- Email: [email protected]
A procedure for the enrichment and isolation of Halobacterium
FEMS Microbiology Letters 173 (1999) 353^358
A procedure for the enrichment and isolation of Halobacterium Aharon Oren a; *, Carol D. Litch¢eld a
b
Division of Microbial and Molecular Ecology, The Institute of Life Sciences, and The Moshe Shilo Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel b Department of Biology, George Mason University, Fairfax, VA 22030-4444, USA Received 15 December 1998; received in revised form 15 February 1999; accepted 17 February 1999
Abstract A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of L-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Halobacterium; Enrichment; Anaerobic growth ; Arginine
1. Introduction Among the genera within the family Halobacteriaceae the genus Halobacterium is the best known. Halobacterium salinarum (previously designated Halobacterium salinarium and Pseudomonas salinaria) was the ¢rst halophilic Archaeon described [1], and thus the genus Halobacterium became the type genus of the family [2,3]. Halobacterium is also the most widely studied genus within the Halobacteriaceae, especially after the discovery of the unique properties of the light-driven proton pump bacteriorhodopsin in the purple membrane of Halobacterium halobium [4]. * Corresponding author. Tel.: +972 (2) 6584951; Fax: +972 (2) 6528008; E-mail: [email protected]
Three Halobacterium species were described in the past (not taking into account those that were later transferred to other genera) [3]: Halobacterium salinarium, Halobacterium halobium, and Halobacterium cutirubrum. They were isolated from such environments as salted cow hide and cured cod¢sh. A comparative study of a large number of isolates showed them all to be very similar in their properties [5], a conclusion supported by later phylogenetic analyses. Presently only a single species of Halobacterium is recognized: Halobacterium salinarum [6]. Analysis of natural communities of halophilic Archaea such as that occur in hypersaline lakes and saltern ponds shows that Halobacterium is not the dominant genus in such environments and is outnumbered by members of other genera, some of which may still await isolation. This was shown
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both by characterization of strains isolated as cultures on agar plates [7], by analysis of the polar lipids extracted from the communities [8,9], and by sequencing of the 16S rDNA genes directly ampli¢ed from such environments [10]. Halobacterium cells are generally not detected. Enrichment cultures also rarely yield Halobacterium strains, as Halobacterium is in most cases outgrown by other halophilic Archaea with simpler nutritional requirements and higher growth rates. It was ¢rst reported in 1980 that Halobacterium halobium can grow anaerobically by fermentation of L-arginine to ornithine, CO2 and NH3 [11,12]. In-depth enzymatic studies and molecular analyses of the genes involved in the fermentation pathway [13,14] have greatly increased our understanding of the process. In the present study we tested a large number of species of halophilic Archaea, belonging to di¡erent genera, for their ability to grow fermentatively on arginine, and we examined the possibility of using the property of arginine fermentation to design a speci¢c enrichment procedure for Halobacterium strains inhabiting hypersaline environments.
2. Materials and methods 2.1. Culture conditions and archaeal strains used Halophilic archaeal strains were grown aerobically in 100 ml portions in 250 ml Erlenmeyer £asks in a rotatory shaker at 35 or 38³C. The following media were employed (concentrations in g l31 ): A: NaCl, 250; KCl, 5; MgCl2 W6H2 O, 5; NH4 Cl, 5; and yeast extract, 10. B: NaCl, 200; KCl, 1; MgSO4 W7H2 O, 20; FeSO4 W7H2 O, 0.02; Tri-Na-citrate, 3; casamino acids, 7.5; and yeast extract, 0.5. C: NaCl, 175; MgCl2 W6H2 O, 20; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; and yeast extract, 5. D: NaCl, 206; MgSO4 W7H2 O, 36; KCl, 0.37; CaCl2 W2H2 O, 0.5; MnCl2 , 0.013; and yeast extract, 5. E (a medium for high magnesium requiring isolates from the Dead Sea): NaCl, 125; MgCl2 W6H2 O, 160; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; yeast extract, 1; casamino acids, 1; and starch, 2. All media were adjusted to pH 7.0 with NaOH. The following archaeal strains were used (the letters in brackets indicating the growth media em-
ployed): Halobacterium salinarum NRC 817 (A, B), Halobacterium cutirubrum (salinarum) NRC 34001 (A, B), Halobacterium salinarum strain 5 [15] (A, B), Halobacterium halobium (salinarum) R1 (A, B), Halorubrum saccharovorum ATCC 29252T (B, C), Halorubrum sodomense ATCC 33755T (E), Halobaculum gomorrense DSM 9297T (E), Haloferax volcanii ATCC 29605T (C), Haloferax mediterranei ATCC 35300T (C), Haloferax gibbonsii ATCC 33959T (C), Haloferax denitri¢cans ATCC 35960T (C), Haloarcula marismortui ATCC 43049T (D), Haloarcula vallismortis ATCC 29715T (D), Haloarcula quadrata DSM 11927 (strain 801030/1, a square motile from a brine pool in Sinai) [16,17] (D), `Haloarcula californiae' ATCC 33799 (D), `Haloarcula sinaiiensis' ATCC 33800 (D), Halogeometricum borinquense ATCC 700274T (B), and Natrialba asiatica JCM 9576T (B). To test for anaerobic growth on arginine, 15 ml screw-capped glass test tubes were ¢lled to the top with the appropriate growth medium, supplemented with 5 g l31 L-arginineWHCl, inoculated with 0.15 ml of aerobic, mid-exponential growth phase culture, and closed. After 5^7 days incubation in the dark the turbidity of the cultures was compared to tubes without added arginine. Strains were considered positive for arginine fermentation when the turbidity (OD600 ) of the arginine containing culture exceeded 0.05, and was at least twice that of the control without arginine. 2.2. Field samples and enrichment cultures Brine samples were collected from saltern crystallizer ponds of the Israel Salt Company, Eilat (May 1998 and November 1988, pond no. 302) [8] and the Cargill Solar Salt Plant, Newark, CA (July 1998, ponds no. 10A and 11) [18]. Anaerobic enrichment cultures for arginine fermenters were set up by inoculating 10 ml portions of brine in 150 ml stoppered glass bottles completely ¢lled with the growth media described above with the addition of 5 g l31 arginine, and incubating the bottles in the dark at 35^38³C. Additional media used for enrichment contained brine from the respective sampling sites, from which most of the bacteria were removed by centrifugation (15 min, 6000Ug), amended with 2.5 g l31 yeast extract or casamino acids and 5 g l31 arginine (media F and G, respectively). Similar media were prepared in
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Fig. 1. Composite picture of representative results of thin layer chromatography analyses of polar lipids extracted from enrichments for halophilic Archaea in the presence of L-arginine, using crystallizer brines from the Cargill Solar Salt Plant (lanes 1^10) and the Eilat Salt Company, Eilat (lanes 11^15) as inoculum, under anaerobic (lanes 1^5 and 11^15) and aerobic conditions (lanes 6^10). Lanes 16, 17, and 18 show polar lipid chromatography patterns obtained after extraction of the biomass collected from the crystallizer ponds of the Cargill (July 1998) and Eilat ponds (May 1998 and November 1998, respectively) without prior enrichment. Media used in the enrichments were A (lanes 1, 6, 11), B (lane 12), C (lane 2), D (lane 13), E (lane 14), F (lanes 3 and 7), G (lane 8), H (lanes 4 and 9), and I (lanes 5, 10, and 15). Media used in the enrichments shown in lanes 11, 12, and 13 contained in addition 25 Wg ml31 penicillin G. Polar lipid patterns of reference strains Halobacterium salinarum (lane 19), Haloarcula vallismortis (lane 20), Haloferax mediterranei (lane 21), Halorubrum sodomense (lane 22), and Natrialba asiatica (lane 23) are also shown. Glycolipids are shown in black. The tentative identi¢cation of the lipid spots, based on literature data [18,21^24], is indicated.
which the brines cleared by centrifugation were diluted with sterile distilled water to 80% of their original salinity (media H and I). Aerobic control experiments were set up by incubating identical enrichments (100 ml portions), with or without arginine, in 250 ml Erlenmeyer £asks with shaking. 2.3. Lipid analyses Bacteria were collected from enrichment cultures, reference cultures of halophilic Archaea and brine samples by centrifugation (15 min, 12 000Ug). Cell pellets were suspended in 1 ml H2 O, and extracted with 3.75 ml methanol-chloroform 2:1 (by volume) for 4 h. The extract was collected by centrifugation, and the pellet reextracted with 4.75 ml methanolchloroform-water (2:1:0.8). Chloroform and water (2.5 ml each) were added to the combined supernatants to achieve phase separation, and after centrifugation the chloroform phase was collected and dried. Lipids were redissolved in a small volume of chloroform, applied to silica gel plates (Sigma-Aldrich Z12,271-8 or Whatman LK6D, 20U20 cm), and sep-
arated by single development with chloroform methanol-acetic acid-water (85:22.5:10:4, vol/vol) [19]. Lipid spots on the plates were detected by spraying with the following reagents: (1) 0.5% K-naphthol in 50% methanol, followed by 5% H2 SO4 in ethanol, and heating of the plates at 150³C (allowing particularly detection of glycolipids); (2) 0.2% CeSO4 in 1 N H2 SO4 , followed by heating at 150³C; (3) orcinol spray reagent (Sigma) followed by heating at 100³C (both general lipid stains, allowing di¡erentiation of glycolipids from other lipids by color); and (4) ammonium molybdate reagent for the detection of phospholipids.
3. Results and discussion Of all 18 strains tested, belonging to seven genera, only the four Halobacterium strains (now all classi¢ed within the species Halobacterium salinarum) showed anaerobic growth in the presence of L-arginine. Other H. salinarum strains also were reported to grow fermentatively on L-arginine, including the
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type strain [14], strain L33 [14], and strain R1 M1 [11]. Anaerobic growth on arginine, a test included in the proposed minimal standards for description of new taxa in the order Halobacteriales [20], is thus not widespread among the neutrophilic halophilic Archaea, and appears to occur only in isolates belonging to the genus Halobacterium. Little is known about the ecological importance of arginine fermentation for Halobacterium populations in nature. It is feasible that when colonizing proteinrich environments such as salted ¢sh and hides halobacteria may reach high cell densities, leading to depletion of oxygen (enhanced by the low solubility of oxygen in concentrated salt solutions), and that their proteolytic enzymes may then maintain a supply of arginine as energy source. Therefore it is interesting to note that, in contrast to most other halophilic Archaea in culture that were isolated from hypersaline aquatic habitats, the reference strains of Halobacterium all came from protein-rich environments (salted ¢sh and hides). As of all the neutrophilic halophilic Archaea tested only members of the genus Halobacterium showed anaerobic growth on arginine, we examined whether this property may be used for the selective enrichment of Halobacterium strains from hypersaline environments. As inoculum we used crystallizer brines collected from salterns at two geographically remote locations: the Red Sea coast of Israel and San Francisco Bay, California. Polar lipid analysis of the archaeal communities inhabiting these brines showed no sign of a massive presence of Halobacterium cells: the dominant glycolipid in both locations was chromatographically identical to S-DGD-1, the sole or main glycolipid found in the genera Haloferax, Halococcus, and Halobaculum. In addition, the phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidylglycerophosphate (MePGP), and phosphatidylglycerosulfate (PGS) were detected (Fig. 1, lanes 16^18). This lipid pattern is similar to that described earlier for the Eilat crystallizer ponds [8]. A lipid chromatogram of the sample collected from the Eilat crystallizer pond in November 1998 showed also a minor spot of S2 -DGD-1, the bis-sulfated glycolipid described from Natrialba asiatica [21]. The Cargill samples contained an additional glycolipid that may be identical to one of the sulfated diglycosyl diether lipids reported in
di¡erent Halorubrum species [3,22,23]. No trace was found of the two glycolipids that characterize the genus Halobacterium: S-TGD-1 (1-O-[L-D-galactose-(3P-SO3 H)-(1PC6P)-K- D-mannose-(1PC2P)-K-Dglucose]-2,3-di-O-phytanyl-sn-glycerol) [24] and STeGD (1-O-[L-D-galactose-(3P-SO3 H)-(1PC6P)-K-Dmannose-(3P61P)-K- D -galactofuranose)-(1PC2P)-KD-glucose]-2,3-di-O-phytanyl-sn-glycerol) [3,23,24]. The small contribution of Halobacterium species to the biota of saltern crystallizers was con¢rmed at a di¡erent geographical location (the Mediterranean coast of Spain), using techniques such as characterization of colonies isolated on suitable media (only 12 out of the 327 isolates of halophilic Archaea recovered belonged to the species Halobacterium) [7], and no sequences characteristic of Halobacterium were recovered during sequencing of the 16S rDNA genes directly ampli¢ed from the archaeal community in the crystallizer ponds [10]. Aerobic enrichment cultures, set up with brines from the two sites tested and using di¡erent growth media designed for the cultivation of halophilic Archaea, failed to yield cultures with the characteristic polar lipid pattern of the genus Halobacterium. Most such enrichments yielded cultures containing PG, Me-PGP, PGS and glycolipids resembling SDGD-1 of Haloferax and/or one of the Halorubrum glycolipids (Fig. 1, lanes 6^10). In a number of cases bacterial lipids (phosphatidylcholine, phosphatidylethanolamine) were also found (Fig. 1, lane 10). Results were similar whether or not arginine was included in the aerobic enrichment media. Enrichments set up in completely ¢lled bottles in the presence of arginine generally showed growth after 7^10 days. Microscopic examination showed the presence of long motile rod-shaped bacteria, measuring around 1U4^8 Wm, similar in appearance to Halobacterium salinarum cultures. For successful enrichment it was not necessary to remove all oxygen at the beginning of the experiment or to use strictly anaerobic techniques; respiration by the community originally present appeared su¤ciently e¡ective in establishing anaerobic conditions, whereafter only those bacteria able of fermentative growth could multiply. In every case in which Archaea developed, S-TGD-1, the main glycolipid of the genus Halobacterium, was found as the main or only glycolipid, in most cases accompanied by S-TeGD-1 (Fig. 1, lanes
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1, 3^5, 11^15). Again some of the enrichments with Cargill brines yielded bacteria instead of, or in addition to, Archaea (Fig. 1, lane 2). This can possibly be avoided by including L-lactam antibiotics and other inhibitors in the enrichment medium that do not a¡ect halophilic Archaea. By plating samples from the enrichment cultures on suitable media, Halobacterium colonies recovered with the highest frequency. Other types (e.g. cells with the lipid signature of Haloferax) were occasionally encountered. In contrast to other genera of halophilic Archaea such as Haloferax and Haloarcula that can grow on inorganic media containing a single carbon and energy source, Halobacterium has complex nutritional requirements. Chemically de¢ned media designed for their growth contain 10^15 di¡erent amino acids, a number of nucleotides, and other compounds [25,26]. In comparison to many other halophilic archaeal species, growth of Halobacterium strains is relatively slow. Our results show that, although they form a small minority in the archaeal biota of the crystallizer ponds, they are present, and may be isolated following the procedure described. The method proposed here provides the ¢rst protocol enabling the selective isolation of one of the genera of the noncoccoid halophilic Archaea with the exclusion of others (it was earlier reported that the coccoid Halococcus can be isolated from seawater, because the cells do not lyse at such low salinity [27]). While most Halobacterium strains in culture contain refractile gas vesicles, gas vesicles were only occasionally observed in our anaerobic enrichment cultures. Whether the gas vesicle-less isolates obtained should be classi¢ed within the species Halobacterium salinarum or may belong to new, yet undescribed species, remains to be determined. Especially interesting is the recovery of Halobacterium cells from the Eilat saltern using medium E, a medium that contains an exceptionally high magnesium concentration (0.8 M), considered to inhibit most halophilic Archaea. These organisms have been recovered and puri¢ed, and they are now being studied in greater depth.
Acknowledgments We thank the Israel Salt Company, Eilat and the
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Cargill Solar Salt Plant, Newark, CA, for enabling access to their saltern ponds, and Ronald S. Oremland (United States Geological Survey, Menlo Park, CA) for the use of laboratory facilities. Halobacterium salinarum strain NRC 817 was a gift from R.F. Shand (Northern Arizona University, Flagsta¡, AZ). This study was supported by grant no. 95-00027 from the United States-Israel Binational Science Foundation (BSF, Jerusalem).
References [1] Harrison, F.C. and Kennedy, M.E. (1922) The red discoloration of cured cod¢sh. Trans. R. Soc. Can. Sect. III 16, 101^ 152. [2] Grant, W.D. and Larsen, H. (1990) Extremely halophilic archaeobacteria, order Halobacteriales ord. nov. In: Bergey's Manual of Systematic Bacteriology, Vol. 3 (Staley, J.T., Bryant, M.P., Pfennig, N. and Holt, J.G., Eds.), pp. 2216^2233. Williams and Wilkins, Baltimore. [3] Tindall, B.J. (1992) The family Halobacteriaceae. In: The Prokaryotes. A Handbook of Bacteria: Ecophysiology, Isolation, Identi¢cation, Applications, Vol. 1 (Balows, A., Truëper, H.G., Dworkin, M., Harder, W. and Schleifer, K.H., Eds.), pp. 768^ 808. Springer-Verlag, New York. [4] Oesterhelt, D. and Stoeckenius, W. (1971) Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature New Biol. 233, 149^152. [5] Colwell, R.R., Litch¢eld, C.D., Vreeland, R.H., Kiefer, L.A. and Gibbons, N.E. (1979) Taxonomic studies of red halophilic bacteria. Int. J. Syst. Bacteriol. 29, 379^399. [6] Ventosa, A. and Oren, A. (1996) Halobacterium salinarum nom. corrig., a name to replace Halobacterium salinarium (Elazari-Volcani) and to include Halobacterium halobium and Halobacterium cutirubrum. Int. J. Syst. Bacteriol. 46, 361. [7] Rodriguez-Valera, F., Ventosa, A., Juez, G. and Imho¡, J.F. (1985) Variation of environmental features and microbial populations with salt concentration in a multi-pond saltern. Microb. Ecol. 11, 107^115. [8] Oren, A. (1994) Characterization of the halophilic archaeal community in saltern crystallizer ponds by means of polar lipid analysis. Int. J. Salt Lake Res. 3, 15^29. [9] Oren, A. (1994) The ecology of the extremely halophilic Archaea. FEMS Microbiol. Rev. 13, 415^440. [10] Benlloch, S., Martinez-Murcia, A.J. and Rodriguez-Valera, F. (1995) Sequencing of bacterial and archaeal 16S rRNA genes directly ampli¢ed from a hypersaline environment. Syst. Appl. Microbiol. 18, 574^581. [11] Hartmann, R., Sickinger, H.-D. and Oesterhelt, D. (1980) Anaerobic growth of halobacteria. Proc. Natl. Acad. Sci. USA 77, 3821^3825. [12] Oesterhelt, D. (1982) Anaerobic growth of halobacteria. Methods Enzymol. 88, 417^420. [13] Bickel-Sandkoëtter, S., Gartner, W. and Dane, M. (1996) Con-
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[14]
[15]
[16]
[17]
[18]
[19]
A. Oren, C.D. Litch¢eld / FEMS Microbiology Letters 173 (1999) 353^358 version of energy in halobacteria: ATP synthesis and phototaxis. Arch. Microbiol. 166, 1^11. Ruepp, A. and Soppa, J. (1996) Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium) : genes, gene products, and transcripts of the arcRACB gene cluster. J. Bacteriol. 178, 4942^4947. Simon, R.D. (1980) Interactions between light and gas vacuoles in Halobacterium salinarium strain 5: e¡ect of ultraviolet light. Appl. Environ. Microbiol. 40, 984^987. Oren, A., Ventosa, A., Gutieèrrez, M.C. and Kamekura, M. (1999) Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt). Int. J. Syst. Bacteriol., in press. Alam, M., Claviez, M., Oesterhelt, D. and Kessel, M. (1984) Flagella and motility behaviour of square bacteria. EMBO J. 3, 2899^2903. Litch¢eld, C.D., Irby, A. and Vreeland, R.H. (1999) The microbial ecology of solar salt plants. In: Microbiology and Biogeochemistry of Hypersaline Environments (Oren, A., Ed.), pp. 39^52. CRC Press, Boca Raton. Torreblanca, M.F., Rodriguez-Valera, F., Juez, G., Ventosa, A., Kamekura, M. and Kates, M. (1986) Classi¢cation of nonalkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst. Appl. Microbiol. 8, 89^ 99.
[20] Oren, A., Ventosa, A. and Grant, W.D. (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int. J. Syst. Bacteriol. 47, 233^238. [21] Matsubara, T., Iida-Tanaka, N., Kamekura, M., Moldoveanu, N., Ishizuka, I., Onishi, H., Hayashi, A. and Kates, M. (1994) Polar lipids of a non-alkaliphilic extremely halophilic archaebacterium strain 172 : A novel bis-sulfated glycolipid. Biochim. Biophys. Acta 1214, 97^108. [22] Kamekura, M. (1992) Lipids of extreme halophiles. In: The Biology of Halophilic Bacteria (Vreeland, R.H. and Hochstein, L.I., Eds.), pp. 135^161. CRC Press, Boca Raton. [23] Kamekura, M. and Kates, M. (1988) Lipids of halophilic archaebacteria. In: Halophilic Bacteria, Vol. II (RodriguezValera, F., Ed.), pp. 25^54. CRC Press, Boca Raton. [24] Kates, M. and Deroo, P.W. (1973) Structure determination of the glycolipid sulphate from the extreme halophile Halobacterium cutirubrum. J. Lipid Res. 14, 438^445. [25] Dundas, I.D., Srinivasan, V.R. and Halvorson, H.O. (1963) A chemically de¢ned medium for Halobacterium salinarium strain 1. Can. J. Microbiol. 9, 619^624. [26] Grey, V.L. and Fitt, P.S. (1976) An improved synthetic growth medium for Halobacterium cutirubrum. Can. J. Microbiol. 22, 440^442. [27] Rodriguez-Valera, F., Ruiz-Berraquero, F. and Ramos-Cormenzana, A. (1979) Isolation of extreme halophiles from seawater. Appl. Environ. Microbiol. 38, 164^165.
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A procedure for the enrichment and isolation of Halobacterium Aharon Oren a; *, Carol D. Litch¢eld a
b
Division of Microbial and Molecular Ecology, The Institute of Life Sciences, and The Moshe Shilo Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel b Department of Biology, George Mason University, Fairfax, VA 22030-4444, USA Received 15 December 1998; received in revised form 15 February 1999; accepted 17 February 1999
Abstract A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of L-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Halobacterium; Enrichment; Anaerobic growth ; Arginine
1. Introduction Among the genera within the family Halobacteriaceae the genus Halobacterium is the best known. Halobacterium salinarum (previously designated Halobacterium salinarium and Pseudomonas salinaria) was the ¢rst halophilic Archaeon described [1], and thus the genus Halobacterium became the type genus of the family [2,3]. Halobacterium is also the most widely studied genus within the Halobacteriaceae, especially after the discovery of the unique properties of the light-driven proton pump bacteriorhodopsin in the purple membrane of Halobacterium halobium [4]. * Corresponding author. Tel.: +972 (2) 6584951; Fax: +972 (2) 6528008; E-mail: [email protected]
Three Halobacterium species were described in the past (not taking into account those that were later transferred to other genera) [3]: Halobacterium salinarium, Halobacterium halobium, and Halobacterium cutirubrum. They were isolated from such environments as salted cow hide and cured cod¢sh. A comparative study of a large number of isolates showed them all to be very similar in their properties [5], a conclusion supported by later phylogenetic analyses. Presently only a single species of Halobacterium is recognized: Halobacterium salinarum [6]. Analysis of natural communities of halophilic Archaea such as that occur in hypersaline lakes and saltern ponds shows that Halobacterium is not the dominant genus in such environments and is outnumbered by members of other genera, some of which may still await isolation. This was shown
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 0 9 5 - 6
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both by characterization of strains isolated as cultures on agar plates [7], by analysis of the polar lipids extracted from the communities [8,9], and by sequencing of the 16S rDNA genes directly ampli¢ed from such environments [10]. Halobacterium cells are generally not detected. Enrichment cultures also rarely yield Halobacterium strains, as Halobacterium is in most cases outgrown by other halophilic Archaea with simpler nutritional requirements and higher growth rates. It was ¢rst reported in 1980 that Halobacterium halobium can grow anaerobically by fermentation of L-arginine to ornithine, CO2 and NH3 [11,12]. In-depth enzymatic studies and molecular analyses of the genes involved in the fermentation pathway [13,14] have greatly increased our understanding of the process. In the present study we tested a large number of species of halophilic Archaea, belonging to di¡erent genera, for their ability to grow fermentatively on arginine, and we examined the possibility of using the property of arginine fermentation to design a speci¢c enrichment procedure for Halobacterium strains inhabiting hypersaline environments.
2. Materials and methods 2.1. Culture conditions and archaeal strains used Halophilic archaeal strains were grown aerobically in 100 ml portions in 250 ml Erlenmeyer £asks in a rotatory shaker at 35 or 38³C. The following media were employed (concentrations in g l31 ): A: NaCl, 250; KCl, 5; MgCl2 W6H2 O, 5; NH4 Cl, 5; and yeast extract, 10. B: NaCl, 200; KCl, 1; MgSO4 W7H2 O, 20; FeSO4 W7H2 O, 0.02; Tri-Na-citrate, 3; casamino acids, 7.5; and yeast extract, 0.5. C: NaCl, 175; MgCl2 W6H2 O, 20; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; and yeast extract, 5. D: NaCl, 206; MgSO4 W7H2 O, 36; KCl, 0.37; CaCl2 W2H2 O, 0.5; MnCl2 , 0.013; and yeast extract, 5. E (a medium for high magnesium requiring isolates from the Dead Sea): NaCl, 125; MgCl2 W6H2 O, 160; K2 SO4 , 5; CaCl2 W2H2 O, 0.1; yeast extract, 1; casamino acids, 1; and starch, 2. All media were adjusted to pH 7.0 with NaOH. The following archaeal strains were used (the letters in brackets indicating the growth media em-
ployed): Halobacterium salinarum NRC 817 (A, B), Halobacterium cutirubrum (salinarum) NRC 34001 (A, B), Halobacterium salinarum strain 5 [15] (A, B), Halobacterium halobium (salinarum) R1 (A, B), Halorubrum saccharovorum ATCC 29252T (B, C), Halorubrum sodomense ATCC 33755T (E), Halobaculum gomorrense DSM 9297T (E), Haloferax volcanii ATCC 29605T (C), Haloferax mediterranei ATCC 35300T (C), Haloferax gibbonsii ATCC 33959T (C), Haloferax denitri¢cans ATCC 35960T (C), Haloarcula marismortui ATCC 43049T (D), Haloarcula vallismortis ATCC 29715T (D), Haloarcula quadrata DSM 11927 (strain 801030/1, a square motile from a brine pool in Sinai) [16,17] (D), `Haloarcula californiae' ATCC 33799 (D), `Haloarcula sinaiiensis' ATCC 33800 (D), Halogeometricum borinquense ATCC 700274T (B), and Natrialba asiatica JCM 9576T (B). To test for anaerobic growth on arginine, 15 ml screw-capped glass test tubes were ¢lled to the top with the appropriate growth medium, supplemented with 5 g l31 L-arginineWHCl, inoculated with 0.15 ml of aerobic, mid-exponential growth phase culture, and closed. After 5^7 days incubation in the dark the turbidity of the cultures was compared to tubes without added arginine. Strains were considered positive for arginine fermentation when the turbidity (OD600 ) of the arginine containing culture exceeded 0.05, and was at least twice that of the control without arginine. 2.2. Field samples and enrichment cultures Brine samples were collected from saltern crystallizer ponds of the Israel Salt Company, Eilat (May 1998 and November 1988, pond no. 302) [8] and the Cargill Solar Salt Plant, Newark, CA (July 1998, ponds no. 10A and 11) [18]. Anaerobic enrichment cultures for arginine fermenters were set up by inoculating 10 ml portions of brine in 150 ml stoppered glass bottles completely ¢lled with the growth media described above with the addition of 5 g l31 arginine, and incubating the bottles in the dark at 35^38³C. Additional media used for enrichment contained brine from the respective sampling sites, from which most of the bacteria were removed by centrifugation (15 min, 6000Ug), amended with 2.5 g l31 yeast extract or casamino acids and 5 g l31 arginine (media F and G, respectively). Similar media were prepared in
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Fig. 1. Composite picture of representative results of thin layer chromatography analyses of polar lipids extracted from enrichments for halophilic Archaea in the presence of L-arginine, using crystallizer brines from the Cargill Solar Salt Plant (lanes 1^10) and the Eilat Salt Company, Eilat (lanes 11^15) as inoculum, under anaerobic (lanes 1^5 and 11^15) and aerobic conditions (lanes 6^10). Lanes 16, 17, and 18 show polar lipid chromatography patterns obtained after extraction of the biomass collected from the crystallizer ponds of the Cargill (July 1998) and Eilat ponds (May 1998 and November 1998, respectively) without prior enrichment. Media used in the enrichments were A (lanes 1, 6, 11), B (lane 12), C (lane 2), D (lane 13), E (lane 14), F (lanes 3 and 7), G (lane 8), H (lanes 4 and 9), and I (lanes 5, 10, and 15). Media used in the enrichments shown in lanes 11, 12, and 13 contained in addition 25 Wg ml31 penicillin G. Polar lipid patterns of reference strains Halobacterium salinarum (lane 19), Haloarcula vallismortis (lane 20), Haloferax mediterranei (lane 21), Halorubrum sodomense (lane 22), and Natrialba asiatica (lane 23) are also shown. Glycolipids are shown in black. The tentative identi¢cation of the lipid spots, based on literature data [18,21^24], is indicated.
which the brines cleared by centrifugation were diluted with sterile distilled water to 80% of their original salinity (media H and I). Aerobic control experiments were set up by incubating identical enrichments (100 ml portions), with or without arginine, in 250 ml Erlenmeyer £asks with shaking. 2.3. Lipid analyses Bacteria were collected from enrichment cultures, reference cultures of halophilic Archaea and brine samples by centrifugation (15 min, 12 000Ug). Cell pellets were suspended in 1 ml H2 O, and extracted with 3.75 ml methanol-chloroform 2:1 (by volume) for 4 h. The extract was collected by centrifugation, and the pellet reextracted with 4.75 ml methanolchloroform-water (2:1:0.8). Chloroform and water (2.5 ml each) were added to the combined supernatants to achieve phase separation, and after centrifugation the chloroform phase was collected and dried. Lipids were redissolved in a small volume of chloroform, applied to silica gel plates (Sigma-Aldrich Z12,271-8 or Whatman LK6D, 20U20 cm), and sep-
arated by single development with chloroform methanol-acetic acid-water (85:22.5:10:4, vol/vol) [19]. Lipid spots on the plates were detected by spraying with the following reagents: (1) 0.5% K-naphthol in 50% methanol, followed by 5% H2 SO4 in ethanol, and heating of the plates at 150³C (allowing particularly detection of glycolipids); (2) 0.2% CeSO4 in 1 N H2 SO4 , followed by heating at 150³C; (3) orcinol spray reagent (Sigma) followed by heating at 100³C (both general lipid stains, allowing di¡erentiation of glycolipids from other lipids by color); and (4) ammonium molybdate reagent for the detection of phospholipids.
3. Results and discussion Of all 18 strains tested, belonging to seven genera, only the four Halobacterium strains (now all classi¢ed within the species Halobacterium salinarum) showed anaerobic growth in the presence of L-arginine. Other H. salinarum strains also were reported to grow fermentatively on L-arginine, including the
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type strain [14], strain L33 [14], and strain R1 M1 [11]. Anaerobic growth on arginine, a test included in the proposed minimal standards for description of new taxa in the order Halobacteriales [20], is thus not widespread among the neutrophilic halophilic Archaea, and appears to occur only in isolates belonging to the genus Halobacterium. Little is known about the ecological importance of arginine fermentation for Halobacterium populations in nature. It is feasible that when colonizing proteinrich environments such as salted ¢sh and hides halobacteria may reach high cell densities, leading to depletion of oxygen (enhanced by the low solubility of oxygen in concentrated salt solutions), and that their proteolytic enzymes may then maintain a supply of arginine as energy source. Therefore it is interesting to note that, in contrast to most other halophilic Archaea in culture that were isolated from hypersaline aquatic habitats, the reference strains of Halobacterium all came from protein-rich environments (salted ¢sh and hides). As of all the neutrophilic halophilic Archaea tested only members of the genus Halobacterium showed anaerobic growth on arginine, we examined whether this property may be used for the selective enrichment of Halobacterium strains from hypersaline environments. As inoculum we used crystallizer brines collected from salterns at two geographically remote locations: the Red Sea coast of Israel and San Francisco Bay, California. Polar lipid analysis of the archaeal communities inhabiting these brines showed no sign of a massive presence of Halobacterium cells: the dominant glycolipid in both locations was chromatographically identical to S-DGD-1, the sole or main glycolipid found in the genera Haloferax, Halococcus, and Halobaculum. In addition, the phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidylglycerophosphate (MePGP), and phosphatidylglycerosulfate (PGS) were detected (Fig. 1, lanes 16^18). This lipid pattern is similar to that described earlier for the Eilat crystallizer ponds [8]. A lipid chromatogram of the sample collected from the Eilat crystallizer pond in November 1998 showed also a minor spot of S2 -DGD-1, the bis-sulfated glycolipid described from Natrialba asiatica [21]. The Cargill samples contained an additional glycolipid that may be identical to one of the sulfated diglycosyl diether lipids reported in
di¡erent Halorubrum species [3,22,23]. No trace was found of the two glycolipids that characterize the genus Halobacterium: S-TGD-1 (1-O-[L-D-galactose-(3P-SO3 H)-(1PC6P)-K- D-mannose-(1PC2P)-K-Dglucose]-2,3-di-O-phytanyl-sn-glycerol) [24] and STeGD (1-O-[L-D-galactose-(3P-SO3 H)-(1PC6P)-K-Dmannose-(3P61P)-K- D -galactofuranose)-(1PC2P)-KD-glucose]-2,3-di-O-phytanyl-sn-glycerol) [3,23,24]. The small contribution of Halobacterium species to the biota of saltern crystallizers was con¢rmed at a di¡erent geographical location (the Mediterranean coast of Spain), using techniques such as characterization of colonies isolated on suitable media (only 12 out of the 327 isolates of halophilic Archaea recovered belonged to the species Halobacterium) [7], and no sequences characteristic of Halobacterium were recovered during sequencing of the 16S rDNA genes directly ampli¢ed from the archaeal community in the crystallizer ponds [10]. Aerobic enrichment cultures, set up with brines from the two sites tested and using di¡erent growth media designed for the cultivation of halophilic Archaea, failed to yield cultures with the characteristic polar lipid pattern of the genus Halobacterium. Most such enrichments yielded cultures containing PG, Me-PGP, PGS and glycolipids resembling SDGD-1 of Haloferax and/or one of the Halorubrum glycolipids (Fig. 1, lanes 6^10). In a number of cases bacterial lipids (phosphatidylcholine, phosphatidylethanolamine) were also found (Fig. 1, lane 10). Results were similar whether or not arginine was included in the aerobic enrichment media. Enrichments set up in completely ¢lled bottles in the presence of arginine generally showed growth after 7^10 days. Microscopic examination showed the presence of long motile rod-shaped bacteria, measuring around 1U4^8 Wm, similar in appearance to Halobacterium salinarum cultures. For successful enrichment it was not necessary to remove all oxygen at the beginning of the experiment or to use strictly anaerobic techniques; respiration by the community originally present appeared su¤ciently e¡ective in establishing anaerobic conditions, whereafter only those bacteria able of fermentative growth could multiply. In every case in which Archaea developed, S-TGD-1, the main glycolipid of the genus Halobacterium, was found as the main or only glycolipid, in most cases accompanied by S-TeGD-1 (Fig. 1, lanes
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1, 3^5, 11^15). Again some of the enrichments with Cargill brines yielded bacteria instead of, or in addition to, Archaea (Fig. 1, lane 2). This can possibly be avoided by including L-lactam antibiotics and other inhibitors in the enrichment medium that do not a¡ect halophilic Archaea. By plating samples from the enrichment cultures on suitable media, Halobacterium colonies recovered with the highest frequency. Other types (e.g. cells with the lipid signature of Haloferax) were occasionally encountered. In contrast to other genera of halophilic Archaea such as Haloferax and Haloarcula that can grow on inorganic media containing a single carbon and energy source, Halobacterium has complex nutritional requirements. Chemically de¢ned media designed for their growth contain 10^15 di¡erent amino acids, a number of nucleotides, and other compounds [25,26]. In comparison to many other halophilic archaeal species, growth of Halobacterium strains is relatively slow. Our results show that, although they form a small minority in the archaeal biota of the crystallizer ponds, they are present, and may be isolated following the procedure described. The method proposed here provides the ¢rst protocol enabling the selective isolation of one of the genera of the noncoccoid halophilic Archaea with the exclusion of others (it was earlier reported that the coccoid Halococcus can be isolated from seawater, because the cells do not lyse at such low salinity [27]). While most Halobacterium strains in culture contain refractile gas vesicles, gas vesicles were only occasionally observed in our anaerobic enrichment cultures. Whether the gas vesicle-less isolates obtained should be classi¢ed within the species Halobacterium salinarum or may belong to new, yet undescribed species, remains to be determined. Especially interesting is the recovery of Halobacterium cells from the Eilat saltern using medium E, a medium that contains an exceptionally high magnesium concentration (0.8 M), considered to inhibit most halophilic Archaea. These organisms have been recovered and puri¢ed, and they are now being studied in greater depth.
Acknowledgments We thank the Israel Salt Company, Eilat and the
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Cargill Solar Salt Plant, Newark, CA, for enabling access to their saltern ponds, and Ronald S. Oremland (United States Geological Survey, Menlo Park, CA) for the use of laboratory facilities. Halobacterium salinarum strain NRC 817 was a gift from R.F. Shand (Northern Arizona University, Flagsta¡, AZ). This study was supported by grant no. 95-00027 from the United States-Israel Binational Science Foundation (BSF, Jerusalem).
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