International Journal of Systematic and Evolutionary Microbiology (2002), 52, 729–738 DOI: 10.1099/ijs.0.01999-0

Haloferax alexandrinus sp. nov., an extremely halophilic canthaxanthin-producing archaeon from a solar saltern in Alexandria (Egypt)

1 Food Science and Dalal Asker1,2 and Yoshiyuki Ohta2 Technology, Faculty of Agriculture, Alexandria University, Egypt Author for correspondence: Yoshiyuki Ohta. Tel: j81 824 24 7923. Fax: j81 824 24 7923. 2 Faculty of Applied e-mail: kohta!hiroshima-u.ac.jp Biological Science, Hiroshima University, 1-4-4 Kagamiyama, An extremely halophilic red micro-organism designated strain TMT was isolated Higashi-Hiroshima from a solar saltern in Alexandria, Egypt. The micro-organism stains Gram- 739-8528, Japan negative, is very pleomorphic, non-motile and strictly aerobic and requires at least 10 g NaCl lN1 for growth. The growth optimum is 250 g NaCl lN1. Growth is N1 also observed over a wide range of MgSO4 concentrations (10–40 g l ). Aerobic reduction of nitrate without gas production was detected. Cells grew aerobically in a minimal salts medium containing ammonium chloride and glucose. Strain TMT produced acid from fructose, glucose, rhamnose, maltose and glycerol. The GMC content of the DNA was 595O03 mol%. On the basis of polar lipid analysis, the isolate belonged to the genus . Analysis of the 16S rDNA sequence showed the highest similarity (S 99%) to be to the type strain . Although the spectrum of antibiotic susceptibility was similar to that of validly described species of the genus Haloferax, the strain could be distinguished from them by its different response to josamycin and rifampicin. Strain TMT is unique within the genus Haloferax in producing canthaxanthin. Comparative analysis of phenotypic properties and DNA–DNA hybridization between strain TMT and Haloferax species supported the conclusion that TMT is a novel species within this genus, for which the name Haloferax alexandrinus sp. nov. is proposed. The type strain is TMT (l JCM 10717T l IFO 16590T).

Keywords: extremely halophilic bacteria, Haloferax alexandrinus sp. nov., , carotenoids, canthaxanthin

INTRODUCTION Halorubrum (McGenity & Grant, 1995), Halobaculum (Oren et al., 1995), Natrialba (Kamekura & Dyall- Extremely halophilic archaea are chemo-organo- Smith, 1995), (Kamekura et al., 1997), trophic organisms that satisfy some of their energy Halogeometricum (Montalvo-Rodrı!guez et al., 1998), requirements with light. These archaea are classified in Natrinema (McGenity et al., 1998), Haloterrigena one order, , and one family, Halobac- (Ventosa et al., 1999), Natronorubrum (Xu et al., 1999) teriaceae (Grant & Larsen, 1989). Recently, 16S rDNA and Halorhabdus (Wainø et al., 2000). sequencing, DNA–DNA hybridization, polar lipid analysis and other studies have recognized 15 genera. Members of the family are charac- The currently recognized genera are Halobacterium, terized by red-coloured cells, the colour mainly being Haloarcula, Haloferax, Halococcus, Natronobac- due to the presence of C&!-carotenoids (bacterio- terium, Natronococcus (Grant & Larsen, 1990; Tindall ruberins) as the major carotenoids (Kushwaha et al., et al., 1984; Tindall, 1992; Torreblanca et al., 1986), 1974; Rønnekleiv & Liaaen-Jensen, 1992, 1995). Some

...... members of the genera Halobacterium and Haloarcula Abbreviation: S-DGD-1, sulfated diglycosyl diether. have been reported to partially produce C%!-caro- The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence tenoids and ketocarotenoids such as β-carotene, ly- of Haloferax alexandrinus strain TMT (l JCM 10717T l IFO 16590T)is copene, 3-hydroxy echinenone and trans-astaxanthin AB037474. as the minor carotenoids (Kelly et al., 1970; Kushwaha

01999 # 2002 IUMS Printed in Great Britain 729 D. Asker and Y. Ohta et al., 1972, 1974, 1982; Kushwaha & Kates, 1973; Morphological, biochemical and physiological characteriza- Calo et al., 1995). Recently, the biotechnological tion. The phenotypic tests were performed with strain TMT potential of these members of the Archaea has in- in accordance with the proposed minimal standards for the creased because of their unique features, which fa- description of new taxa in the order Halobacteriales (Oren et cilitate many industrial procedures. For example, no al., 1997). All tests were carried out using the optimal growth sterilization procedures are required, because of the conditions defined in our previous work (Asker & Ohta, 1999). In brief, the micro-organism was identified on the extremely high NaCl concentration used in the growth basis of colony morphology by streaking on a standard medium; this is useful for preventing contamination growth agar medium, incubated at 37 mC for 7 d. Motility by other organisms. In addition, no cell-disrupting was studied by using the ‘hanging drop’ technique and the devices are required, as cells lyse spontaneously in deep-agar method. Anaerobic growth was tested in the " fresh water, and these micro-organisms are able to standard growth medium in the presence of 5 g l− nitrate, utilize single carbon sources such as sugars, acetate or -arginine. HCl or DMSO in completely filled stoppered succinate for growth (Rodrı!guez-Valera et al., 1980; tubes. Controls without additives were included, and all Kauri et al., 1990; Calo et al., 1995; Asker & Ohta, incubations were performed in the dark. Haloferax denitri- ficans was used as a positive control for the formation of 1999). In summer at El-Mallahet, a solar saltern near T Alexandria City in Egypt, the temperature ranges from nitrite and gas from nitrate, and N. pellirubrum JCM 10476 37 to 40 C. As a result, the concentration of the total served as a positive control for the anaerobic growth on m arginine. The Voges–Proskauer test was performed; in dissolved salts increases to saturation at pH 7n2. It is addition, tryptophan deaminase and the utilization of citrate noteworthy that under these extreme conditions the were tested for by using API-20 E (bioMe! rieux). Arginine surface of the saltern is characterized by a reddish dihydrolase, lysine and ornithine decarboxylase were tested purple colour corresponding to the growth of red for by using the method of Skerman (1967), modified " halophilic members of the Archaea; other organisms by the addition of the following (in g l− ): NaCl, 250; cannot survive there. In a previous work, an attempt MgSO%.7H#O, 40; KCl, 2; and trisodium citrate, 3. Phos- was made to find a new biological source of can- phatase activity was tested for by adding 1% (w\v) aqueous thaxanthin by isolating 31 red, extremely halophilic phenolphthalein diphosphate solution to the standard microbes from this saltern. Of the strains isolated, growth medium; β-galactosidase activity was detected using strain TMT produced the highest levels of carotenoids ONPG. Variation of pigmentation at different salt concen- −" trations was determined on standard growth agar media at [2n06 mg (g dry cells) ], including β-carotene, 3- −" hydroxy echinenone, bacterioruberins, and a remark- NaCl concentrations of 100, 150, 200 and 250 g l . Hy- −" drolysis of Tween 80 and gelatin was tested as described by able amount of canthaxanthin [700 µg (g dry cells) ] Gutierrez & Gonzalez (1972). Gram staining was carried out (Asker & Ohta, 1999); however, among the Archaea, as described by Dussault (1955). Other bacteriological tests production of canthaxanthin has not been reported were carried out as described by Gibbons (1957). The previously. To the authors’ knowledge, no other production of acids from different sugars was tested for by ecological studies on the halobacteria of this saltern using the API 50 CHE test (bioMe! rieux) in the standard have been performed, although this saltern may be a growth medium except that the yeast extract was omitted, " rich source diverse halophilic bacteria. the concentration of Casamino acids was reduced to 5 g l− , −" T and 0n18 g phenol red l was added. The final pH was In the present work, strain TM was further charac- adjusted to 7n2, and incubation was done at 37 mC for 2–4 d. terized. On the basis of the 16S rRNA gene sequence, the polar lipid composition, physiological analysis and To test for the ability to grow on single carbon sources, a DNA–DNA hybridization, the creation of a new chemically defined medium was used. This medium was species for the genus Haloferax appears to be justified. prepared by omitting the yeast extract and the Casamino acids from the standard growth medium and adding the " carbon sources being tested, as follows (in g l− ): glycerol, METHODS 0 5; sodium succinate, 4 5, or glucose, 10; supplemented by n " n 0 27gl− NH Cl. The production of poly β-hydroxy butyrate Strains and culture conditions. The reference strains used n % were obtained from the Japan Collection of Microorganisms was examined by growing the cells in the chemically defined (JCM) and are listed in Table 3. These strains were grown at medium, in which the NH%Cl content was reduced to 0n005% −" (w\v). Furthermore, 1% (w\v) glucose was added. The 37 mC in a complex medium containing (in g l unless otherwise indicated): Bacto Casamino acids (Difco), 5; presence of poly β-hydroxy butyrate was tested according to Gerhardt et al. (1981). Tolerance for high concentrations of Bacto yeast extract (Difco), 5; sodium glutamate, 1; triso- # the Mg + cation was determined at 123n2, 197n2 and dium citrate, 3; MgSO%.7H#O, 20; KCl, 2; NaCl, 200; −" −" −" 394n4gl and up to the saturation of MgSO%.7H#O in the FeCl#.4H#O, 36 mg l and MnCl#.4H#O, 0n36 mg l . The −" pH was adjusted to 7n2 with 1 M NaOH. standard growth medium, containing 150 or 250 g NaCl l . Strain TMT was grown in a standard growth medium (Asker Susceptibility to antibiotics was tested by spreading 100 µl " & Ohta, 1999) containing (in g l− ): yeast extract, 10; exponential-phase cultures (3–4 d) on the standard growth Casamino acids, 7n5; NaCl, 250; MgSO%.7H#O, 40; KCl, 2; agar medium plate and applying antibiotic discs (Becton trisodium citrate, 3; and trace-elements solution, 10 ml. The Dickinson microbiology system: chloramphenicol, 30 µg; trace-elements solution contained the following (in mg erthromycin, 15 µg; neomycin, 30 µg; josamycin, 30 µg; −" 100 ml): FeCl#.4H#O, 2n3; CaCl#.7H#O, 7; MnSO#.H#O, rifampicin, 5 µg; novobiocin, 30 µg; bacitracin, 10 µg and 0n3; ZnSO%,0n44; CuSO%.5H#O, 0n050). The pH was adjusted tetracycline, 30 µg). Zones of inhibition were recorded after to 7n2 with 1 M NaOH. The culture was incubated on a incubation at 37 mC for 6 d. Novobiocin, bacitracin, rifampi- shaker at 37 mC for 7 d. cin and josamycin were further tested in broth at the

730 International Journal of Systematic and Evolutionary Microbiology 52 Haloferax alexandrinus sp. nov.

Table 1. Primers used in the amplification and sequencing of 16S rDNA of strain TMT

Primer name* Sequence (5h 4 3h) Position† Reference

Forward F1‡ attccggttgatcctgccgg 1–20 Kamekura & Seno (1992) F2‡ tccgacagtgagggacgaaa 688–707 Kamekura & Seno (1992) TMF3 cgcagcaggcgcgaaacctt 329–348 This study TMF4 gcaacgagcgagacccg 1039–1055 This study TMF5 gagaggaggtgcatggccg 983–1001 This study Reverse R1‡ cccgggtatctaatccggtt 720–739 Kamekura & Seno (1992) R2‡ aggaggtgatccagccgcag 1453–1472 Kamekura & Seno (1992) TMR3 gtattaccgcggcggctg 457–474 This study TMR4 gcgacgggcggtgtgtgc 1340–1357 This study

* All of the primers listed above were used to sequence the complete 16Sr DNA in this study. † Numbers correspond to positions in the sequence with GenBank accession number D11107. ‡ Two sets of forward and reverse primers (F1jR1 and F2jR2) were used to amplify two PCR products of approximately 740 bp (1–739) and 784 bp (688–1472), respectively, of strain TMT 16S rDNA.

following concentrations: 5, 10, 20, 30, 40, 50, 60, 100, 150, 15:4, by vol.) (Montalvo-Rodrı!guez et al., 1998). Glycolipid −" 180 and 200 µgml . spots were detected by spraying the plates with 0n5% α- naphthol in 50% (v\v) methanol and then 50% (w\v) Phase-contrast microscopy and scanning electron micro- H SO in ethanol, before heating them at 150 C (Torre- scopy. Micrographs were prepared from exponential-phase # % m blanca et al., 1986), or by spraying them with 0 1% CeSO in cells grown in the standard growth medium. Drops of the n % culture were mixed on a microscope slide with an equal 1MH#SO%, followed by heating at 150 mC for 5 min (i.e. a volume of melted 2% (w\v) agarose containing 25% (w\v) general lipid stain, allowing differentiation of glycolipids NaCl, and covered with a cover-slip. For examination with from other lipids by colour) (Kates, 1972). Ammonium scanning electron microscopy, 4 µl culture was treated with molybdate\sulfuric acid reagent was used for the detection 2%(w\v) glutaraldehyde solution and transferred to plastic of phospholipids. slides. These specimens were subsequently dehydrated Whole-cell protein profiles. Colonies were taken from through an ethanol gradient of 30, 50, 75 and 95%, then standard growth agar plates, and whole-cell proteins were subjected to an overnight dehydration in 100% ethanol. extracted by boiling in SDS-PAGE sample buffer for 15 min. Drying was accomplished by using an Hitachi HCP-2 Five microlitres of the solution mixture was separated by Critical Point Dryer. The specimens were placed on self- SDS-PAGE, according to the procedure described by adhesive conducting aluminium tape and gold-coated using Laemmli (1970), using an ATTO AE-7300 compact PAGE an Hitachi E-1030 Spatter Ion. The specimen was examined unit at a constant current of 20 mA per gel for approximately using a JEOL JSM-5800LV electron microscope at 20 kV. 1 h. Proteins were stained with 0n5% Coomassie brilliant Micrographs were recorded at magnifications of 3500. blue and destained in an aqueous solution of 10% (v\v) Images were scanned and electronically enhanced using the acetic acid and 25% (v\v) methanol. High-molecular-mass    and  editing programs. and low-molecular-mass markers (Amersham Pharmacia Lipid analysis. Core lipids were isolated and analysed by Biotech) were applied. The gel was scanned and analysed using TLC as described by Minnikin et al. (1975) and Ross using Scanning Gel (version 1.1,  , Diversity et al. (1981). Freeze-dried cells (100 mg) were hydrolysed in Database; pdi). All separated band weights were calculated methanol\toluene\concentrated H#SO% (3:3:0n1, by vol.) at from those data. 55 mC for 15–16 h; this was followed by extraction with 1 ml Plasmid analysis. A rapid procedure for the detection of large hexane. The hexane extract was concentrated in a vacuum, and small plasmids was performed (Kado & Liu, 1981). then chromatographed on silica gel 60 F#&% glass-packed thin-layer plates (20i20 cm; Merck), using petroleum DNA base composition and 16S rDNA sequence analysis. ether\diethyl ether (85:15, v\v) as the developing solvent. Determination of the GjC content of the DNA was The lipids were visualized by spraying with 10% (w\v) performed by using HPLC (Katayama-Fujimura et al., ethanolic dodecamolybdophosphoric acid, followed by heat- 1984). Exponential-phase cells (72 h) grown in the standard ing at 160 mC for 15 min. Polar lipids were extracted from 1 g growth medium at 37 mC on a rotary shaker were lysed in freeze-dried cells with methanol\chloroform\saline (2:1: distilled water, and total DNA was extracted and purified 0n8, by vol.) as described by Kates (1986). The polar lipids using the method of Marmur (1961). The purified DNA was −" were separated using TLC on silica gel plates (10i20 cm; dissolved in distilled water (1 mg ml ) and then was heated Merck), using single development as described by Torre- at 100 mC for 15 min before being cooled rapidly in an ice blanca et al. (1986) and Oren et al. (1996, 1999). The bath. To the denatured DNA (10 µg) was added 10 µl " following two different solvent systems were used: chloro- nuclease P1 solution (2 U ml− 40 mM sodium-acetate buffer −% form\methanol\acetic acid\water (85:22n5:10:4 or 80:12: containing 2i10 M ZnCl#,pH5n3) before incubation at http://ijs.sgmjournals.org 731 D. Asker and Y. Ohta

50 mC for 1 h. Standard solution and the hydrolysate were (a) subjected separately to HPLC. The 16S rDNA was amplified using the PCR method of Embley (1991), modified by McGenity & Grant (1993). Four primers (forward, F1 and F2; reverse, R1 and R2) (Table 1) were designed from very conserved regions of 16S rDNA of halophilic archaea (Hui & Dennis, 1985; Kamekura & Seno, 1992). These primers are numbered according to Haloferax mediterranei 16S rDNA (accession no. D11107), and were applied in two independent PCR reactions, resulting in two PCR products comprising positions 1–740 and 688–1472 of the 16S rDNA sequence. Purified PCR products were sequenced auto- (b) matically using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) as directed by the manufacturer’s protocol, employing nine primers (Table 1). Sequencing reactions were electrophoresed using the Ap- plied Biosystems 377 ABI PRISM DNA Sequencer (Applied Biosystems). A total of 1472 bases were identified during these analyses. The phylogenetic position of the resulting sequence of strain TMT was determined using the EMBL database and  (Pearson & Lipman, 1988). Similarity values were calculated from the alignment of the strain TMT sequence with sequences from the following organisms: Haloferax volcanii ATCC 29605T (accession no. K00421T), T Haloferax mediterranei ATCC 33500 (D11107), Haloferax (c) denitrificans ATCC 35960T (D14128) and Haloferax gib- bonsii ATCC 33959T (D13378), using   (version 1.8) multiple sequence alignments (Thompson et al., 1994). Alignment gap base positions were not taken into con- sideration for the calculation. Each 1467-base sequence of all five strains was used for the calculation. The evolutionary distance matrix was used to determine the similarity (Ki- mura, 1980). The programs used for this analysis can all be found in the  program (version 3.5.1) (Felsenstein, 1993).

Preparation of labelled DNA, and DNA–DNA hybridization. $# The DNA of strain TMT was nick-translated with [α- P]- ...... dCTP by using a nick-translation kit (Boehringer Mann- T heim). The labelled DNA was purified with Quick Spin Fig. 1. Morphology of strain TM . (a) Scanning electron micrograph, (b) phase-contrast photomicrograph and (c) light- Columns, Sephadex G-25 (Boehringer Mannheim) and etha- microscope photomicrograph, showing the pleomorphic forms nol precipitation. The mean specific activity obtained with ) " including cocci, ovals, triangles, squares and rods. Bars, 5 µm. this procedure was 3i10 d.p.m. (µg DNA)− . The labelled DNA was denatured prior to hybridization, by heating at 100 mC (block incubator; ASTEC) for 5 min, and then placed on ice. DNA–DNA hybridization was performed by using RESULTS the competition procedure of the membrane method (Johnson, 1994). Competitor DNAs were sonicated at 50 W Morphological, biochemical, cultural and T for two 15 s time intervals. Membrane filters (HAHY; physiological characteristics of strain TM −# Millipore) containing reference DNA (25 mg cm ) were Colonies of strain TMT on standard growth agar placed in 100 ml screw-cap vials that contained the labelled, medium were circular, convex, entire, translucent, sheared, denatured DNA and the denatured, sheared com- smooth and red. The colonies appeared as a very small petitor DNAs. The ratio of the concentrations of competitor to labelled DNA was at least 150:1. The final volume points within 2–3 d, and the size increased up to 0 5–1 mm in diameter after 6 d incubation. The pig- and concentration were adjusted to 5 ml, 2i SSC and 30% n ment intensity of the colonies was affected by the salt formamide. Hybridization was performed for 18 h in a −" hybridization incubator (HB-100; TAITEC) at 56 mC, which concentration: at 250 g NaCl l , the colonies were orange-red, but reducing the concentration to 150 g is within the temperature limits for the method (De Ley −" & Tijtgat, 1970). After hybridization, the filters were washed NaCl l increased the pigment intensity to deep red. in 2i SSC at 56 mC. The radioactivity bound to the filters Gas vacuoles were never observed. The cells stained was measured in a liquid-scintillation counter (Beckman negative with Gram staining and were pleomorphic Instruments), and the percentage homology was calculated (irregular cocci, short and long rods, squares, triangles according to Johnson (1994). At least three independent and ovals) when grown on the standard growth determinations were performed. medium at the optimum growth conditions (Fig. 1).

732 International Journal of Systematic and Evolutionary Microbiology 52 Haloferax alexandrinus sp. nov.

Table 2. Characteristics that distinguish strain TMT from the validy described species within the genus Haloferax ...... Strains: 1, TMT;2,Haloferax volcanii NCBM 2012T;3,Haloferax denitrificans ATCC 35960T;4,Haloferax mediterranei ATCC 33500T;5,Haloferax gibbonsii ATCC 33959T. Data on the validy described species of the genus Haloferax were obtained from Calo et al. (1995), Grant & Larsen (1990), Juez et al. (1986), Mullakhanbhai & Larsen (1975), Nieto et al. (1993), Oren et al. (1995), Rodrı!guez-Valera et al. (1980, 1983), Rønnekleiv & Liaaen-Jensen (1995), Tindall et al. (1989), Tindall (1992), Tomlinson et al. (1986), Torreblanca et al. (1986). j, Positive; k, negative; p, varied; , not determined.

Characteristic 1 2 3 4 5

Gas vacuoles (production) kkkjk Anaerobic growth in the presence of nitrate kkjjk Starch hydrolysis kkkjk Tween 80 hydrolysis jkkjj Gelatin hydrolysis jkjjj Casein hydrolysis kkkjj Gas produced from nitrate kkjjk H#S produced from cysteine kjkkk # " Mg + requirements for growth (g l− ) 10–40 4n9–9n9  4n9–9n92n5–4n9 " NaCl range for growth (g l− ) 100–320 k 88–263 76–270 100–320 " Optimum NaCl for growth (g l− ) 250 146 110–180 160 200–250 " Cell stability (NaCl, g l− ) 100 30 88 30 30–40 Acid produced from: Mannose k  k  j Galactose kjj j Lactose kjkj " Resistant to antibiotics (µgml− ) Josamycin (180) jjkkk Rifampicin (100) jkkkk Carotenoid composition† 1, 3, 4, 5 2, 5 5 3, 5 5 " * Only sparse growth occurs at 233 g l− (Mullakhanbhai & Larsen, 1975). † The carotenoid composition is as follows: 1, β-carotene; 2, lycopene; 3, 3-hydroxy-echinenone; 4, canthaxanthin; 5, bacterioruberins.

Cell dimensions varied from 1n1to1n5 µmi1n6–2n0 negative. Poly β-hydroxybutyrate was not detected. In µm; rod-shaped cells were 1 1–1 5 3 5–4 µm in size. the standard growth medium, a high growth rate was n n i n " " This strain was not motile. Upon gradual dilution of observed at 250 g NaCl l− and at 123 2 g MgSO l− . " n % the culture in 250 g NaCl l− with water, the cells At the same concentration of NaCl, the strain tolerated −" changed in shape from pleomorphic forms to spheres, elevated concentrations of MgSO% up to 394n4gl , and the spheres then underwent lysis below 100 g but the lag phase was extended for several days. " NaCl l− . Unlike Haloferax volcanii, cells did not However, with the reduction in NaCl concentration " become sphaeroplasts in the presence of low concen- to 125 g l− in the same medium, strain TMT could −" trations of magnesium and calcium (Cohen et al., tolerate up to 493 g MgSO% l . Amino acids and yeast 1983). It was a strict aerobe and was unable to grow extract in the complex medium supported growth. anaerobically by using alternative electron acceptors Growth was not supported by polypeptone, tryptone such as nitrate or DMSO, or by fermenting -arginine. or casein. The strain was able to grow in chemically However, the aerobic reduction of nitrate and nitrite defined media containing ammonium chloride and without gas production was detected. The Voges– single carbon sources such as glucose, glycerol and Proskauer test was negative. Catalase and oxidase succinate. However, the addition of a mixture of biotin " " activity tests were positive. The cells hydrolysed gelatin (1 µgml− ) and thiamine (8 µgml− ) to the chemically and Tween 80 but not starch or casein. Indole was defined medium (with succinate and glycerol as carbon formed from tryptone. H#S was produced from sodium sources) could replace yeast extract and Casamino thiosulfate. The arginine dihydroxylase was negative; acids (complex medium) in supporting growth of the in the absence of this enzyme, the mechanism sup- culture. Acid production from a variety of sugars porting anaerobic growth on arginine cannot operate (fructose, glucose, rhamnose, maltose, -arabinose, (Hartmann et al., 1980). The strain was positive for β- -xylose, ribose, sucrose, N-acetyl gluconate and galactosidase, phosphatase and tryptophan deaminase glycerol) was observed. Lactose, galactose, mannose, activities. Tests for lysine decarboxylase and ornithine citrate and starch were not utilized. Strain TMT was " decarboxylase were negative. The urease test was susceptible to 30 µg novobiocin ml− and 30 µg baci- http://ijs.sgmjournals.org 733 D. Asker and Y. Ohta

kDa 12 345 678910 Lipid analysis T 212 Strain TM appeared to contain the diphytanyl diether 170 moieties (C#!–C#!) as core diether lipids, tetraether 116 kDa lipids being absent. The polar lipids detected were 94 phosphatidylglycerol, phosphatidylglycerophosphate- 76 methyl ester, sulfated diglycosyl diether (S-DGD-1) 67 and diglycosyl diether. On the TLC, S-DGD-1 of T 53 strain TM runs similarly to S-DGD-1 from species of Haloferax (S-DGD-1 is the glycolipid marker of 43 Haloferax). The sulfated tetraglycosyl diether of N. pellirubrum JCM 10476T, the triglycosyl diether of Haloarcula vallismortis JCM 8877T and the S-DGD-3 30 of Halorubrum saccharovorum JCM 8865T were not detected. Moreover, strain TMT does not contain phosphatidylglycerosulfate, which is present in Halo- rubrum saccharovorum JCM 8865T, N. pellirubrum ...... T T Fig. 2. SDS-PAGE gel of whole-cell proteins. Lanes: 1, high- JCM 10476 and Haloarcula vallismortis JCM 8877 . molecular-mass marker; 2, strain TMT ;3,Haloferax volcanii JCM 8879T ;4,Haloferax denitrificans JCM 8864T ;5,Haloferax Whole-cell protein profiles mediterranei JCM 8866T ;6,Haloferax gibbonsii JCM 8863T ; T 7, Halorubrum saccharovorum JCM 8865T ;8,Natrinema Similarities between strain TM and Haloferax species pellirubrum JCM 10476T ;9,Haloarcula vallismortis JCM 8877T ; were also observed in the whole-cell protein profiles, as 10, low-molecular-mass marker. The high-molecular-mass mar- determined by SDS gel electrophoresis (Fig. 2). On the ker contained the following (approx. molecular masses in kDa): other hand, these protein profiles clearly differentiated myosin (212); α2-macroglobulin (170); β-galactosidase (116), T transferrin (76) and glutamic dehydrogenase (53). The low- strain TM from members of the genera Natrinema, molecular-mass marker contained the following (kDa): phos- Halorubrum and Haloarcula. Moreover, among the phorylase b (94), BSA (67), ovalbumin (43), carbonic anhydrase Haloferax species, strain TMT could be differentiated (30), soybean trypsin inhibitor (20) and α-lactoalbumin (14). by a distinct 66n51 kDa band of polypeptide. A quan- titative analysis of the relatedness of the protein patterns indicated that strain TMT was closely related to species of Haloferax. " tracin ml− , but was resistant to 10–20 µg bacitracin " ml− ,30µg chloramphenicol, 15 µg erthromycin, 30 µg Characterization of a plasmid in strain TMT neomycin, 180 µg josamycin, 100 µg rifampicin and Two small plasmids with of 3 and 2 kbp were identi- 30 µg tetracycline. Furthermore, the susceptibility to fied in cells of strain TMT after DNA extraction and antibiotics in comparison with that of four reference agarose gel electrophoresis. strains was tested: N. pellirubrum JCM 10476T, Halo- ferax mediterranei and Halorubrum saccharovorum T DNA base composition and 16S rDNA sequence JCM 8865 were susceptible to 5 µg rifampicin; only analysis Haloferax mediterranei is susceptible to 15 µg erthro- T mycin. Table 2 shows the comparisons of the pheno- The GjC content of strain TM was 59n5p0n3mol%, typic features of strain TMT and those of the type as determined by HPLC (mean of three independent strains of Haloferax species. determinations). The GjC content is within the range

Table 3. Levels of DNA–DNA relatedness between strain TMT and other halobacterial species ...... Values are means of three experiments, which did not differ by more than 5%. T, Type strain.

Source of unlabelled DNA DNA hybridization (%) with 32P-labelled DNA from TMT

Strain TMT (l JCM 10717T l IFO 16590T) 100 Haloferax volcanii JCM 8879T (l ATCC 29605T)49 Haloferax denitrificans JCM 8864T (l ATCC 35960T)27 Haloferax mediterranei JCM 8866T (l ATCC 33500T)21 Haloferax gibbonsii JCM 8863T (l ATCC 33959T)36 Halorubrum saccharovorum JCM 8865T (l ATCC 29252T)0 Natrinema pellirubrum JCM 10476T (l NCIMB 786T)0 Haloarcula vallismortis JCM 8877T (l ATCC 29715T)0

734 International Journal of Systematic and Evolutionary Microbiology 52 Haloferax alexandrinus sp. nov. reported for Haloferax species (59n5–66, Table 2). The the same manner, Haloferax volcanii and Haloferax T complete 16S rDNA sequence of strain TM was denitrificans shared 99n5% similarity. Hezayen et al. determined. The sequence was compared with the (2001) have reported a high degree of similarity published 16S rDNA sequences of representative (! 99%) between the 16S rDNA sequences of the two members of the Archaea. The sequence showed a high species. Therefore, it was necessary to carry out degree of similarity (99n7%) to the sequence of DNA–DNA hybridization and phenotypic analysis to Haloferax volcanii (K00421), demonstrating that the investigate whether strain TMT belongs to Haloferax two strains are indeed closely related but not identical. volcanii or to a new species. Strain TMT was related to Haloferax denitrificans (99n3% similarity), Haloferax gibbonsii (99n2% simi- At the level of phenotypic properties (Table 1), strain T larity) and Haloferax mediterranei (98n2% similarity). TM exhibits the typical pleomorphic flattened shape It is noteworthy to mention that the similarity values of Haloferax species (Mullakhanbhai & Larsen, 1975; between four distinguished species of the genus Halo- Rodrı!guez-Valera et al., 1983; Juez et al., 1986), but ferax are 98n3–99n5%, when the alignment gap base displays a distinctly higher requirement for MgSO%. positions are not taken into consideration for the Two members of this genus (Haloferax mediterranei calculation. Because of the high degree of 16S rDNA and Haloferax denitrificans) might be differentiated sequence similarity between strain TMT and Haloferax from strain TMT: Haloferax mediterranei contains gas volcanii as well as other members of this genus, no vacuoles and is capable of anaerobic growth in the attempt was made to reconstruct a phylogenetic tree. presence of nitrate with gas production; Haloferax denitrificans is not gas-vacuolated, but produces gas T DNA–DNA hybridization studies and grows anaerobically with nitrate. Strain TM neither contains gas vacuoles nor is capable of an- Data on the DNA–DNA hybridization between the aerobic growth in the presence of nitrate. Strain TMT reference strains and strain TMT are shown in Table 3. differed from Haloferax gibbonsii by its inability to The relatedness obtained between DNAs from the produce acid from galactose or mannose and by its $# reference strains and the P-labelled DNA from strain ability to hydrolyse casein. No growth of strain TMT T TM showed that there was a low level of hybridization was observed at pH values above 7n5. In contrast, among them, the values ranging from 49 to 0%. Thus, Haloferax gibbonsii has the ability to grow at pH the DNA–DNA hybridization confirmed that strain values above 8. In addition, Haloferax gibbonsii is TMT is differentiated at the species level from the other motile by means of a polar flagellum. The most representatives of the genus Haloferax. important features distinguishing strain TMT from Haloferax volcanii were its extreme requirement for NaCl and its good growth at saturating salt concen- DISCUSSION " trations (303 g l− ); in contrast, Haloferax volcanii has The presence of the glycerol diether moeities and the a moderate salt requirement and is strongly inhibited absence of glycerol ester lipid indicate that strain TMT at that concentration. To maintain cell stability, is an archaeon (Ross et al., 1981; Torreblanca et al., Haloferax volcanii requires a lower salinity (30 g " 1986). The high salt requirement (at least 100 g NaCl l− ) than strain TMT (Mullakhanbhai & Larsen, " NaCl l− ) for growth, the lysis at low salinity, the 1975). The above features clearly emphasize the great resistance to chloramphenicol, erthromycin, neo- environmental differences between the isolation source mycin, josamycin, rifampicin and tetracycline (antibio- of strain TMT (a solar saltern) and Haloferax volcanii tics which inhibit the growth of halophilic eubacteria) (the Dead Sea). Moreover, strain TMT differed from and the susceptibility to novobiocin and bacitracin are Haloferax volcanii by its lipolytic and gelatinase all characteristics of the family Halobacteriaceae (Tin- activities, by its inability to produce H#S from cysteine, dall, 1992). Strain TMT is characterized by the lack of and by its ability to utilize galactose and lactose. phosphatidylglycerosulfate and the presence of S- Although the spectrum of antibiotic susceptibility was DGD-1 and C#!–C#! in the absence of C#!–C#& core similar to those of other Haloferax spp., the strain was diether lipids. These features of strain TMT are much more resistant to josamycin than were Haloferax consistent with those of the genus Haloferax (Tindall, mediterranei, Haloferax denitrificans and Haloferax 1992; Tindall et al., 1989; Torreblanca et al., 1986; gibbonsii: whilst strain TMT was able to grow at 180 µg " Kamekura & Dyall-Smith, 1995). Electrophoresis of josamycin ml− , the minimal inhibitory concentration the whole-cell protein profile has been used to differen- of this antibiotic against these other Haloferax species " tiate between taxa of the Halobacteriales (Hesselberg was 31 2 µgml− (Nieto et al., 1993). Strain TMT was n " & Vreeland, 1995; McGenity et al., 1998). The electro- also resistant to 100 µg rifampicin ml− . In contrast, phoresis demonstrated that strain TMT is related to the other Haloferax species were sensitive to 15–31 2 µg " n genus Haloferax. On the basis of its 16S rDNA rifampicin ml− (Torreblanca et al., 1986; Nieto et al., sequence, strain TMT clearly demonstrated its affilia- 1993). We found that this antibiotic has a strong " tion with representatives of the genus Haloferax. The inhibitory effect on Haloferax mediterranei (5 µgml− ). greatest similarity percentages were obtained with 16S The different responses of the genus Haloferax to some rDNA sequence from Haloferax volcanii and Halo- antibiotics could be used for taxonomic purpose (Nieto T ferax denitrificans (99n7 and 99n3%, respectively). In et al., 1993). Thus, it could be said that strain TM http://ijs.sgmjournals.org 735 D. Asker and Y. Ohta differed from the distinguished species of the genus nithine decarboxylase tests are negative. The strain can Haloferax by its different response to the antibiotics utilize various complex carbon and nitrogen sources listed above. Strain TMT differed from Haloferax such as amino acids and yeast extract. The strain is volcanii and other Haloferax spp. by its ability to unable to utilize peptone, casein or starch. The major produce bicyclic β-carotene as well as canthaxanthin isoprenoid neutral lipids are the carotenoids (bacteri- (Table 1). Strain TMT was screened with respect to the oruberins, β-carotene and 3-hydroxy echinenone), production of canthaxanthin – a feature which made it including a large amount of canthaxanthin [700 µg(g " unique within the genus Haloferax and among other dry cells)− ]. Acid hydrolysis of whole cells releases a members of the Archaea. The low DNA–DNA hybri- single diether component identical to 2,3-di-O-phy- dization values between strain TMT and Haloferax tanyl-sn-glycerol. The major polar lipids are the diether volcanii (49%) proved that strain TMT and Haloferax analogues of phosphatidylglycerol, phosphatidylgly- volcanii are not identical and belong to different cerolphosphate-methyl ester, a diglycosyl glycerol species. In addition, the DNA–DNA hybridization diether and sulfate diglycosyl diether. The GjC T values between strain TM and the other Haloferax content of the DNA is 59n5p0n3 mol%. The type T T T T spp. indicated that strain TM represents a new species strain is TM (l JCM 10717 l IFO 16590 ). within the genus Haloferax. Accordingly, on the basis of chemotaxonomy, phenotypic characteristics and low DNA–DNA hybridization with the type strains ACKNOWLEDGEMENTS of Haloferax species, we suggest that strain TMT is sufficiently different from the currently recognized We would also like to thank Professor Ahmed R. El-Mahdy species in the genus Haloferax to warrant designation of Alexandria University for useful suggestions and Dr as a new species, namely Haloferax alexandrinus strain Tarek Awad for his critical reading of the manuscript. This TMT. work was financially supported by the Japan Ministry of Education (Monbusho).

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