International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2271–2280 DOI: 10.1099/ijs.0.02324-0 Characterization of a novel halophilic archaeon, Halobiforma haloterrestris gen. nov., sp. nov., and transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb. nov. 1 Institut fu$ r Mikrobiologie F. F. Hezayen,1 B. J. Tindall,2 A. Steinbu$ chel1 and B. H. A. Rehm1 der Westfa$ lischen Wilhelms-Universita$ t Mu$ nster, Corrensstr. 3, Author for correspondence: Bernd Rehm. Tel: 49 251 8339848. Fax: 49 251 8338388. D-48149 Mu$ nster, j j Germany e-mail: rehm!uni-muenster.de 2 DSMZ–Deutsche Sammlung fu$ r Mikroorganismen und Strain 135T, a novel red-pigmented, aerobic, extremely halophilic member of Zellkulturen GmbH, the Archaea showing rod, coccus and slightly pleomorphic morphology, was Mascheroder Weg 1b, D-38124 Braunschweig, isolated from hypersaline soil close to Aswan (Egypt). This organism is Germany neutrophilic, motile and requires at least 22 M NaCl, but no MgCl2, for growth and exhibits optimal growth at 42 mC. Polar lipid analysis revealed the presence of sulfated triglycosyl diether and triglycosyl diether as the sole glycolipids as well as the absence of the glycerol diether analogue of phosphatidyl glycerosulfate. C20:C20 and C20:C25 core lipids are present in almost equal proportions. The GMC content of the DNA is 669 mol%. 16S rDNA analysis revealed that strain 135T was a member of the phyletic group defined by the family Halobacteriaceae, but there was a low degree of similarity to other members of this family. Highest similarity values of 964 and 938–943% were obtained to the 16S rDNA of Natronobacterium nitratireducens and Natronobacterium gregoryi, Natronococcus occultus and Natronococcus amylolyticus. Strain 135T is able to accumulate polyhydroxybutyrate as intracellular reserve material. On the basis of the data presented, strain 135T should be placed in a new genus, Halobiforma gen. nov. as Halobiforma haloterrestris sp. nov. The type strain is strain 135T (l DSM 13078T l JCM 11627T). Moreover, the transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb. nov. is proposed. Keywords: Halobiforma haloterrestris, nitratireducens, halophilic, archaea, polyhydroxybutyrate INTRODUCTION Halobacteriaceae are red-pigmented due to the pres- ence of carotenoids (Kamekura & Dyall-Smith, 1995; The aerobic, extremely halophilic archaea that belong Hezayen et al., 2001; Grant et al., 2001). Before the to the family Halobacteriaceae, order Halobacteriales, 1970s, halobacterial taxonomy was based mainly on class Haloarchaea, are chemo-organotrophic or- standard biochemical tests and morphology (Gibbons, ganisms that use amino acids or carbohydrates to grow 1974). The current taxonomy is based largely on 16S and need at least 1n5 M NaCl for growth; most exhibit rDNA sequences and chemotaxonomic criteria, par- optimal growth at 3n5–4n5 M NaCl (Grant et al., 2001). ticularly polar lipid composition, which has induced a Except for some members of the genus Natrialba, further reclassification of some uncertain halophilic which are non-pigmented, all members of the family species and the creation of new genera (Grant & ................................................................................................................................................. Larsen, 1989; Ross & Grant, 1985; Kamekura & Abbreviations: PHB, poly-β-hydroxybutyrate; VPMR, Voges–Proskauer Dyall-Smith, 1995; Kamekura et al., 1997). The methyl red. availability of complete 16S rDNA sequences of many The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of haloarchaea has resulted in the recognition of even strain 135T is AF333760. more diversity at the genus level within the family 02324 # 2002 IUMS Printed in Great Britain 2271 F. F. Hezayen and others Halobacteriaceae (Kamekura & Dyall-Smith, 1995; previously (Hezayen et al., 2001). Unless indicated other- McGenity & Grant, 1995). This has resulted in a wise, the pH was adjusted to 7n5 with 1 M NaOH and media dramatic increase in the number of these halophilic were sterilized by autoclaving. Agar plates were prepared by taxa. Fifteen genera of the family Halobacteriaceae adding 2% (w\v) agar before autoclaving. havebeendescribed:Halobacterium,Haloarcula,Halo- Light microscopic examination. Gram staining was per- ferax, Halococcus, Halorubrum, Halogeometricum, formed by using acetic-acid-fixed samples as described by Haloterrigena, Halobaculum, Halorhabdus, Natrialba, Dussault (1955). Wet mounts were prepared to detect Natrinema, Natronobacterium, Natronococcus, Natro- motility, cell shape and cell dimensions from various growth norubrum and Natronomonas (Tindall et al., 1984; stages from both broth and solid S–G medium at different NaCl concentrations and were examined with an Ortholux Torreblanca et al., 1986; Kamekura & Dyall-Smith, II microscope (Leitz). 1995; McGenity & Grant, 1995; Oren et al., 1995; Kamekura et al., 1997; McGenity et al., 1998; Physiological and biochemical characterization. The growth Montalvo-Rodrı!guez et al., 1998; Xu et al., 1999; response to NaCl was examined in liquid and solid S–G medium by using serial NaCl concentrations ranging from " Ventosa et al., 1999; Wainø et al., 2000; Grant et al., 50 to 320 g l− and to pH by testing growth at pH 5–10. The 2001). However, differences in the evolutionary rates growth response to temperature was examined by testing in various groups of organisms prevent the use of growth in liquid and in S–G agar at up to 60 C. The # m phylogenetic parameters alone in delineating taxa; requirement for Mg + for growth was tested qualitatively by consequently, the use of 16S rDNA sequences alone to growing the strain in S–G liquid medium with and without describe taxa is not enough and the combination of MgSO% ; 7H#O. Growth was monitored in liquid culti- genotypic and phenotypic characteristics (polyphasic vations by Klett photometer. Catalase and oxidase activities taxonomy) constitutes a practical solution to the were tested according to Gerhardt et al. (1994). Hydrolysis delineation of taxa (Oren et al., 1997). of starch, gelatin, casein and lipids, esterase and urease activities, nitrate reduction, indole formation from tryptone, In this paper, 1457 bp of the 16S rDNA sequence from hydrogen sulfide production, citrate utilization and Voges– the halophilic organism strain 135T is reported as well Proskauer and methyl red (VPMR) reactions were per- as polar lipid analysis and physiological and bio- formed according to Gerhardt et al. (1994) as mentioned chemical characteristics. Based on the data presented, previously (Hezayen et al., 2001). Production of acid from we found that strain 135T is not identical to any of the sugars was tested as described previously (Hezayen et al., 2001). Utilization of n-butyric acid, xylose, arabinose, present species and is sufficiently different from them fructose, glucose, galactose, sucrose, maltose, lactose, to justify classification as a novel species within a new starch, mannitol, sorbitol, citrate, gluconate, acetate, py- genus. In addition, our findings also support the ruvate, glycerine, bacto-peptone, gelatin, nutrient broth, removal of Natronobacterium nitratireducens (Xin et glycine, proteose peptone, Casamino acids and tryptone as al., 2001) from the genus Natronobacterium and its sole nitrogen and\or carbon and energy sources was placement in the same genus as the novel isolate, as performed in the salt-minerals medium supplemented with Halobiforma nitratireducens comb. nov. 1% (w\v) substrate. In the case of non-nitrogenous com- pounds, 0n2%(w\v) NH%Cl was supplied as the sole nitrogen source and the test was also performed in the basal medium. METHODS In the case of sugars, the test medium was buffered with T T T 20 mM PIPES. Growth was monitored with a Klett pho- Archaeal strain. Strain 135 (l DSM 13078 l JCM 11627 ) tometer. was isolated from a soil sample that was collected from the surface of a hypersaline soil (Aswan, Egypt), using S–G Antibiotic susceptibility. The susceptibility of the strain to " medium [containing (l− ): 250 g NaCl, 2 g KCl, 20 g antibiotics was determined as described previously (Hezayen MgSO% ; 7H#O, 10 g Casamino acids, 7n5 g yeast extract, 3 g et al., 2001). The following antibiotics were tested (µg per sodium citrate; Sehgal & Gibbons, 1960]. Soil particles were disc unless indicated otherwise): penicillin G (5), chloram- spread on the surface of S–G agar plates and were then phenicol (10), tetracycline (30), streptomycin (25), nitro- incubated in sealed plastic bags at 40 mC. After 2 weeks furantoin (300), bacitracin (10 U), novobiocin (5), fusidic incubation, several pigmented colonies appeared on the agar acid (10), anisomycin (20), trimethoprim (2n5), rifampicin plates. A representative red colony for strain 135T was (30), nystatin (100 U), nalidixic acid (30) and erythromycin transferred to S–G broth medium and was grown at 40 mC. (15). To ensure the selection of a pure colony, serial dilutions were Carotenoid extraction. Carotenoids were extracted with made from liquid growth and spread on agar plates. After 2 methanol\acetone (1:1, v\v) as described by Gochnauer et weeks incubation at 40 mC, a single colony was picked and al. (1972) from cells that were grown on agar plates and in was used for further study. S–G broth. Carotenoids were also tested in the water lysate Media and growth conditions. The isolation medium S–G from cells grown in S–G broth and the absorption spectrum was also