INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1997, p. 853-857 Copyright 0 1997, International Union of Microbiological Societies 0020-7713/97/$04.00+ 0 Vol. 47, No. 3 Diversity of Alkaliphilic Halobacteria: Proposals for Transfer of Na tronobacterium vacuola tum , Na tronoba cterium magadii, and Natronobacterium pharaonis to Halorubrum, Natrialba, and Natronomonas gen. nov., Respectively, as Halorubrum vacuolatum comb. nov., Natrialba magadii comb. nov., and Natronomonas pharaonis comb. nov., Respectively MASAHIRO KAMEKURA,l* MICHAEL L. DYALL-SMITH,2 VIVEK UPASANI,3 ANTONIO VENTOSA,4 AND MORRIS KATES' Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken 278, Japan '; Department of Microbiology, University of Melbourne, Parkville 3052, Australia2; M. G. Science Institute, Navrangpura, Ahmedabad 380 009, Gujarat, India3; Department of Microbiology and Parasitology, University of Sevilla, 41 012 Seville, Spain4; and Department of Biochemistry, University of Ottawa, Ottawa, Ontario KIN 6N5, Canada' The 16s rRNA genes of three species of the genus Natronobacterium (Natronobacteriumgregoryi, Natronobac- terium pharaonis, and Natronobacterium vacuolatum) were sequenced and compared to that of the previously sequenced species Natronobacterium magadii. The sequences revealed that Natronobacterium pharaonis was phylogenetically distinct from the other members of the genus and also from other recognized genera of the family Halobacteriaceae. However, Natronobacterium vacuolatum and Natronobacterium magadii were found to be most closely related to the genera Halorubrum and Natrialba, respectively. An unidentified haloalkaliphile, strain SSL1, was also closely related to Natronobacterium magadii and Natrialba asiatica. On the basis of phylogenetic tree reconstructions, signature bases specific for individual genera, and sequences of spacer regions between 16 and 23s rRNA genes, we propose the following changes: Natronobacterium pharaonis to be transferred to Natronomonas gen. nov. as Natronomonas pharaonis gen. nov., comb. nov.; Natronobacterium vacuolatum to be transferred to the genus Halorubrum as Halorubrum vacuolatum comb. nov.; and Natronobac- terium magadii to be transferred to the genus Natrialba as Natrialba magadii. The extremely halophilic, aerobic Archaea are classified within the family Halobacteriaceae, which currently contains nine valid genera: Halobacterium, Halococcus, Haloarcula , Haloferax, Halorubrum, Halobaculum , Natrialba , Natronobac- terium, and Natronococcus (3, 5, 7, 13, 16, 17). The alkaliphilic members of the Halobacteriaceae form a distinct physiological group as they require not only high NaCl concentrations but also high pH (between 8.5 and 11) and low Mg2+ concentrations (less than 10 mM) for growth. They have been isolated from a variety of alkaline, hypersaline lakes and soils (3, 15, 18, 20-22). Microscopically, the initial isolates consisted of rods and cocci and were accordingly separated into two genera, Natronobacterium and Natronococcus, by Tin- dall et al. (21). Natronobacterium currently contains four rec- ognized species, Natronobacterium gregoryi, Natronobacterium magadii, Natronobacterium pharaonis, and Natronobacterium vacuolatum, with Natronobacterium gregoryi as the type species. The present classification of these isolates is based largely on chemotaxonomic criteria, particularly phenotypic properties and lipid composition. Glycolipid analysis, which has been of great use in the taxonomy of the neutrophilic halobacteria, has had little impact in the classification of the natronobacteria, as these lack major amounts of glycolipids in their membranes (3, 15). The complete sequence of the 16s rRNA gene of only one species, Natronobacterium magadii, has been reported, and * Corresponding author. Mailing address: Noda Institute for Scien- tific Research, 399 Noda, Noda-shi, Chiba-ken 278, Japan. Phone: 81-47 1-23-5573. Fax: 81-47 1-23-5 959. E-mail: mkamekur @ supernig .nig.ac.jp. comparison to other halobacteria has clearly shown that it represents a distinct genus (7, 12). However, the sequences of the other three species of Natronobacterium (Natronobacterium gregoryi, Natronobacterium pharaonis, and Natronobacterium vacuolatum) have not been published, so phylogenetic support for their inclusion in this genus is lacking. DNA-DNA hybrid- ization of the four species indicated that they have very little sequence homology (31 to 38%) (15), and while this is good evidence for their classification as separate species, it does not preclude their representing different genera. The aim of this study was to determine the phylogenetic relationships among the natronobacteria and in particular to ascertain whether the four members of the genus Natronobac- terium were indeed close relatives. For this purpose, 16s rRNA sequences of the three unsequenced species of Natronobacte- rium and of one unclassified haloalkaliphilic strain, SSL1, were determined, as well as sequences from four other strains of halobacteria. These sequences were incorporated into phylo- genetic tree reconstructions of the family Halobacteriaceae. MATERIALS AND METHODS Bacterial strains and culture conditions. The following strains of the genus Natronobacterium were used in this study: Natronobacteriurn gregolyi NCIMB 2189T, Natronobacterium pharaonis JCM 8858T (derived from DSM 2160T via I F 0 14720T), Natronobacterium vacuolatum JCM 9O6OT (deposited by W. D. Grant), and strain SSLl (microscopically long rods, originally isolated from an alkaline saline brine from Sambhar Salt Lake, India, and deposited with the American Type Culture Collection as Natronobacterium sp. strain ATCC 43988) (22, 23). These strains were grown at 37°C in haloalkaliphile medium as previ- ously described (20, 23). Halobacterium trapanicum NCIMB 767 was obtained from the National Col- lection of Industrial and Marine Bacteria in 1994 and deposited as JCM 8979. Three species of the genus Halococcus (Halococcus saccharolyticus ATCC 49257, 853