International Journal of Systematic and Evolutionary Microbiology (2001), 51, 1693–1698 Printed in Great Britain Natrialba hulunbeirensis sp. nov. and Natrialba chahannaoensis sp. nov., novel haloalkaliphilic archaea from soda lakes in Inner Mongolia Autonomous Region, China 1 Institute of Microbiology, Yi Xu,1 Zhenxiong Wang,1 Yanfen Xue,1 Peijin Zhou,1 Yanhe Ma,1 Chinese Academy of 2 3 Sciences, Zhongguancun Antonio Ventosa and William D. Grant district, Beijing 100080, China Author for correspondence: Yanhe Ma. Tel: 86 10 62553628. Fax: 86 10 62560912. 2 j j Department of e-mail: mayh!sun.im.ac.cn Microbiology and Parasitology, Faculty of Pharmacy, University of T T Sevilla, 41012 Sevilla, Spain Two haloalkaliphilic archaeal strains, X21 and C112 , were isolated from soda lakes in Inner Mongolia Autonomous Region, China. Their morphology, 3 Department of Microbiology and physiology, biochemical features, polar lipid composition and 16S rRNA genes Immunology, University of were characterized in order to elucidate their taxonomy. According to these Leicester, Leicester data, strains X21T and C112T belong to the genus Natrialba, although there are LE1 9HN, UK clear differences with respect to their physiology and polar lipid composition between the two strains and the type species, Natrialba asiatica. On the basis of low DNA–DNA hybridizations, these two strains should be considered as new species of genus Natrialba. The names Natrialba hulunbeirensis sp. nov. (type strain X21T l AS 1.1986T l JCM 10989T ) and Natrialba chahannaoensis sp. nov. (type strain C112T l AS 1.1977T l JCM 10990T ) are proposed. Keywords: Natrialba hulunbeirensis, Natrialba chahannaoensis, archaea, haloalkaliphiles INTRODUCTION Smith, 1995; Kamekura et al., 1997; Montalvo- Rodrı!guez et al., 1998; McGenity et al., 1998; Ventosa The family Halobacteriaceae was proposed by et al., 1999; Xu et al., 1999; Wainø et al., 2000). Most Gibbons (1974) to accommodate the rods and cocci halobacteria are neutrophilic, but some of these genera that required high concentrations (more than 12%, include species that are haloalkaliphilic, requiring not w\v) of sodium chloride for growth, and included two only high NaCl concentrations but also high pH and # genera, Halobacterium and Halococcus. The isolation low Mg + concentrations for growth. of haloalkaliphilic and pleomorphic halobacteria necessitated the extension of this scheme to include six The phenotypic characteristics of haloalkaliphilic genera: Halobacterium, Halococcus, Haloarcula, Halo- archaea that permit their differentiation are com- ferax, Natronobacterium and Natronococcus (Grant & paratively very limited, so it is relatively difficult to Larsen, 1989; Torreblanca et al., 1986). Currently, classify them on the basis of their phenotypic features members of the aerobic, extremely halophilic archaea alone. In addition, their phospholipid composition is are classified in 15 genera: Halobacterium, Halococcus, highly conserved and glycolipids are absent from the Haloarcula, Haloferax, Halorubrum, Halobaculum, majority of strains. Thus, chemotaxonomy on the Natrialba, Natronomonas, Natronobacterium, Natro- basis of lipid composition is difficult. Therefore, in nococcus, Halogeometricum, Natrinema, Haloterri- combination with other phenotypic features such as gena, Natronorubrum and Halorhabdus (McGenity & morphology, physiology and biochemistry, the phylo- Grant, 1995; Oren et al., 1995; Kamekura & Dyall- genetic inference of 16S rRNA sequences and DNA– DNA hybridization play important roles in the classi- fication of haloalkaliphilic archaea. In 1997, the ICSB ................................................................................................................................................. Subcommittee on the Taxonomy of Halobacteria Abbreviations: PG, phosphatidylglycerol; PGP-Me, phosphatidylglycero- proposed minimum standards for the classification of phosphate methyl ester; S2-DGD-1, 2,6-HSO3-Manp-α(1 ! 2)-Glcp-α(1 ! 1)- sn-glyceroldiether. halobacterial archaea and suggested that the classi- The GenBank accession numbers for the 16S rDNA sequences of strains X21T fication of halobacteria should be polyphasic and and C112T are respectively AF262026 and AJ004806. consistent with the phylogenetic analysis of 16S rRNA 01718 # 2001 IUMS 1693 Y. Xu and others $# gene sequences (Oren et al., 1997). Recently, great [α- P]dCTP using a nick-translation kit (Boehringer progress has been made in the classification of halo- Mannheim). alkaliphilic archaea: Kamekura et al. (1997) classified 16S rRNA gene sequence and phylogenetic analysis. The some species previously included in the genus Natrono- methods used for DNA preparation, PCR amplification of bacterium into three different genera: Natronomonas, 16S rRNA genes and gene sequencing were described Natrialba and Halorubrum. Additionally, Xu et al. previously (Zhou et al., 1994). (1999) proposed a new genus, Natronorubrum, with Multiple sequence alignments were performed using two new haloalkaliphilic species, Natronorubrum version 1.8 (Thompson et al., 1994). Phylogenetic bangense and Natronorubrum tibetense. analysis of multiple sequence alignments was performed with version 1.3b (Van de Peer & De Wachter, In this study, we describe two novel soda lake isolates, T T 1994). Phylogenetic tree construction was carried out by the X21 and C112 . We have determined the complete neighbour-joining method with Kimura’s two-parameter 16S rDNA sequences of these isolates, as well as their calculation model in version 1.3b. polar lipid compositions. In addition, we have per- formed DNA–DNA hybridization experiments with RESULTS other members of the genus Natrialba in order to determine the taxonomic positions of strains X21T and Morphology T C112 . The two strains X21T and C112T were Gram-negative, rod-shaped and non-motile. Cells were 0n4–0n6i METHODS 1–2n5 µm and lysed in distilled water. On agar plates, both strains formed red, circular and smooth Bacterial strains and culture conditions. Strain X21T was T colonies, 0n5 mm in diameter for strain X21 and isolated by enrichment from a sediment sample (pH 10) T collected from Chahannao soda lake in the Inner Mongolia 2n0 mm in diameter for strain C112 after 1 week at Autonomous Region of China, whereas strain C112T was 37 mC. isolated from a sediment sample (pH 9n5) of an unnamed soda lake in the Hulunbeir prefecture of the Inner Mongolia Physiological characterization Autonomous Region, China. The medium and methods for enrichment and isolation were described previously (Tindall Growth of both strains occurred in the pH range T et al., 1980). In addition, the following strains of the genus 8n5–10n5, with optimum growth at pH 9n0. Strain X21 Natrialba were used in this study: Natrialba magadii NCIMB grew in media containing between 12 and 30% (w\v) T T 2190 and Natrialba asiatica strains JCM 9576 and JCM NaCl, with an optimum at 20% (w\v) NaCl. Strain 9577. These strains were cultivated aerobically at 37 mCas C112T grew in media containing between 10 and 30% described previously (Tindall, 1992). The growth medium −" (w\v) NaCl, with an optimum at 15% (w\v) NaCl. for the following studies contained (l ): 7n5 g Casamino Growth of both strains occurred in the temperature acids (Difco), 10 g yeast extract (Difco), 3n0 g trisodium #+ citrate, 0n3 g MgSO% ; 7H#O, 2n0 g KCl, traces of Fe and range 20–55 mC, with respective optima at 50 and 45 mC #+ T T Mn , 200 g NaCl and 8n0gNa#CO$. for strains X21 and C112 . Phenotypic characterization. Phenotypic tests on strains Both strains were catalase- and oxidase-positive and X21T and C112T were carried out in accordance with the strictly aerobic. Both strains reduced nitrate to nitrite recommended standard methods for halobacteria (Oren et anaerobically and produced H#S from cysteine but not al., 1997). Cell motility and shape were examined by phase- from Na#S#O$. They were sensitive to some antibiotics contrast microscopy without fixation and by Gram staining (such as erythromycin, rifampicin and bacitracin) but with acetic acid fixation. The optimal conditions of growth, insensitive to tetracycline hydrochloride. Both strains reduction of nitrate and utilization of different carbon hydrolysed gelatin and did not hydrolyse Tween 80. sources were determined as described previously (Grant & Strain X21T hydrolysed Tween 40 but did not hy- Tindall, 1980; Mwatha & Grant, 1993). Tests for catalase T and oxidase activities and hydrolysis of starch, gelatin, drolyse starch or casein. Strain C112 hydrolysed casein and Tween 80 were performed as described previously starch and casein but did not hydrolyse Tween 40. (Gonzalez et al., 1978). Antibiotic susceptibility and H#S Both strains utilized some sugars (fructose and malt- production was tested as described previously (Colwell et al., ose) and amino acids (asparagine, arginine and pro- 1979). Growth was monitored by turbidity at 660 nm. line) as sole carbon sources for growth. They were unable to use sucrose, lactose, mannitol, serine, thre- Lipid composition. Total lipids were extracted and analysed T on silica-gel plates (Kieselgel 60 F#&%; Merck) by one- and onine or isoleucine. In addition, strain C112 utilized glucose, acetate and lysine as sole carbon sources for two-dimensional TLC (Ross et al., 1981; Ross, 1982). The T core lipids were analysed by TLC, as described by Ross et al. growth, but strain X21 did not. Acid production by (1981). both strains was observed from fructose and maltose, DNA base composition and DNA–DNA hybridization.