Taxonomic Study of Extreme Halophilic Archaea Isolated from the “Salar De Atacama”, Chile

System. Appl. Microbiol. 24, 464Ð474 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Taxonomic Study of Extreme Halophilic Archaea Isolated from the “Salar de Atacama”, Chile

CATHERINE LIZAMA1,2, MERCEDES MONTEOLIVA-SÁNCHEZ1, BERNARDO PRADO2, ALBERTO RAMOS-CORMENZANA1, JURGEN WECKESSER3 and VICTORIANO CAMPOS2

1Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja Granada, Spain 2Laboratory of Microbiology, Institute of Biology, Faculty of Basic and Mathematics Sciences, Catholic University of Valparaíso, Val- paraíso, Chile 3Institute of Biology and Microbiology, Freiburg, Germany

Received July 1, 2001

Summary

A large number of halophilic bacteria were isolated in 1984–1992 from the Atacama Saltern (North of Chile). For this study 82 strains of extreme halophilic archaea were selected. The characterization was performed by using the phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility test. The results, together with those from reference strains,

were subjected to numerical analysis, using the Simple Matching (SSM) coefficient and clustered by the unweighted pair group method of association (UPGMA). Fifteen phena were obtained at an 70% similarity level. The results obtained reveal a high diversity among the halophilic archaea isolated. Represen- tative strains from the phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains. The 16S rRNA studies showed that some of these strains constitutes a new taxa of extreme halophilic archaea.

Key words: Atacama Saltern – Tebenquiche Lake – Extreme Halophilic Archaea – Numerical Taxonomy

Introduction

The extreme halophilic archaea require at least 1.5 M promise of providing valuable molecules for biotechno- NaCl. Most strains grow best at 3.5–4.5 M NaCl. They logical use in industry (HORIKOSHI, 1997). have been isolated from different habitats including alka- In a previous study carried out on the Atacama saltern line and salt lakes, marine salterns, the Dead Sea and (MORAGA et al., 1974) was showed that the characteris- saline soils. Traditionally the family Halobacteriaceae tics of this habitat, in relation with geographic, climatic contains six genera: Halobacterium, Haloarcula, Halofer- ax, Halococcus and two alkalophilic genera, Natrono- bacterium and Natronococcus (HOLT et al., 1994). Re- Abbreviations: PG – phosphatidylglycerol (diether analogue); cently, applying molecular techniques nine new genera PGP – phosphatidylglycerolphosphate (diether analogue); PGS – were described: Halorubrum (MCGENITY et al., 1995), phosphatidylglycerolsulfate (diether analogue); S-DGD-1 – α → Halobaculum (OREN et al., 1995), Natrialba (KEMEKURA monosulphated diglycosylglyceroldiether (6-HSO3-Man p- 1 et al., 1995), Natronomonas (KAMEKRA et al., 1997), 2-Glc p-α1 → 1-glyceroldiether); DGD-1 – diglycosylglyc- α → α → Halogeometricum (MONTALVO-RÓDRIGUEZ et al., 1998), eroldietther (Man p- 1 2-Glc p- 1 1-glyceroldiether); S-DGD-3 – monosulphated diglycosylglyceroldiether (2-HSO - Natrinema (MCGENETY et al., 1998), Haloterrigena 3 Man p-α1 → 2-Glc p-α1 → 1-glyceroldiether); S-TGD-1 – sul- (VENTOSA et al., 1999), Natronorubrum (XU et al., 1999) β → fated triglycosylglyceroldiether (3-HSO3-Gal p- -1 6-Man and Halorhabdus (WAINØ et al., 2000) extending the di- p-α1 → 2-Glc p-α1- glyceroldiether); TGD-1 – triglycosylglyc- versity of the taxonomy of halophilic archaea. Halophilic eroldiether (Gal p-β-1 → 6-Man p-α1 → 2-Glc p-α1- glyc- microorganisms offer important insights into the biology eroldiether); TGD-2 – triglycosylglyceroldiether (Glc p-β-1 → and evolution of many organisms and also hold the 6-Man p-α1 → 2-Glc p-α1- glyceroldiether).

0723-2020/01/24/03-464 $ 15.00/0 Extreme halophilic archaea from “Salar de Atacama” 465 and chemical composition, revealed it to be an ideal envi- For the nutritional test, a modified minimal medium de- ronment for the development of extreme halophilic mi- scribed by RODRÍGUEZ-VALERA et al. (1980), was used: 20% croorganisms. In previous works were showed new iso- (w/v) total salts; 0.2% NH4Cl and 0.1% KH2PO4. The pH was lates of moderately halophilic microrganisms as gram- adjusted to 7.0 with NaOH 4 M. The media was filtered before sterilization. The substrates were used in the following concen- negative rods (PRADO et al., 1991) as well as gram posi- trations: 0.2% for carbohydrates, and 0.1% for alcohols and or- tive cocci (VALDERRAMA et al., 1991). The purpose of this ganic acids. The media were sterilized by heating at 90 ºC for 15 study was to widens the knowledge of the biodiversity of minutes. For all the tests, inocula with young bacterial suspen- extremely halophilic archaea on the Atacama saltern . sions were used and maintained for 24 h. in saline solutions, in order to eliminate possible nutritional reserves that could be cause erroneous results. Minimal media without substrate and Materials and Methods media HE at 25% (w/v) total salts were used as controls. Tur- bidity of the positive tubes was determined after seven days of Isolation and culture conditions incubation at 40 ºC. The 82 strains studied were isolated from water and sedi- The utilization of amino acids as carbon and nitrogen ment of Lake Tebenquiche, located in the northern part of Ata- sources was determined using the method described previously cama Saltern, and from the sediment of Poligonal Zone. Water by VENTOSA et al. (1982), with a basal medium in which was samples of 100, 50 and 10 ml were filtered in situ using special deleted the inorganic nitrogen sources. The composition was as membranes (Sartorius 11406/0.45/324/1) which were placed on follows (g/l): NaCl 200; KCl 2; MgSO4 · 7 H2O 0.2; KH2PO4 plates. The media used for the enrichment cultures were the fol- 0.5. The pH were take to 7.0 before sterilization. The amino lowings: Eimhjellen medium: Yeast extract (Difco), 5.0 g; acids were added for filtration to the sterile basal medium at MgSO4 · 7 H2O, 2.0 g; CaCl2 · 2 H2O, 0.5 g; NaCl, 25 g; dis- 0.1% final concentration. tilled water, 100 ml (EIMHJELLEN, 1965); Sehgal and Gibbons The susceptibility to antimicrobial agents was tested on medium: Yeast extract (Difco), 1.0 g; Casamino acids (Difco), media HE 25% (w/v)total salts with antibiotic disks (Difco), by 0.75 g; Na citrate, 0.3 g; MgSO4 · 7 H2O, 2.0 g; KCl, 0.2 g; the method of Bauer et al. (1966). The antibiotic tested were: FeCl2, 0.0023 g; NaCl, 25 g; distilled water, 100 ml (SEHGAL penicillin G (10 units), kanamycin (30 mg), tetracycline (30 mg), AND GIBBONS, 1960); MH medium: Proteose-peptone nº 3 erythromycin (15 mg), streptomycin (10 mg), bacitracin (10 (Difco), 0.5 g; Yeast extract (Difco), 1.0 g; glucose, 0.1 g with units), novobiocin (30 mg), polymyxin B (300 units), ampicillin 25% (w/v) of total salts (VENTOSA et al., 1982); and the HE (10 mg), neomycin (30 mg), chloramphenicol (30 mg), gen- medium: Yeast extract (Difco), 0.5 g; glucose, 0.1 g, with 25% tamycin (10 mg) and carbenicillin (50 mg). (w/v) of total salts (TORREBLANCA et al., 1986). The MH and HE media were supplemented with penicillin (500 units/ml) The Numerical analysis stock of total salts at 30% was prepared as described SUBOV 67 differential characters were coded in binary form; positive (1931): NaCl, 23.4 g; MgCl2 · 6 H2O, 4.2 g; MgSO4 · 7 H2O, and negative results were coded as 1 and 0 respectively and non- 6.0 g; CaCl2 · 2 H2O, 0.1 g; KCl, 0.6 g; NaCO3H, 0.02 g; NaBr, comparable or missing data were coded as 9. Strain similarities 0.07 g, FeCl , 0.0005 g; distilled water, 100 ml. All the media 3 were estimated by simple matching (Ssm) (SOKAL et al., 1958), were solidified with 20 g/l Bacto-Agar (Difco) and were adjusted coefficients and clustered by the unweighted par-group method to pH 7.3 with 1 N KOH before autoclaving. The plates were of association (UPGMA) (SNEATH et al., 1973). The cophenetic incubated four weeks at 40 ºC, in sealed plastic bags and correlation was also obtained for each method. The test error checked at the end of the first and second weeks. Colonies were was estimated by examining 10 strains in duplicate (SNEATH et re-isolated by streaking on a fresh plates with the same medium. al., 1972). The computation was performed by the NT-SYS pro- Pure cultures were finally transfer to agar slants of medium with gram, using an VAX-785 computer at the Computer Center, 5 g/l of yeast extract in 25% (w/v) total salts supplemented with University of Sevilla, Sevilla, Spain. 0.1% glucose. The pH was adjusted to 7.0 with NaOH 4 M. The reference strains used in this study were the followings: Haloarcula sinaiiensis ATCC 33800, Haloarcula marismortui Electron microscopy DSM 3752, Haloarcula hispanica DSM 4426, Haloarcula cali- Transmission electron microscopy was used to exam cell forniae ATCC 33799, Haloarcula japonica DSM 6131, Haloar- morphology of representative strains from each phenon. Sam- cula vallismortis DSM 3756, Halobacterium halobium CCM ples from liquid culture were stained with 2% uranyl acetate (30 2090, Halobacterium halobium CECT 396, Halobacterium sali- sec) and then washing with 0.3% acetic acid. A TEM 902 narum CCM 2148, Halobacterium salinarum DSM 3754, (Zeiss) high resolution transmission electron microscope at 80 Halorubrum sodomense DSM 3755, Halorubrum saccharovo- Kw was used. rum DSM 1137, Halorubrum lacusprofundi DSM 5036, Halorubrum coriense DSM 10284, Halorubrum trapanicum Lipid composition CECT 397, Haloferax mediterranei ATCC 33500, Haloferax Lipid were extracted from wet cells with chloroform- gibbonsii DSM 4427 Haloferax volcanii DSM 3757, Haloferax methanol (2:1 v/v) as described by KATES (1972). The lipids were denitrificans DSM 4425 and Halococcus morrhuae NCMB 757. separated by thin-layer chromatography by single development on silica gel plates (60 F254, Merk) in chloroform-methanol- Characterization of isolates acetic acid-water (85:22:5:10:4 v/v). In addition, two-dimen- 121 phenotype characters including morphological, physiologi- sional chromatography was performed by using chloroform- cal, biochemical, nutritional and antimicrobial susceptibility tests, methanol-water (65:25:4 v/v) in the first and chloroform- were determined for each strain. The procedures were described methanol-acetic acid-water (80:12:15:4 v/v) in the second di- by TOMLINSON and HOCHSTEIN (1976); TOMLINSON et al. (1986); mensions. Phospholipids were visualized with ammonium RODRÍGUEZ-VALERA et al. (1980, 1982, 1983); TINDALL et al. molybdate-sulfuric acid spray (KATES, 1972). Glycolipids were (1987); GONZALEZ et al. (1978); JUEZ et al. (1986); TORREBLANCA detected by spraying the plates with 0.5% α-naphtol in 50% et al. (1986). HE Medium at 25% (w/v) supplemented with 0.1% methanol and then with 5% H2SO4 in ethanol and heating to glucose was used as the basal media (TORREBLANCA et al., 1986). 150 ºC for 10 minutes (OREN et al., 1996, TINDALL et al., 1987). 466 C. LIZAMA et al.

G+C content of DNA CATTG-3′) and the reverse primer R1462 (5′-ATCCAGC- Representative strains from each phenon were used for this CGCAGATTCCCCTAC-3) corresponding to positions 8–30 study. Their DNA was isolated and purified by the method de- and 1462–1441, respectively. The oligonucleotides were pro- scribed by LING et al. (1986). The guanine-plus-cytosine (G+C) duced by Pharmacia Biotech and then were diluted to 100 content of the DNA was determined from the midpoint value™ pmol/µl. The PCR was performed in Thermal Cycler (480 of the thermal denaturation profile (MARMUR and DOTY, 1962) model, Perkin Elmer) for 30 cycles starting with 1 min of denat- with a Perkin Elmer UV-Vis Lambda3B spectrophotometer at uration at 94 ºC, which was followed by 1 min of annealing at 260 nm, programmed for temperature increases of 1.0 ºC/min. 55 ºC and 2 min of elongation at 72 ºC. The mixture reaction in Tm was determined by the graphic method described by FER- total volume 100 µl contained: 2 µl of genomic DNA, 10 µl of RAGUT and LECLERC (1976) and the G+C content was calculated Buffer (100 mM tris-HCl, pH 8.3; 500 mM KCl), 4 µl MgCl2 from this temperature using OWEN and HILL´s equation (1979). (25 mM), 1 µl of dNTPs mixture (dATP, dCTP, dGTP, dTTP at The Tm value of reference DNA from Escherichia coli NCTC 10 mM concentration, Ultrapure dNTPs set of Pharmacia 9001 was taken to be 74.6 ºC in 0.1 × SSC (OWEN and PITCHER, Biotech) and 1 µl of Taq DNA polymerase (Amplitaq DNA 1985). At least three independent determinations were carried polymerase, Perkin Elmer). Each primer was used at a concen- out for each experiment and the means were calculated. tration of 20 pmoles/µl. The PCR products were analyzed by electrophoresis in 1.7% agarose gels in TAE buffer using the λ Preparation of labelled DNA and DNA-DNA hybridization PST1 as the size marker. This products were purified with Mi- Reference DNA was double-labelled by nick translation crocon-100 concentrators (Amicon). using digoxigenin-11-2′-deoxy-uridin-5′-triphosphate (DIG-11- Four sequencing primers were used to determine the com- ′ dUTP) and biotin-16-2′-deoxyuridin-5′-triphosphate (biotin-16- plete 16S rRNA sequence: r1 (5 -TACCGTGAGGCGTCCTGT ′ ′ ′ dUTP) (Boehringer Mannheim). The optimun ratio of the nu- TAA-3 ); r2 (5 -TTGTCTCGACCATTGTAGCC-3 ); r3 (CC ′ ′ cleotide mixture was 0.75 vol. DIG-11-duTP: 0.25 vol. Biotin- CGCCAATTCCTTTAAGTTTC-3 ) and r4 (5 -TGGCACCG ′ 16-dUTP. 0.5 µg of DNA was labelled according to the protocol GTCTTGCCCAG-3 ) were designed to complement regions of the manufactured and resuspended in 100 µl distilled water. from position 1011–1032, 1197–1178, 870–849 and 458–440, One microlitre was used to measure the efficiency of the la- respectively. The purified PCR products were sequenced directly belling reaction. using an Strech Applied Biosystems ABI 373 DNA Sequencer r DNA-DNA hybridization was performed following the and the manufacturer’s protocols for AmpliTaq FS with fluores- TM methods of LIND and URSING (1986), and the modifications of cent dye-labelled dideoxynucleotides (Kit ABI PRISM BigDye ZIEMKE et al. (1998). Between 40 ng and 60 ng of labelled DNA Terminator Cycle Sequency Ready Reaction of Perkin-Elmer) as was mixed with 15 mg of unlabelled DNA in 0.1 ml 0.28 M described Sanger et al., (1977). The sequences were analyzed for tm phosphate buffer (PB), pH 6.8. This mixture was denatured at ABIPrism 373 xl Collection 2.0 and Sequencing Analysis 3.3 100 ºC for 10 min and subsequently cooled in a bath of ice programs in the Macintosh G3. water. The mixture was then incubated for 16 h at 30 ºC below the TM of the homologous DNA. Single- and double-stranded Phylogenetic analysis on the basis of 16S rRNA sequence DNA were eluted on hydroxyapatite (HA) (BRENNER et al., The sequences obtained were compared with previously de- 1969). Rehydrated HA (DNA grade Bio-Gel HTP; Bio-Rad) was scribed 16S rRNA sequences of halophilic archaea deposited in equilibrated with 0.14 M PB. The preparation was mixed thor- the EMBL database. The sequences were aligned by using oughly and incubated for 15 min at 5 ºC below the temperature Clustal W 1.74 (THOMPSON et al., 1994), and phylogenetic tree used in the reassociation. The single- and double-stranded DNA were constructed by using programs in version 3.5.1 of PHYLIP were than eluted using four washed of 0.25 ml 0.14 M PB, 0.2% (FELSENSTEIN, 1993). We determinated a matrix of evolutionary (w/v) SDS and two washes of 0.2 ml, 0.4 M PB respectively. distances from the sequence alignment data with the Jukes-Can- To detect the eluted DNA, 200 µl were incubated for 2 h on tor model (JUKES and CANTOR, 1969). A phylogenetic tree was streptavidine-coated microtitre plates (Boehringer Mannheim) constructed from the distance matrix by using the program with 0.1% (w/v) BSA (Sigma). The double-stranded DNA was FITCH, which uses the least-squares algorithm (FITCH and MAR- denatured and chilled on ice before incubation. Whases with GOLIASH, 1967). PBS were followed by 1 h incubation with anti-DIG antibodies conjugated with alkaline phosphatese (Boehringer Mannheim) in 200 µl PBS and 0.1% (w/v) BSA. Both incubations were at Results room temperature with agitation. The wells were then washed with PBS. Detection was performed with 250 µl p-nitrophenyl phosphate at 37 ºC without agitation. Colour development was Numerical analysis measured at 405 nm (TILSSEN, 1985). The degree of reassociation (Binding ratio) was expressed as All strains studied were grouped in 15 phena at a 70% the percentage of labelled DNA released with 0.4 M PB com- similarity level (Fig. 1). Only two of the reference strains pared to the total laballed DNA released. The relative binding clustered within these phena. The cophenetic correlation ratio of heterologous DNA was expressed as the percentage of value was 0.78% and the estimated test error 0.4%. All homologous binding. Pooled standard deviations (SD) of all ex- extreme halophilic archaea in study were gram-negative periments ranged from 1% to 3%. pleomorphic bacteria that showed growth at 30% (w/v) total salts. They had no magnesium requirement at satu- PCR amplification of the 16S rRNA gene coding sequence rated NaCl concentration. The results of pH spectrum and sequencing trend to alkaline and all the strains did growth between Purified genomic DNA of selected strains were used for PCR amplification of the 16S rDNA gene. Two primers were de- 7.0–10.0. Acid was not produced from: lactose, D(+) cel- signed to complement the highly conserved regions of the lobiose, starch, inuline, (L) sorbose, D(+) rafinose, L(+) Halobacterium salinarum and Halorubrum spp. 16S ribosomal rhamnose, D(–) mannitol and glycerol. They not used as DNA. The forward primer F8 (5′-TTGATCCTGCCGGAGGC sole source of carbon, nitrogen and energy the organic Extreme halophilic archaea from “Salar de Atacama” 467 acids: succinate, formiate and benzoate thus as the amino lipids but variation appear in the minor phospholipid acids ; L-lysine, L-aspartic acid, L-cysteine, L-cystine, D- PGS and in the glycolipids. Table 1 shows the G+C con- glutamic acid and L-tryptophan. All were Dnase negative tent of the DNA and the main glycolipids of the represen- and susceptible to novobiocin (30 µg) and bacitracin (10 tative strains of each phenon. The most significant fea- U). All strains contained PGP and PG as major phospho- tures of each phenon, based on those tests for which at

Fig. 1. Simplified dendrogram showing clustering of 82 strains of extreme halophilic archaea isolated from “Salar de Atacama” (Chile), based on the Simple Matching (Ssm) coefficient and the unweighted-pair-group method with average (UPGMA) clustering. 468 C. LIZAMA et al.

Table 1. G+C content and main glycolipids of representative and arabinose. Glucose, fructose, maltose, starch, tre- strain of each phenon. halose; and glycerol, mannitol, sorbitol as well as acetate and pyruvate were used as the only sources carbon. The Strain Phenon G+C Main GlycolipidGenus representative strain TeSe-77 had a G+C content of content 58.3%. The main glycolipids was TGD-2 These organ- (mol%) isms were included in genus Haloarcula. TeSe-1 A 60.5 S-DGD-3 Halorubrum Phenon F: Two strains were included at 78% similari- Te-Se-45 B 61.2 S-TGD-1,TGD-1 Halobacterium ty. They growth between 15–30% (w/v) total salts and ALT-51 C 61 S-TGD-1,TGD-1 Halobacterium were catalase positive. They hydrolyzed esculin and TeSe-64 D 57.2 TGD-2 Haloarcula tweens 20, 40, 60 and 80. They reduced nitrate and used TeSe-77 E 58.3 TGD-2 Haloarcula as the only source of carbon: glucose, fructose, galactose, TeSe-31 F 60.6 S-DGD-3 Halorubrum cellobiose, mannose, salicine, trehalose, glycerol and ALT-42 G 54.6 TGD-2 Haloarcula pyruvate. Strain TeSe-31, taken as representative, had a TeSe-38 H 63 DGD-1,S-DGD-1 Haloferax G+C content of 60.6%. The major glycolipid was S- ALT-6 I 53.2 S-DGD-3 Halorubrum TeSe-19 J 62.1 S-DGD-3 Halorubrum DGD-3. This organisms were included in the genus TeSe-66 K 61.1 S-DGD-3 Halorubrum Halorubrum. TeSe-73 L 61.2 S-DGD-3 Halorubrum Phenon G: This phenon included three strains at 75% TeSe-69 M 62.1 S-DGD-3 Halorubrum of similarity level, growth between 35–50 ºC of tempera- ALT-9 N 60.7 S-DGD-3 Halorubrum ture and were catalase positive. They hydrolyzed gelatin TeSe-21 O 60.8 S-DGD-3 Halorubrum and esculin. They produced H2S from cystein and reduced nitrate. Acids were produced from arabinose and they used maltose, mannose, trehalose, glycerol and pyru- least 80% of the strains were positive or negative, are de- vate as only carbon source. The G+C content of strain scribed bellow. ALT-42, chosen as representative of this phenon, was Phenon A: This phenon included two strains clustered 54.6%. The lipids of this group were characterized by the at 75% similarity. They had reduced metabolic activity. presence of TGD-2 as the main glycolipid. The member

They hydrolyzed esculine; produced H2S from cysteine, of this phenon could be related to the genus Haloarcula. nitrate reduced, acid produced from xylose and were cata- Phenon H: It clustered two strains with similarity level lase negative. The representative strain TeSe-1 had a G+C of 79%. They were catalase positive and growth between content of 60.5% and the main glycolipid was S-DGD-3. 35–50 ºC. Tween 20 and esculin were hydrolyzed. They These strains were assigned to genus Halorubrum. produced H2S from cysteine and reduced nitrate. Acid Phenon B: Two strains were clustered in this phenon at was produced from xylose and fructose. They used a 91% similarity, the highest similarity value obtained in great variety of organic compounds as only sources of this study. They showed growth between pH 6.0-10.0, carbon: glucose, xylose, fructose, maltose, arabinose, cel- and 35-50 ºC. Both strains were oxidase positive. They lobiose, salicine, trehalose, rhamnose, glycerol, acetate, hydrolyzed gelatin and reduced nitrate. Strain TeSe-45, malate, pyruvate, citrate, fumarate and tartrate. The G+C taken as representative, had a G+C content of 61.2%. content was 63%, the main glycolipids were DGD-1 and The major glycolipids were TGD-1 and S-TGD-1. These S-DGD-1. The strain Haloferax gibbonsii DSM 4427 was organism were included in the genus Halobacterium. included in this phenon. Phenon C: This phenon included two strains, one of Phenon I: Three strains were clustered at 72% of simi- them was Halobacterium salinarum DSM 3754. They hy- larity. They growth between 15–30% (w/v) total salts and drolyzed gelatin and used acetate and pyruvate as sole between 35–50 ºC, of temperature. They were catalase source of carbon. The main glycolipids were S-TGD-1 positive. H2S was produced from cystein and they re- and TGD-1, the G+C content was 61%. duced nitrate . They used as only source of carbon and Phenon D: This phenon included 15 strains at 72% energy; glucose, fructose, galactose, mannose, trehalose, similarity. These strains growth between 35–50 ºC, and acetate and pyruvate. The representative strain ALT-6 were catalase positive. They hydrolyzed gelatin, esculin had a G+C content of 63.2% and the main glycolipid was and tween 20, 40, 60 and 80. H2S was produced from S-DGD-3. These strains were assigned to the genus cysteine and the strains reduced nitrate. Acid was pro- Halorubrum. duced from glucose, fructose and arabinose. They used Phenon J: It clustered three strains at 71% similarity glucose, fructose, maltose, mannose, trehalose, glycerol, than growth 15–30% (w/v) total salts and 35–50 ºC of mannitol, sorbitol, acetate and pyruvate as sole sources temperature. They were catalase positive. H2S was pro- of carbon. The G+C content of strain TeSe-64, chosen as duced from cystein and they reduced nitrate. They used representative of this phenon, was 57.2%. The lipids of glucose, trehalose, pyruvate and serine as only source of group are characterized by the presence of TGD-2 as carbon and energy: The representative strain TeSe-19 had main glycolipid. The member of this phenon could be re- a G+C content of 62.1%. The main glycolipids was S- lated to the genus Haloarcula. DGD-3. These organisms were included in genus Phenon E: Nine strains clustered at 70% similarity Halorubrum. level. They growth at 35–50 ºC, they were catalase posi- Phenon K: This phenon included three strains at 74% tive and reduced nitrate. Acid was formed from fructose similarity. They growth between 35–50 ºC of tempera- Extreme halophilic archaea from “Salar de Atacama” 469 ture. Both were catalase and oxidase positive. Esculin and phenon C). These results confirmed that both phena are strach were hydrolyzed. H2S was produced from cystein. related with H. salinarium. The representative strain of Glucose, maltose, trehalose, acetate, malate and pyruvate phenon H, TeSe-38, give a high value of similarity were used as only source of carbon and energy. Strain (83.44%) with H. gibbonsii DSM 4427. The strains was TeSe-66, taken as representative, had a G+C content of designated as Haloarcula, and selected as representatives 61.1%. The major glycolipid was S-DGD-3. These organ- of phena E,D and G were respectively TeSe-77, TeSe-64 isms were included in the genus Halorubrum. and ALT-42. The results showed a considerable degree of Phenon L: It clustered four strains at 74% similarity DNA-DNA similarity between these strains and also that growth between 35–50 ºC and were catalase posi- from TeSe-77 with H. hispanica DSM 4426 (90.33%). tive. They reduced nitrate, tweens 20 and 80 were hy- The main number of phena (A, I, J, K, L, M, N, O) were drolyzed and H2S was produced from cystein. Glucose, assigned to Halorubrum genus by chemotaxonomic stud- galactose, acetate and pyruvate were used as sole source ies, and the strains representatives of these phena showed of carbon and energy. The G+C content of strain TeSe-73, variable values of similarity in relation with the labeled chosen as representative of this phenon, was 61.2%. The strain (TeSe-66), and also this strain showed a DNA- lipids of this group are characterized by the presence of S- DNA-similarity with the reference strains significantly DGD-3 as main glycolipid. The member of this phenon lower. could be related to the genus Halorubrum. Phenon M: Five strains were included in this phenon at 72% similarity level. They growth between 35–50 ºC, Table 2. DNA-DNA homology between representative strains and were catalase positive. They used as only source of from each phenon and some related Halobacterium, Haloarcula, carbon and energy: glucose, galactose, acetate and pyru- Haloferax and Halorubrum species. vate. H2S was produced from cystein. The representative strain TeSe-69 had a G+C content of 62.1% and the main Strain Phenon Genus Similar- glycolipid was S-DGD-3. These strains were assigned to designated ity (%) the genus Halorubrum. Phenon N: Two strains were included in this phenon at TeSe-45* B Halobacterium 100 76% similarity level. They were catalase positive and re- ALT-51 C Halobacterium 85.14 duced nitrate. Esculin and tweens 20, 40, 60, and 80 H. salinarum CCM 2148 97.52 H. halobium CCM 2090 81.28 were hydrolyzed. They used glycerol, acetate and pyru- H. halobium CECT 396 70.18 vate as only source of carbon and energy. Strain ALT-9, H. salinarum DSM 3754 7344 taken as representative, had a G+C content of 60.7%. The major glycolipid was S-DGD-3. These organisms TeSe-38* H Haloferax 100 were included in the genus Halorubrum. H. mediterranei ATCC 33500 43.6 Phenon O: It clustered two strains at 74% similarity. H. volcanii DSM 3757 33 They growth between 35–50 ºC of temperature. They H. denitrificans DSM 4425 16.09 were catalase positive. Tweens 20, 40, 60 and 80 were H. gibbonsii DSM 4427 83.44 hydrolyzed. They reduced nitrate and produced acids TeSe-77* E Haloarcula 100 from arabinose, acetate, maltose and pyruvate as only TeSe-64 D Haloarcula 73.17 source of carbon and energy. The G+C content of strain ALT-42 G Haloarcula 82.92 TeSe-21, chosen as representative of this phenon was H. sinaiiensis ATCC 33800 12.81 60.8%. The lipids of this group are characterized by the H. marismortui DSM 3752 57.72 presence of S-DGD-3 as main glycolipid. The member of H. californiae ATCC 33799 14.83 this phenon could be related to the genus Halorubrum. H. japonica DSM 6131 44.81 H. hispanica DSM 4426 90.33 H. vallismortis DSM 3756 54.42 DNA-DNA homology TeSe-66* K Halorubrum 100 As stated in experimental procedures representative TeSe-1 A Halorubrum 31.51 strains were selected to carry out the DNA-DNA hy- ALT-6 I Halorubrum 77.15 bridization experiments (Table 2). In this sense it is TeSe-69 M Halorubrum 73.50 showed genomic similarities among the reference strains TeSe-31 F Halorubrum 13.21 and the representative strains of some phenon. Our re- TeSe-19 J Halorubrum 95.62 sults are in agreement with the indication that levels of TeSe-73 L Halorubrum 72.89 hybridization equal or greater than 70% may be consid- ALT-9 N Halorubrum 35.81 TeSe-21 O Halorubrum 29.63 ered indicative of same species (GUTIERREZ et al., 1989, 1990). H. saccharovorum DSM 1137 40.84 H. sodomense DSM 3755 55.5 A high degree of DNA-DNA similarity (97.52%) was H. lacusprofundi DSM 5036 44.06 observed between the strain TeSe-45(representative of H. trapanicum CECT 397 42.45 phenon B) and the reference strain H. salinarium CCM H. coriense DSM 10284 43.14 2148. Also strain TeSe-45 showed a high degree of similarity (95.14%) with strain ALT-51, (representative of *Labeled strain 470 C. LIZAMA et al.

Fig. 2. Phylogenetic tree showing the position of the strains ALT-6, TeSe-19, TeSe-66, TeSe-69 and TeSe-73 among the species representatives of genus Halorubrum and some genera of extreme halophilic archaea. The sequence data used were obtained from the EMBL database as following (accesion numbers are give in parentheses): Halobacterium salinarum DSM 671 (M38280), Halococcus morrhuae ATCC 17082 (X00662), Haloferax mediterranei ATCC 33500 (D11107), Haloferax volcanii ATCC 29715 (K00421), Halorubrum trapanicum NRC 34021 (X82168), Halorubrum vacuolatum JCM 9060 (D87972), Halorubrum lacusprofundii ACAM 34 (X82170), Halorubrum saccharovorum NCIBM 2081 (X82167), Halorubrum sodomense ATCC 33755 (X82169), Halorubrum distributum VKMB 1733 (D63572), Halorubrum coriense JCM 9275 (L00922), Haloarcula hispanica ATCC 33960 (U68541), Haloarcula vallismortis ATCC 29715 (D50851), Halogeometricum borinquense ATCC 700274 (AF002984), Halobacu- lum gomorrense DSM 9297 (L37444), Halorabdus utahensis DSM 12940 (AF071880), Natronococcus occultus NCIMB 2192 (Z28378), Natronomonas pharaonis JCM 8858 (D87971), Natrialba asiatica JCM 9576 (D14123), Natrinema pellirubrum NCIMB 786 (AJ002947), Natronobacterium gregoryi NCIMB 2189 (D87970). The sequence of Methanospirillum hungatei DSM 864 (M 60880) was used as the outgroup. Bootstrap values (greater than 50) are shown at nodes. Bar, 0.1 substitution per nucleotide position. Extreme halophilic archaea from “Salar de Atacama” 471

Phylogenetic analysis Discussion

A nearly complete 16S rRNA gene sequence was ob- During the last years extreme environments have been tained for the strains ALT-6, TeSe-19, TeSe-66, TeSe-69 extensively studied, and the extreme halophilic bacteria and TeSe-73. The phylogenetic tree (Fig. 2) constructed has been found in a wide range of saline environments. for the Jukes-Cantor model and the least-squares algo- The Atacama Saltern is a extreme environment in the east rithm of Fich and Margoliash confirmed that these part of the Antofagasta district of Chile and it is situated strains were closely related at Halorubrum cluster. The at 2300 m above sea level, at 20º 30′S and 68º 15′W. This evolutionary distance of our strains with others members environment have extremely conditions so special that of Halorubrum genus was low and confirmed its affilia- distinguish it with another hypersaline environments, tion as new taxa of genus Halorubrum.. The phenotypic such as: high salinity, high radiations (U.V), changes in characteristics of these strains are shown in Table 3. The temperatures and dryness. At its edges are many small EMBL accession number for the 16S rRNA nucleotide se- salt water ponds which obviously receive streams of fresh quence of strain ALT-6, selected as representative of the water from the sub-surface. In its interior, there is a num- new taxa of genus Halorubrum, is AJ276887. ber of small shallow lakes with high concentration of

Table 3. Phenotypic characteristics of the strains ALT-6, TeSe-19, TeSe-66, TeSe-73 and TeSe-69; isolated from Atacama saltern and representatives of new taxa of genus Halorubrum.

Strain Alt-6 TeSe-19 TeSe-66 TeSe-73 TeSe-69 Phenon (I) (J) (K) (L) (M)

Growth at % (w/v) total salts 10 –––+– 15 ++–+– Growth at 50 °C + + + + + Growth at pH 6 – – – – – Catalase + + + + + Oyidase + + + + + Tween 20 hydrolysis – – – + – Tween 40 hydrolysis – – – – – Tween 60 hydrolysis – – – – – Tween 80 hydrolysis – – – + – Esculin hydrolysis + – + – – Nitrate reduction + + – + – Starch hydrolysis – – + – –

Utilization as sole source of carbon and energy of: Glucose + + + + + Fructose + – – – – Galactose + – – + + Maltose – – + – + Cellobiose – – – – – Mannose + – – – – Arabinose – – – – – Trehalose + + + – – Sorbitol – – – – – Glycerol – – – – – Pyruvate + + + + + Acetate + – + + + Malate – – + – – Citrate – – – – – Fumarate – – – – –

Utilization as sole source of carbon, nitrogen and energy of: Histidine – – – – – Alanin – – – – – Asparagine – – – – – Serine – + – – – Ornitine – – – – – Glutamine – – – – – 472 C. LIZAMA et al. salts; these also receive streams of fresh sub-surface conclusion, this study is the first description of taxonom- water. “Tebenquiche” is the biggest of these lakes and is ic position of halophilic archaea on the Atacama saltern situated in the northern part of the salar. Lake “Teben- and our results support the hypothesis that the different quiche” is hypersaline, with pH close to neutral and prac- environmental conditions in each hypersaline habitat tically anoxic. The ionic composition of Lake “Teben- may well influence the types of halophilic archaea present quiche” waters is dominated by sodium and chloride ions and a considerable portion of culturable extreme but with a high sulphate concentration in which ionic halophilic achaea remains unknown. More geno-and dominance are Na>K>Mg>Ca:Cl>SO4>HCO3+CO3 chemotaxonomic studies will be of interest to elucidate (ZUÑIGA et al., 1991). the position of these strains. In our knowledge this is the first taxonomical study about the extreme halophilic archaea isolated from the Lake “Tebenquiche” on the Atacama Saltern . In the first Acknowledgements time, studies on extremely halophilic bacteria employed This work was partially supported by grants from the Junta complex media supplemented with aminoacids (EIMH- de Andalucía, Spain, to the Investigation Unit “Microorganis- mos Halófilos” number CVI190; and from the Stiftung Volk- JELLEN, 1965). We used these type of media in our study, swagen, project 1/64.465. but the strains isolated from “Salar de Atacama” were also able to growth very well in the simple medium HE, as described TORREBLANCA et al. (1986). 2+ Studies have showed that Mg content increase with References the halophilic character in non-alcalophilic extreme halophilic bacteria that growth at neutral pH (DE MEDI- BAUER, A.W., SERRIS, J.C., TURCK, M., KIRBY, W.M.: Antibiotic CIS et al., 1986). However, the strains studied in the pre- susceptibility testing by a standardized single disc method. 2+ sent work have not shown specific requirements for Mg Am. J. Clin. Path. 45, 493–496 (1966). at saturated NaCl concentrations. In relation with this re- BRENNER, D.J., FANNING, G.R., RAKE, A. V., JOHNSON, K.E.: sult it is important to consider that the Mg2+ concentra- Bath procedure for thermal elution of DNA from hydroxy- tions in the Atacama Saltern are significantly less that de- apatite. Anal. Biochem. 28, 447–459 (1969). scribed in another hypersaline environments in relation to DE MEDICIS, E., PAQUETTE, J., GAUTHIER, J.J., SHAPCOTT, D. : concentration of ions as Na+ or Cl-. 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