INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1983, p. 381-386 Vol. 33, No. 2 0020-7713/83/020381-06$02.00/0 Copyright 0 1983, International Union of Microbiological Societies Halobacterium sodomense sp. nov. a Dead Sea Halobacterium with an Extremely High Magnesium Requirement AHARONOREN Department of Microbiology, University of Illinois, Urbana, Illinois 61801 A strain of Halobacterium was isolated from the Dead Sea. This isolate differs from the previously isolated halobacteria in (i) its requirement for sodium ions, which is lower than that of most other halobacteria (20.5 M), (ii) its requirement for divalent cations (Mg2+ or Ca2+),which is higher than that of most other halobacteria (optimal growth was obtained in the presence of 0.6 to 1.2 M Mg2+), (iii) its requirement for either starch or clay minerals (bentonite) for growth in the standard growth medium used, and (iv) its synthesis of purple membrane at low oxygen tensions in the light. This organism has been designated Halobacterium sodomense sp. nov. The type strain is strain ATCC 33755. The genus Halobacterium includes red-col- 1980 from the Dead Sea, about 8 km east of Ein Gedi ored, rod-shaped bacteria that require NaCl (station 5 [ll). This strain was used in all experiments. concentrations of 3 M or more for growth (8) and Isolation and culture methods. I used the observation possess special biochemical characteristics, in- of Kritzman (G. Kritzman, M.Sc. thesis, The Hebrew University of Jerusalem, Jerusalem, Israel, 1973; cluding cellular lipids with ether bonds, absence G. Kritzman, P. Keller, and Y. Henis, Abstr. 1st Int. of peptidoglycan, glycoproteins in the cell wall, Cong. Bacteriol., vol2, p. 242,1973) that the inclusion and characteristic base sequences in the ribo- of starch in the medium permitted efficient enumera- somal ribonucleic acid. These properties place tion of Dead Sea halobacteria. Organisms were isolat- the halobacteria in the archaebacteria (18). Hal- ed in medium containing 1 g of peptone (Difco Labora- obacteria are found in many hypersaline bodies tories), 1 g of yeast extract (Difco), 20 g of potato of water, such as salterns, the Great Salt Lake in starch (Baker and Adamson), 800 ml of Dead Sea Utah, and the Dead Sea. water, and 200 ml of distilled water (pH 6.5 to 7.0). Since the pioneering work of Volcani more The isolation plates consisted of two layers; the lower 40 layer contained 2% (wt/vol) agar (Difco), and the than years ago (29), it has been known that upper layer contained 0.5 ml of a water sample or a the Dead Sea is inhabited by red halobacteria, dilution of a sample in autoclaved Dead Sea water colorless bacteria, and unicellular algae. Phase- mixed with 4.5 ml of molten isolation medium contain- contrast microscopy of Dead Sea water samples ing 0.8% agar. After solidification of the upper layer, has shown that the dominant bacteria are cup- or the plates were incubated at 37°C. Colonies were disk-shaped pleomorphic organisms. A minor visible after 2 to 3 weeks. Pure cultures were obtained part of the population consists of long, rod- by repeated streaking onto a 2% agar medium of the shaped bacteria (16, 22). The pleomorphic cup- same composition. The plates were incubated for 2 to shaped, red halobacteria include Halobacterium 3 weeks before the colonies reached a convenient size for transfer. volcanii, which was isolated by Mullakhanbhai Pure cultures were grown in a medium containing and Larsen (21), a strain resembling the lost 125 g of NaCI, 160 g of MgCl2.6H2O, 0.13 g of isolate of “Halobacterium marismortui” of Vol- CaC12 - 2H20, 5.0 g of K2S04,1 g of peptone (Difco), 1 cani (29, 30), which was isolated by Ginzburg et g of yeast extract (Difco), 2 g of soluble starch (BDH), al. (lo), and additional strains isolated by me and distilled water to a final volume of 1 liter; the pH (22). All of these strains have a relatively low of this medium was adjusted to 7.0 with NaOH before requirement for sodium ions and a high magne- autoclaving. Cells were grown in 100-ml Erlenmeyer sium tolerance, making them well adapted for flasks containing 50 ml of medium or 50-ml flasks life in the Dead Sea. Isolation of long, rod- containing 25 ml of medium in a shaking water bath at 35°C. For experiments to determine purple membrane shaped bacteria has not been reported. formation, 150-ml portions were grown in 250-ml Er- I succeeded in isolating a rod-shaped, red lenmeyer flasks in an orbital shaker (Psycrotherm; halobacterium from the Dead Sea by using me- New Brunswick Scientific Co.) illuminated by white dia containing starch. This organism appears to fluorescent light at an intensity of 3 x lo3 ergs/cm2 per be distinct from previously described species. s. To determine growth requirements, the concentra- tions of NaCl, MgCI2 6H20, CaClz 2H20, starch, MATERIALS AND METHODS - - bentonite (Evans), and kaolin (Merck) in the growth Source of organism. Strain RD-26T (type strain) was medium were modified. Growth was measured by isolated from a surface water sample collected in May determining the optical densities of the cultures at 600 381 382 OREN INT. J. SYST.BACTERIOL. FIG. 1. (A) Halobacterium strain RD-26T cells grown in standard liquid medium. Phase-contrast microscopy. Bar = 10 pm. (B) Electron micrograph of a cell grown in standard liquid growth medium. Negatively stained preparation. Bar = 1 pm. nm with a model 300-N spectrophotometer (Gilford Biochemical tests. Gram staining was performed Instrument Laboratories, Inc.), using uninoculated after smears were fixed on slides with acetic acid by growth medium as a blank. Pure cultures were main- the method of Dussault (5). The cellular lipids were tained on slants of growth medium supplemented with partially characterized by the method of Ross et al. 1.5% agar. (24) and were compared with the cellular lipids of Electron microscopy. Bacteria were negatively Escherichia coli, H. volcanii (21), and the Halobacter- stained with 1% aqueous uranyl acetate by washing 1 ium of the Dead Sea described by Ginzburg (7, 9). drop of bacterial suspension with stain. Micrographs Acid production from carbohydrates was deter- were obtained with a Philips model EM-400 electron mined by measuring the pH in starch-free growth microscope operated at 80 kV. medium containing bentonite (0.02%) and filter-steril- Carotenoid extraction. Carotenoids were extracted ized carbohydrates at final concentrations of 1.O%. in methanol-acetone (l:l, vol/vol) (ll),and the absorp- The susceptibility of the organism to antibiotics and tion spectra of these compounds were determined with vibriostatic agent 0029 was tested in growth medium a model 402 spectrophotometer (Perkin-Elmer Corp.), for 4 to 5 days after inoculation. The inhibitors tested using the solvent as a blank. were penicillin G (25 U/ml), kanamycin (30 kg/ml), Purple membrane. The purple membrane assay was novobiocin (10 kg/ml), chloramphenicol succinate (30 performed as described previously (23). pg/ml), streptomycin sulfate (30 pg/ml), bacitracin (5 VOL.33, 1983 HALOBACTERIUM SODOMENSE SP. NOV. 383 it was grown in standard growth medium (Fig. la). When this organism was grown at subopti- mal magnesium concentrations (see below), the cells were shorter, and in extreme cases pleo- morphic rods and spheres were observed. The cells were motile by means of a tuft of polar flagella (Fig. 1b), like Halobacterium halobium (14). Gas vacuoles, which are sometimes found in halobacteria, particularly in fresh isolates (14, 25), were never observed in any of the strains isolated. Upon gradual dilution of a liquid cul- ture with water, the cells were transformed to spherical structures, which lysed when the cul- ture was diluted further. At 35°C optimum growth occurred at an NaCI concentration of about 2 M (in the presence of 0.6 to 1.2 M MgC12); minimal doubling times were 12 h. At an NaCl concentration of 4.3 M growth rates were relatively low. The specific requirement for sodium was exceptionally low, and fair growth was obtained at sodium concen- trations as low as 0.5 M. Increased magnesium FIG. 2. Growth of Halobacterium strain RD-26= concentrations were then required to provide compared with growth of H. volcanii, H. halobium, the necessary osmotic pressure in the medium. and H. salinarium, plotted onto the environmental Magnesium concentrations as high as 0.6 to 1.2 space by the method of Edgerton and Brimblecombe M were required for optimal growth; lower (6). The x-axis (X’) represents the mole fraction of monovalent cations (mNa+ + mK+)/(mNa+ + mK+ + concentrations caused lowered growth rates and mMg2+ + mCa2+),and the y-axis represents the total a change in cell morphology to pleomorphic rods charge concentration (mNa2++ mKf + 2mMgZf)(m, and spheres. Higher sodium concentrations did molal concentration). The values for the optimal not depress the requirement for magnesium. growth conditions for H. volcanii, H. halobiurn, and Calcium at least partly replaced magnesium, and H. salinarium were taken from Edgerton and Brimble- high growth rates and rod-shaped cells were combe (6). obtained in medium containing 1 M CaC12, 15 mM MgCI2, and 2.1 M NaCl. Figure 2 compares the growth requirements of Halobacterium strain RD-26T with respect to mono- and diva- and 20 U/ml), and vibriostatic agent 01129 (2,4-diam- lent cations with the growth requirements of the ino-6,7-diisopropylpteridine phosphate; BDH) (10 and Dead Sea isolate H. volcanii (21) and with the 50 p,g/ml). In addition, I tested susceptibility to penicil- growth requirements of H. halobium and Halo- lin, chloramphenicol, erythromycin, kanamycin, neo- bacterium salinarium.