International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1297–1303 Printed in Great Britain Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China Huawei Xin,1,2 Takashi Itoh,2 Peijin Zhou,1 Ken-ichiro Suzuki,2 Masahiro Kamekura3 and Takashi Nakase2 Author for correspondence: Takashi Itoh. Tel: j81 48 467 9560. Fax: j81 48 462 4617. e-mail: ito!jcm.riken.go.jp 1 The Institute of A novel extremely halophilic archaeon, strain XF10T, was isolated from a salt Microbiology, Chinese lake in China. This organism was neutrophilic, non-motile and pleomorphic, Academy of Sciences (CAS), Beijing 100080, PR China and was rod, coccus or irregularly shaped. It required at least 15 M NaCl for growth and grew in a wide range of MgCl concentrations (0005–05 M). Lipid 2 Japan Collection of 2 Microorganisms, RIKEN extract of whole cells contained two glycolipids with the same (The Institute of Physical chromatographic properties as two unidentified glycolipids found in the two and Chemical Research), described Natrinema species, Natrinema pellirubrum and Natrinema pallidum. Wako-shi, Saitama 351-0198, Japan Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain XF10T clustered with the two described Natrinema species and several 3 Noda Institute for Scientific Research, other strains (strains T5.7, GSL-11 and Haloterrigena turkmenica JCM 9743) 399 Noda, Noda-shi, with more than 981% sequence similarities, suggesting that strain XF10T Chiba 278-0037, belongs to the genus Natrinema. Comparative analysis of phenotypic Japan properties and DNA–DNA hybridization between strain XF10T and the Natrinema species supported the conclusion that strain XF10T is a novel species within the genus Natrinema. The name Natrinema versiforme sp. nov. is proposed for this strain. The type strain is XF10T (lJCM 10478TlAS 1.2365TlANMR 0149T). Keywords: Natrinema versiforme sp. nov., halobacteria, archaea INTRODUCTION Hypersaline environments are commonly found in China. In addition to many coastal salterns, a number Halobacteria (the family Halobacteriaceae; Grant & of salt lakes, soda lakes and salt-rich deserts are Larsen, 1989) are a diverse group of extremely halo- located in the west to northwest part of China, i.e. philic archaea that require at least 1n5 M NaCl for from Inner Mongolia, Qinghai Province, to Xinjiang growth. In the last few years, the numbers of halo- and Tibet Autonomous Regions. From these saline bacterial genera and species have increased rapidly. environments, a number of halobacteria have been Several new genera, e.g. Halogeometricum (Montalvo- isolated, including strains A5 and B2, which have been Rodrı!guez et al., 1998) and Natronorubrum (Xu et al., shown to be related to the genus Haloarcula based on 1999), have been created to accommodate newly polar lipid composition and 16S rDNA sequence isolated strains, and the others have been proposed by (Zhou et al., 1994; Xu et al., 1995), strain HAM-2, re-evaluation of misidentified or insufficiently des- which has been shown to be a close relative of Natrialba cribed strains, e.g. Natrinema (McGenity et al., 1998) magadii on the basis of 16S rDNA sequence (Tian et and Haloterrigena (Ventosa et al., 1999). al., 1997; Xu et al., 1999), two species of the genus Natronorubrum (Natronorubrum bangense and Natro- norubrum tibetense)(Xuet al., 1999), and some ................................................................................................................................................. incompletely characterized strains (Wang et al., Abbreviations: PG, phosphatidylglycerol; PGP-Me, phosphatidylglycero- 1984; Zhou et al., 1990). Isolation of novel strains of phosphate-methyl ester; PGS, phosphatidylglycerosulfate; S -DGD-1, 2,6- 2 the genera Haloarcula, Natrialba and Natrinema (H. HSO3-Manp-α(1 ! 2)-Glcp-α(1 ! 1)-sn-glyceroldiether; TMAO, trimethyl- amine N-oxide. Xin and others, unpublished work) has also exempli- The DDBJ accession number for the 16S rDNA sequence of Natrinema fied the wide diversity of halobacteria in the hyper- versiforme XF10T is AB023426. saline habitats in China. 01224 # 2000 IUMS 1297 H. Xin and others diluted medium or distilled water. Growth ranges and optima of NaCl and MgCl# levels were determined using growth medium containing various concentrations of NaCl (1n2–5n2 M) and MgCl# (0n005–0n5 M), respectively. Various buffer systems were employed in the determination of growth pH (50 mM of each): MES (pH 5n5–6n5), PIPES (pH 6n5–7n5), HEPES (pH 7n0–8n0), Tricine (pH 7n5–9n0) and CHES (pH 9n0–10n0). Growth temperature was determined in the range 4–60 mC by using a temperature gradient incubator (model TN-3; ADVANTEC). Growth rate was determined by measuring culture turbidity at 660 nm. Nutrition. Anaerobic growth in the presence of arginine, DMSO, trimethylamine N-oxide (TMAO) and nitrate (5 g " l− of each) was determined in rubber-stoppered tubes completely filled with the growth medium in the dark for 1–3 weeks and compared to growth on medium without the test ................................................................................................................................................. T compounds. Reduction of nitrate was detected by using the Fig. 1. Phase-contrast micrograph of strain XF10 . Cells were α pleomorphic and rod, coccus or irregularly shaped. Bar, 10 µm. sulfanilic acid and -naphthylamine reagent (Smibert & Krieg, 1981). Formation of gas from nitrate was detected by using Durham tubes under anaerobic conditions. In this paper, a novel halobacterial isolate, XF10T, To estimate the utilization of various carbohydrates as carbon and energy sources, the basal medium [0n1 g yeast which was isolated from a salt lake in the northwest −" −" −" part of China, is described. extract l (Difco), 0n5gNH%Cl l ,0n05 g KH#PO% l ,atpH 7 0 with 50 mM HEPES] was supplemented with 10 g test n " carbohydrate l− . Production of acids from carbohydrates METHODS was tested in the basal medium supplemented with 0n5 g test −" Isolation procedure, strains and growth conditions. Strain substrate l without buffer. The cultures were incubated at XF10T was isolated from clay collected from the shallow 37 mC without shaking for 2 weeks. Growth was determined near-edge floor of Aibi salt lake in Xinjiang Autonomous visually and pH was measured with a pH electrode. Region, China. After enrichment of the sample in Sehgal Biochemical tests. Tests for catalase and oxidase activities, and Gibbons medium (Sehgal & Gibbons, 1960) at 37 mC formation of indole, and hydrolysis of starch, gelatin, casein with shaking for 1–2 weeks, a pure culture was obtained by and Tween 80 were performed according to the standard or plating serial dilutions of enrichment cultures and repeated modified procedures of Oren et al. (1997). Formation of transfers of separate colonies on agar plates of the same sulfide was determined by incubating cells in the growth " medium. The purity of the strain was checked by colony medium supplemented with elemental sulfur (6n4g l− )or −" morphology and randomly amplified polymorphic DNA sodium thiosulfate (Na#S#O$;5H#O, 5n0gl ) without shak- patterns of total DNAs derived from different colonies on ing, and detecting sulfide with a strip of paper impregnated the agar plate. The growth medium used for the following " with 10% (w\v) lead acetate solution. studies contained (l− ): 5 g Casamino acids (Difco); 5 g Sensitivity to antimicrobial agents. Sensitivity to anti- yeast extract (Difco); 1 g sodium glutamate; 3 g trisodium microbial agents was determined in the growth medium citrate dihydrate; 30 g MgCl ;6H O; 5 g K SO ;36mg " # # # % containing each antimicrobial agents at 50 mg l− for at FeCl ;6H O; 036 mg MnCl ;4H O; and 220–250 g NaCl # # n # # least 2 weeks. Antimicrobial agents used were ampicillin, (pH 7 0). Agar slants and plates were prepared by adding n " anisomycin, bacitracin, chloramphenicol, erythromycin, 20 g agar l− . The strain was maintained on agar slants for neomycin, novobiocin, penicillin G and rifampicin. short periods (several months) or stored in growth medium with glycerol (final 20%, v\v) at k80 mC for long-term Lipid analyses. Total lipids were extracted by the modified preservation. Haloterrigena turkmenica JCM 9743 (for genus method of Kamekura (1993) and separated by TLC on and species designation, see Discussion; deposited in JCM Merck Kieselgel 60-HPTLC. For one-dimensional devel- by M. Kamekura in 1995), Haloterrigena turkmenica JCM opment, the solvent was chloroform\methanol\acetic acid\ 9101T (lVKM B-1734T ; transferred from VKM to JCM in water (85:22n5:10:4, v\v). For two-dimensional develop- 1993), Natrinema pallidum JCM 8980T (l NCIMB 777T ; ment, the solvents were chloroform\methanol\water transferred from NCIMB to JCM in 1993) and Natrinema (65:25:4, v\v) followed by chloroform\methanol\acetic pellirubrum JCM 10476T (l NCIMB 786T ; transferred from acid\water (80:12:15:4, v\v). Phospholipids were detected NCIMB to JCM in 1999) were used as reference strains with the Zinzadze reagent of Dittmer & Lester (1964). and cultivated in the above growth medium with 20 g Glycolipids were detected by spraying the plate with 0n5% −" −" MgSO%;7H#Ol and 2 g KCl l (instead of MgCl#;6H#O 1-naphthol in methanol\water (1:1) and then with sulfuric −" and K#SO%), and 200 g NaCl l . If not specified, strains were acid\ethanol (1:1) followed by heating at 120 mC for 5–10 cultivated at 37 mC with shaking at 180 r.p.m. in 500 ml min; all the other polar lipids were detected by further Erlenmeyer flasks containing 100 ml medium. Inoculated heating of the plate at about 250 mC for several minutes agar plates were wrapped in plastic bags and incubated at (Ihara et al., 1997). 37 mC. Sequencing of 16S rDNA. Total DNAs were extracted by the Morphology and growth characteristics of strains. Cells method of Cline et al. (1989). The 16S rRNA genes were were observed under a phase-contrast light microscope amplified by PCR with the following forward and reverse (Optiphot-2; Nikon).