Institute of Genetics and General Biology, Hellbrunnerstr. 34, A- 5020 Salzburg, Austria1
Dept of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK2
Institute of Microbiology and Genetics, University of Vienna, Dr Bohrgasse 9,A-1030 Vienna, Austria3
Department of Microbiology, University of Regensburg, Universit ätsstraße 31, D-93053 Regensburg , Germany4
Botanical Institute of the University of Munich, Menzinger Str. 67, D-80638 Munich, Germany5
Author for correspondence: Helga Stan-Lotter. Tel: +43 662 8044 5756. Fax: +43 662 8044 144. e-mail: helga.stan-lotter{at}sbg.ac.at
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ABSTRACT |
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Keywords: archaea, Halococcus, subterranean microbiology, salt deposits, prokaryotic longevity
Abbreviations: FT-IR, Fourier-transform infrared; PG, glycerol diether of phosphatidylglycerol; PGP-Me, glycerol diether of phosphatidylglycerol methylphosphate; PGS, glycerol diether of phosphatidylglycerol sulphate; SDGD-1, sulphated mannosylglucosylglycerol diether; C20:C20 , 2,3-di-O-phytanyl-sn-glycerol diether; C 20:C25, 2-O-sesterterpanyl-3-O- phytanyl-sn-glycerol diether
The GenBank accession numbers for the nucleotide sequence data reported in this paper are Z28387 (Hc. salifodinae BIp DSM 8989T), AJ238897 (strain Br3), AJ131458 (strain BG2/2), AJ245422 and AJ245423 (strain N1), AJ245424 and AJ245425 (strain H2). Accession numbers for other 16S rRNA sequences used for comparisons in this study have been reported previously (McGenity et al., 1998 ).
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INTRODUCTION |
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METHODS |
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Phenotypic characterization.
Biochemical assays and antibiotic susceptibility tests were described by Denner et al. (1994 ). Hydrolysis of casein, starch, Tween 20 and Tween 80 was tested as described by Smibert & Krieg (1994)
. Additional characterization with respect to the presence of enzymes was carried out with API ZYM (bioMérieux) strips. These were used according to the instructions of the manufacturer, except that cells were suspended in mineral solution I (Tomlinson & Hochstein, 1976
), which contained 200 g NaCl l-1.
Electron microscopy.
Cells were harvested after 1 week of incubation in M2 medium and prepared for scanning or transmission electron microscopy as described previously (Denner et al., 1994 ).
Polar lipid analysis.
Strains were grown on halophile medium agar, until colonies were nearly confluent (~14 d). Colonies were removed from the surface of three agar plates by spreading with 2 ml 20% (w/v) NaCl, and transferring the cell suspension to a centrifuge tube. Cells were harvested by centrifugation and freeze-dried. Approximately 0·5 g freeze-dried cells were stirred with 5 ml chloroform/methanol (1:1, v/v) at 50 °C for 16 h. Extracted lipids were separated from cells by filtration through a 0·2 µm pore-size PTFE filter, and dried under a stream of oxygen-free nitrogen. The dried polar lipids were resuspended in ~0·3 ml chloroform/methanol (1:1, v/v). For two-dimensional thin-layer chromatography the method of Collins et al. (1980) was used. Polar lipid extracts were spotted onto the corner of a thin-layer 10x10 cm silica gel plate (60 F254, Merck), and were developed first in chloroform/methanol/water (65:25:4, by vol.), and then in chloroform/methanol/glacial acetic acid/water (80:12:15:4, by vol.). For one-dimensional development the latter solvent mixture was used. Lipids were visualized by dipping dry TLC plates in 0·1% (w/v) ceric sulphate in 1 M sulphuric acid, and charring at 150 °C for 5 min. Glycolipids appeared as purple spots for up to 1 h after charring, while other lipids appeared brown. The equivalence of spots was determined by co- chromatography in two dimensions and by determining Rf values based on one-dimensional chromatography.
16S rRNA sequences and analysis.
Nucleotide sequences of the 16S rRNA genes of Hc. salifodinae BIp DSM 8989T, and strains BG2/2, N1 and H2, were determined by the Identification Service of the DSMZ; the 16S rRNA gene of strain Br3 was sequenced as described previously (McGenity & Grant, 1995 ). Extraction of the genomic DNA, amplification of the 16S rDNA by PCR and purification of the products were performed as described by Rainey et al. (1996)
. The purified 16S rDNA was sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) as indicated by the manufacturer. The DNA fragments of the sequencing reactions were separated and analysed using an Applied Biosystems 373 DNA Sequencer. The resulting sequences were read into the Alignment Editor ae2 (Maidak et al., 1996
), aligned manually and compared with 16S rRNA gene sequences of representative archaea. Sequences used for comparisons were obtained from the EMBL database or the database of the RDP (Maidak et al., 1996
). The phylogenetic tree was constructed using the neighbour-joining method of Saitou & Nei (1987)
in the PHYLIP program (Felsenstein, 1993
), following transformation of sequence distances according to Jukes & Cantor (1969)
. Confidence of the branching pattern was assessed by 100 bootstrap analyses, using the PHYLIP package.
DNA base composition.
Cells were harvested in the late-exponential phase of growth and G+C content was determined by the Identification Service of the DSMZ. Cells were broken by passage through a French press (Aminco) and DNA was isolated and purified according to the procedures of Cashion et al. (1977) and Visuvanathan et al. (1989)
. Analysis was by HPLC according to Mesbah et al. (1989)
and Tamaoka & Komagata (1984)
.
Detection and sequencing of an insertion in halococcal 5S rRNA genes.
Primers were designed to flank the region of the 5S rRNA gene in which an insertion of 108 bases has been reported in Hc. morrhuae ATCC 17082T (Luehrsen et al., 1981 ; see Fig. 4
). An alignment of 19 halobacterial 5S rRNA sequences demonstrated that there was a highly conserved region towards the 5' end (primer 5SF), but none at the 3' end of the gene, and so the reverse primer was designed from the cysteine tRNA gene just downstream of the 5S rRNA gene (primer cysR). The sequences of the PCR primers were: 5SF, 5'-CGTACCCAT(T/C)CCGAAC-3', and cysR, 5'-CTGCCACCTTGGC(A/G)CA-3'. DNA was extracted by the method of Pitcher et al. (1989)
, using guanidinium thiocyanate. The PCR included BioXact polymerase (2 units) in buffer (Bioline), with the indicated final concentrations of MgCl 2 (2·5 mM), dNTPs (200 µM), BSA (400 ng µl-1), primers (400 nM), and DNA (~10 ng). Cycle conditions were: 2 min denaturation at 94 °C, followed by 30 cycles of denaturation (15 s at 94 °C), annealing (20 s at 55 °C) and extension (1 min at 68 °C). Reactions were carried out in thin- walled tubes in a Progene thermal cycler (Techne). PCR products (47 µl) were separated by electrophoresis at 105 V for 60 min in a 2% (w/v) agarose gel in TAE buffer, and visualized after staining with ethidium bromide using a UV transilluminator (Sambrook et al., 1989
). PCR product was cleaned using a Qiagen column, and sequenced with a primer (5'- AGTACTGGAGTGTGCGA-3') labelled at the 5' end with Cy5 (Pharmacia). For the cycle sequencing reaction and electrophoresis on an ALF-Express (Pharmacia) automated sequencer the procedures described by the manufacturer were followed.
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Other methods.
SDS-PAGE of whole-cell proteins was performed following lysis of archaea as described previously (Stan-Lotter et al., 1989 , 1993
). At least six gels were run with each of the samples. Protein was determined by the Lowry method, with bovine serum albumin as standard.
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RESULTS |
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DNA base composition
The mol% G+C contents of the strains were as follows (means of three determinations are stated,±SEM, except for H2, where the mean of four determinations is given): BG2/2, 63·9±0·1; Br3, 61·0±0·1; N1, 62·2±0·5; H2, 62·8±0·4. These values were similar to that of Hc. salifodinae BIp DSM 8989T (62±1; Denner et al., 1994 ) and generally somewhat higher than that of Hc. saccharolyticus ATCC 49257T (59·5; Montero et al., 1989
).
Gel electrophoresis of whole-cell proteins
SDS gel electrophoresis of whole-cell proteins is a rapid method for distinguishing bacterial species and has a similar level of discrimination as DNADNA hybridization (Jackman, 1987 ). Bacterial cells that are grown under carefully standardized conditions produce constant protein patterns, which greatly facilitates the identification of strains (Vauterin et al., 1993
). This method has been widely used in the systematics of numerous bacterial strains (Kersters & De Ley, 1980
), including the identification of several novel isolates by our laboratories (Denner et al., 1994
; Dang et al. , 1996
; Nguyen et al., 1999
). The whole-cell protein patterns of Hc. salifodinae BIp DSM 8989 T, Br3, BG2/2, N1 and H2, following separation by SDS gel electrophoresis, were very similar (Fig. 2
, lanes 27); they were distinctly different from the protein patterns of Hc. morrhuae DSM 1307T (lane 1) and Hc. saccharolyticus ATCC 49257T (lane 8) (see also Denner et al., 1994
).
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Polar lipid compositions
Several independent analyses of the polar lipids of four of the halococci (Hc. morrhuae NCIMB 787T, Hc. saccharolyticus ATCC 49257T, Hc. salifodinae BIp DSM 8989T, strain Br3) have been reported previously (Ross et al., 1985 ; Montero et al., 1989
; Norton et al., 1993
; Denner et al., 1994
); this is the first description of the polar lipids from strain BG2/2, and the first study in which all strains were cultivated, extracted and run under the same conditions. The five halococci had in greatest abundance the phospholipids PGP-Me and PG, and the sulphated glycolipid SDGD-1; these are shown for strain BG2/2, Hc. salifodinae BIp DSM 8989T and Hc. saccharolyticus ATCC 49257T in Fig. 5
; all lacked PGS. Profiles and relative abundance of polar lipids were almost identical for Hc. salifodinae BIp DSM 8989T, Br3 and BG2/2. Both C20:C 20 and C20:C25 diether core lipids were present in the five halococci and are most readily observed by looking at the double spot obtained for PG (Fig. 5
). The relative proportion of the C20:C20 and C20 :C25 forms of SDGD-1 differed in Hc. saccharolyticus ATCC 49257T, which had almost equal amounts of the two forms, whereas Hc. morrhuae NCIMB 787T , Hc. salifodinae BIp DSM 8989T, and strains Br3 and BG2/2 had significantly more of the C20:C20 diether core lipid. In addition, all strains had some indistinguishable glycolipids which did not migrate far from the origin. Hc. saccharolyticus ATCC 49257T also had a prominent glycolipid that ran just beyond SDGD-1 in both the x and y directions, labelled as P-1 (Fig. 5
).
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DISCUSSION |
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The origins of the halococcal isolates were subterranean salt sediments in different parts of northern Europe: walls from a freshly blasted tunnel in the salt mine near Bad Ischl (Austria), a drilling core from a salt deposit in Berchtesgaden (Germany), and solution-mined brine from Lostock, Cheshire (England). Over the past decade it has become increasingly apparent that micro-organisms survive in a wide variety of sediments, such as sandstones, chalk and palaeosols, often extending to depths of several hundreds of metres (see Amy & Haldeman, 1997 ). The geological age of many of these environments dates back thousands or millions of years. However, the origin and transport of micro-organisms within these sediments remain uncertain, since numerous ubiquitous micro-organisms have been found, which occur also in surface environments. In contrast, halite deposits represent a physically more enclosed environment (see Grant et al. , 1998
); they are characterized in general by the absence of water (except for fluid inclusions; see below), and species variety is limited. The micro-organisms which were previously isolated from surface-sterilized rock salt have been shown to be extremely and obligately halophilic, unable to grow in less than 1·5 M NaCl (Bibo et al., 1983
; Norton et al., 1993
; Denner et al., 1994
; Grant et al., 1998
); our strains of Hc. salifodinae required at least 2·5 M NaCl for growth.
It has been suggested that obligately halophilic micro-organisms isolated from ancient salt deposits are actually the result of laboratory contamination. Our investigation provides the strongest evidence to date that this is not the case. Each isolation was independent; each sample was from a different location; the detailed polyphasic characterization demonstrated that the three salt-deposit strains were almost identical, yet distinct from other halococci. In addition, several very similar strains have been isolated recently from walls of newly blasted tunnels of the Bad Ischl salt mine; two of them, N1 and H2, were characterized in more detail in this study and found to be identical to Hc. salifodinae. Besides the halococci described here, we have isolated approximately 30 more extremely halophilic strains from the same pieces of rock salt, all of which possessed red, pink or orange pigmentation (A. Legat & H. Stan- Lotter, unpublished results), similar to the halophilic isolates from the Winsford salt mine in England (Norton et al., 1993 ).
In order to consider how the strains of Hc. salifodinae came to be inside the salt deposits, it is necessary to understand the depositional setting of the evaporites. The age of the Austrian salt sediments is known from palynological and isotope studies. Klaus (1974) detected plant spores in rock salt from extinct species; the spore types Pityosporites, Gigantosporites and others are characteristic of the Permian period and can be clearly distinguished from Triadosporites, which are found in Triassic evaporites. The formation of most of the Austrian evaporites was thus dated to the Upper Permian, with some originating from the Triassic. By using sulphur isotope ratios, Pak & Schauberger (1981)
confirmed a Permian to Triassic age for the Austrian salt deposits. Therefore, the maximum age of the Bad Ischl salts is 260 million years, and the minimum age is uncertain (based on the International Union of Geological Sciences, 1989 Global Stratigraphic Chart). The salt mine at Berchtesgaden is about 50 km from Bad Ischl and belongs to the same geological formation (Zharkov, 1981
), which implies a similar age of the sediments. The Lostock solution-mined brine originates from rock from the Anisian stage in the Triassic (235240 million years) (Warrington, 1970
). Therefore, the salt sediments of Austria and southern Germany are separated in age from those of the Cheshire Basin by at most 25 million years, and they may be at least contemporaneous. During the time of sedimentation, Northern Europe was closer to the Equator, and the climate was arid and warm. A large hypersaline sea (Zechstein Basin) covered an area of about 250000 km2 over much of northern Europe, and was directly connected to the relatively small Cheshire Basin, which is exploited at Lostock (Zharkov, 1981
). Another hypersaline sea, which gave rise to the Bad Ischl and Berchtesgaden deposits, was present in the Alpine Basin, which extended along the modern Alps and the Carpathian Mountains; it could have been connected to the Zechstein Basin (Zharkov, 1981
), since the formation of the Alps took place later (in the Cretaceous; ~100 million years ago). It is conceivable that the same halobacteria would be widespread throughout the vast hypersaline seas in Europe during the Permo-Triassic, because transport by direct brine movement or wind- blown salt would have been relatively easy. The situation today, however, is very different. The only large hypersaline areas are to be found underground, with no continuous connection. One may argue that it is possible for wind-blown salt to have seeded the salt deposits in recent times; however, this could not be the case for strain BG2/2, which was isolated from a core, and it appears unlikely for strains BIp, N1 and H2, which were isolated from blasted rock salt from the walls of a new tunnel at approximately 650 m depth.
Our working hypothesis is that the halococci from salt deposits represent relict populations from hypersaline Permo-Triassic seas, which became restricted in habitat when the seas evaporated and were buried. A significant question concerns how the halococci could have survived for hundreds of millions of years within salt deposits. Norton & Grant (1988) have shown that several halobacteria trapped inside fluid inclusions in laboratory-grown halite remain viable and thus may be capable of dormancy. In ancient evaporites, between 30 and 1000 p.p.m. of brine is present in the form of fluid inclusions (Roedder, 1984
), within which bacterial cells could have become enclosed. Halobacteria may be adapted better than most micro-organisms to survive long periods of dormancy due to the extremely high (approx. 5 M) concentration of KCl inside their cells, which confers considerable stability to DNA (Marguet & Forterre, 1998
). Grant et al. (1998)
discussed several plausible scenarios for the long-term survival of halobacteria in salt deposits, such as potential energy sources, or the formation of special structures, such as cyst-like resting cells. While cysts or spores have not been detected in Hc. salifodinae, its cells have some unusual features. For example, Fig. 1(b)
shows the presence of a common cell envelope, similar to the common capsule seen in some halobacteria from soils, which was suggested to be involved in survival during unfavourable environmental conditions (Kostrikina et al., 1991
). Therefore, the cell structure of Hc. salifodinae may help its possible long-term survival. Furthermore, studies with Vibrio and other marine bacteria suggested that non-spore-forming cells survive long periods of starvation as well as spores (see Kjelleberg, 1993
; Morita, 1997
). It is uncertain whether the salt-deposit halococci have actually been in a state of dormancy for more than 200 million years, or if extremely slow in situ reproduction (Kennedy et al., 1994
) has occurred. In any case, the fact that the isolates from different geographical areas appear so very similar suggests that perhaps they have not been evolving rapidly. Alternatively, if it is considered that Hc. salifodinae strains and other halophilic archaea somehow entered the salt deposits in recent times, and in different geographical locations, then it would be necessary to propose a suitable mechanism. To date, Hc. salifodinae has not been isolated from surface hypersaline environments, nor from air samples, although further work is required to specifically search for it, using species-specific DNA probes.
It is worthy of note that the pink halococci resembling Hc. salifodinae grow relatively fast, producing colonies in about 34 weeks, but other, red- or mauve-pigmented halobacteria from the same rock-salt samples grow much more slowly (in the order of several months) on agar plates (H. Stan-Lotter & A. Legat, unpublished results). A characterization of these isolates is under way.
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ACKNOWLEDGEMENTS |
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Received 13 January 1999;
revised 30 August 1999;
accepted 10 September 1999.