: D-Arabinose 1116111211 D-Fructose 1111111111 D-Galactose 2222222226 D-Glucose 1161111111 Glycerol 1262226126 Lactose 2222222222 Maltose 2111166611 D-Mannitol 2222222222 Sucrose 6222222126 D-Trehalose 2222222622 D-Xylose 1111111611 aConcentration (%, wt/vol) of total salts. b 1, positive; 2, negative; 6, intermediate. described elsewhere (3, 36, 39). Production of acid from sugars and other com- tubes by using a Perkin-Elmer 480 thermal cycler. Three microliters of 1/1,000- pounds was determined on maintenance medium modified as follows: yeast diluted DNA and 1 mM primers were added to the reaction mixture to a final extract was reduced to 0.5% (wt/vol), and 0.001% (wt/vol) phenol red was added. volume of 82 ml. The following conditions were used for amplification: denatur- The compounds tested were D-arabinose, D-fructose, D-galactose, D-glucose, glyc- ation at 948C for 45 s, annealing at 508C for 45 s, and elongation at 728C for 1.5 erol, lactose, maltose, D-mannitol, sucrose, D-trehalose, and D-xylose. Each of min with an additional 5 s added for each cycle for a total of 30 cycles, followed these compounds was added to a final concentration of 1% (wt/vol) by filtration by a final elongation step at 728C for 15 min. The purity of the amplified product through a 0.45-mm-pore-size membrane filter (Millipore Corp., Bedford, Mass.) was determined by electrophoresis in a 1% agarose gel (FMC Bioproducts). onto the sterile medium. Starch hydrolysis was tested by flooding colonies grown DNA was stained with ethidium bromide and observed under short-wavelength on agar plates with an iodine solution. Plates for the DNase test were flooded UV light. witha1NHClsolution, and the reaction was considered positive if a halo Purification of PCR products. The amplified DNA was purified by precipita- formed around the colonies (3). tion with polyethylene glycol 8000 (16). After removal of Ampliwax, 0.6 volume Extraction of DNA. Cells were cultured to approximately the late exponential of 20% polyethylene glycol 8000 (Sigma) in 2.5 M NaCl was added and the phase and then harvested by centrifugation. The cells were lysed by adding 200 mixture was incubated at 378C for 10 min.