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Natronobacterium Vacuolata Sp. Nov., a Haloalkaliphilic Archaeon Isolated from Lake Magadi, Kenya

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Natronobacterium Vacuolata Sp. Nov., a Haloalkaliphilic Archaeon Isolated from Lake Magadi, Kenya INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, July 1993, p. 401-404 Vol. 43, No. 3 0020-7713/93/030401-04$02.00/0 Copyright 0 1993, International Union of Microbiological Societies Natronobacterium vacuolata sp. nov., a Haloalkaliphilic Archaeon Isolated from Lake Magadi, Kenya WANJIRU E. MWATHA' AND WILLIAM D. GRANT2* Department of Botany, Kenyatta University, Nairobi, Kenya, and Department of Microbiology, University of Leicester, P.O. Box 138, Leicester LEI 9HN, United Kingdom2 A novel haloalkaliphilic archaeon was isolated from Lake Magadi, a Kenyan alkaline soda lake. Cells of the organism contain large gas vacuoles in the stationary phase of growth, and colonies produced by these archaea are bright pink in appearance. The major polar lipids of these organisms are C2,C2, and C20C2sderivatives of phosphatidylglycerol phosphate and phosphatidylglycerol, and the organisms contain an unidentified phospholipid as a minor component. The G+C content of the DNA is 62.7 mol%. The name Natronobucterium vacuofutu sp. nov. is proposed. The type strain is designated NCIMB 13189. The haloalkaliphilic members of the Archaea (15) make up of red or orange typical for natronobacteria and natrono- a distinct physiological group within the halobacterial (ar- cocci. chaeal halophile) lineage (5, 9) in that they are confined to N. pharaonk DSM 2161, N. gregoryi NCMB 2189, and N. soda lakes and are obligately alkaliphilic. These archaea rnagadii NCMB 2190 were available for comparison. They have been isolated from Lake Magadi, Kenya (13); Wadi were maintained and grown in the medium of Tindall et al. Natrun, Egypt (12); Owens Lake, California (7); and Chinese (13)- and Russian soda lakes (16, 17). Like other halobacteria, Light microscopy. Cells were Gram stained by the method these organisms may produce a red coloration in brines of Dussault (2) and observed with a Leitz Orthoplan micro- (in this case, alkaline brines) due to the synthesis of scope. Unstained preparations were observed with phase- C,, carotenoids. Lake Magadi is an exam le of a red soda lake, parts of which harbor lo7 to 10P haloalkaliphiles contrast optics. Biochemical tests. Nitrate reduction was tested by inocu- m1-l. lating strains into liquid medium supplemented with 0.1% The haloalkaliphiles comprise rod-shaped and coccoid KN03 and incubating the strains for 72 h at 37°C. Nitrate isolates that are classified into the genera Natronobacterium reduction was detected by withdrawing 0.5-ml samples and and Natronococcus, respectively. Representatives of the adding naphthylamine and sulfanilic acid reagent as de- genus Natronobacterium are readily distinguishable from scribed by Smibert and Krieg (11). other rod-shaped halobacteria by virtue of their alkaliphily Utilization of different carbon sources was determined and low Mg2+ requirement (3). Natronococci have coccoid with a minimal medium containing 1.0 g of yeast extract per morphologies like Halococcus s p. but are distinguished liter, 1.0 g of KH2P0, per liter, 0.2 g of MgSO, - 7H20 per from them by alkaliphily, low Mg' requirement, and partial liter, 1.0 g of KNO, per liter, 200 g of NaCl per liter, 18 g lysis of cells in distilled water (4). of Na2C03 per liter, and 20 g of agar per liter. Sodium Three species of natronobacteria are currently recognized: chloride and carbonate were sterilized separately and then Natronobacteriumpharaonis, N. magadii, and N. gregoryi mixed with the rest of the ingredients at 60°C just before (14). They are identified on morphological and biochemical the medium was poured into square petri dishes. Filter- grounds and by DNA-DNA homology studies. In addition, sterilized carbohydrates were added to the basal minimal slight differences in polar lipid composition have been shown medium to a final concentration of 0.5% (wtkol) just before to distinguish these species (7). the medium was poured. Plates containing the different Here we report the isolation of a new rod-shaped haloal- carbon sources were inoculated with a multiple inoculator kaliphilic archaeon from Lake Magadi and its assignment as and then incubated at 37°C for 5 days. Growth on different a new species of the genus Natronobacterium. carbon sources was compared with growth on a plate con- taining basal minimum medium only; any enhancement of growth was assumed to be due to carbohydrate utiliza- MATERIALS AND METHODS tion. The pH range for growth was determined after 5 days at 37°C by the pH gradient method of Grant and Tindall(6) on Medium and culture procedures. Strain M24 was isolated square plates containing the agar medium of Tindall et al. by spreading on agar media, on site, dilutions of red pig- (13) but at pH 7, with an Na2C03-NaOH agar layer at one mented brine taken from the side of the main causeway end (6). across Lake Magadi. Dilution medium and agar medium The salt range for growth was determined by estimating were described by Tindall et al. (13). The plates were growth after incubation at 37°C for 5 days on the agar incubated in sealed plastic bags at 37°C for 7 to 10 days. M24 medium of Tindall et al. (13) but with salt concentrations of was selected as producing bright pink colonies, whereas the 0,5,10,15,20,25, and 30% (wtkol). The temperature range other pigmented colonies on this agar medium were shades for growth was determined by estimating growth on the agar medium of Tindall et al. (13) after incubation at 15, 20, 25, 30, 35, 40, 45, 50, and 55°C for 5 days. * Corresponding author. Antibiotic sensitivity. Mastring Antibiotic Assay Discs 401 402 MWATHA AND GRANT INT. J. SYST. BACTERIOL. FIG. 1. Cells of strain M24 after 7 days at 37°C in the medium of Tindall et al. (14). This image was obtained with phase-contrast optics. Bar, 10 p,m. (Mast Laboratories, Bootle, Merseyside, United Kingdom) RESULTS AND DISCUSSION were placed on agar media previously spread with 150 pl of late-exponential-phase cultures of the strain being tested. Isolate M24 was a nonmotile short rod, cells of which were Each disc contained one of the following antibiotics: chlor- 0.5 to 0.7 by 1.5 to 3.0 pm in size in liquid medium when in amphenicol, 25 pg; erythromycin, 5 pg; methicillin, 10 pg; exponential phase. After 3 days at 37"C, as the cells reached novobiocin, 5 pg; penicillin G, 1 U; streptomycin, 10 pg; stationary phase, the cells became coccoid and gas vacuolate tetracycline, 25 pg; ampicillin, 25 pg; trimethoprim, 2.5 pg; with one or more gas vacuoles (Fig. 1).Cells taken from agar sulfamethoxazole, 50 pg; nalidixic acid, 30 pg; fusidic acid, cultures had the appearance shown in Fig. 1. The periphery 10 pg; and gentamicin, 10 pg. Zones of inhibition were of the cells could not be clearly seen because of the intense recorded after incubation at 37°C for 2 to 5 days. refractility of the gas vacuoles. Attempts to flatten cells in The vibriostat reagent 0/129 (2,4-diamino-6,7-diisopropy- order to improve the contrast of the cell periphery were not lpteridine) was applied to the plates in crystalline form, and successful because of the fragility of the cells. the plates were incubated as described above. On agar media, M24 formed small, circular, bright pink, Lipid analyses. Polar lipids and archaeal core lipids were convex colonies with an entire margin (1 to 2 mm) after 6 isolated and analyzed by thin-layer chromatography as de- days at 37°C. Cells in unshaken liquid media in stationary scribed by Ross et al. (10). Quinones were extracted and phase floated at the surface of the media. analyzed by thin-layer chromatography as described by Alkaliphily, G+ C content, quinone composition, and ar- Collins (1). chaeal core lipid composition indicated that M24 was a DNA-DNA homology studies. DNA was extracted, nick member of the genus Natronobacten'um, but biochemical translated, and hybridized as described by Tindall et al. (14). tests together with morphological details distinguished M24 G+C analyses. Cells were harvested in the exponential from the known species of natronobacteria (Table 1). In phase of growth, and DNA was extracted in Tris buffer (pH particular, the isolate was gram variable and utilized a wide 8.2) as outlined by Tindall et al. (14). Purified DNA was range of organic compounds. dissolved in 0.1~standard saline citrate buffer (8). The Examination of the polar lipid composition showed that moles percent G+C was determined by the thermal denatur- M24 had, in common with other natronobacteria, a charac- ation method (T,) with a Philips PU 8720 spectrophotometer teristic, comparatively simple pattern (14). All natronobac- fitted with a temperature program accessory. The formula teria characterized to date have C2,C2, and C2,C2, diether moles percent G+C = 2.O8Tm - 106.40 was used to convert derivatives of phosphatidylglycerol phosphate and phos- melting point to moles percent (8). Escherichia coli LE392 phatidylglycerol together with minor amounts of unidentified DNA was included as the standard DNA. phospholipids. Morth and Tindall(7) have shown differences VOL. 43, 1993 NATRONOBACTERIUM VACUOLATA SP. NOV. 403 TABLE 1. Characteristics of natronobacteria isolated from Lake Magadi Characteristic ~ M24 N. pharaonis N. gregoyi N. magadii Gram stain V" - - - pH range 8.5-10.5 8.0-11.0 8.5-11.0 8.0-11.5 Salt concn range (%) 15-30 12-30 12-30 12-30 Quinone(s) MK8 MK8, MK8(H2)b MK8, MK8(H2)6 MK8, MK8(H2)b Mol% G+C 62.7 64.3 65.0 63.0 Motility - + - Nitrate reduction + + Gelatin liquefaction - + Presence of gas vacuoles + - Sensitivity to erythromycin -c - + + Stimulation of growth by: Glucose + + Fructose - + Fumarate + + Succinate + + Citrate + + Acetate + + Sucrose + - Galactose + + Mannitol - + Pyruvate - + Proline + - Lysine + * V, gram variable.
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