Download Molecular ecology of extremely halophilic Archaea and Bacteria

FEMS Microbiology Ecology 39 (2002) 1^7
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MiniReview
Molecular ecology of extremely halophilic Archaea and Bacteria
Aharon Oren *
Division of Microbial and Molecular Ecology, The Institute of Life Sciences, and The Moshe Shilo Minerva Center for Marine Biogeochemistry,
The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
Received 13 August 2001 ; received in revised form 29 October 2001; accepted 29 October 2001
First published online 13 December 2001
Abstract
Water bodies with NaCl concentrations approaching saturation are often populated by dense microbial communities. Red halophilic
Archaea of the family Halobacteriaceae dominate in such environments. The application of molecular biological techniques, in particular
the use of approaches based on the characterization of ribosomal RNA sequences, has greatly contributed to our understanding of the
community structure of halophilic Archaea in hypersaline ecosystems. Analyses of lipids extracted from the environment have also provided
useful information. This article reviews our present understanding of the community structure of halophilic Archaea in saltern crystallizer
ponds, in the Dead Sea, in African hypersaline soda lakes, and in other hypersaline water bodies. It was recently shown that red
heterotrophic Bacteria of the genus Salinibacter, which are no less salt-dependent and salt-tolerant than the most halophilic among the
Archaea, may coexist with the halophilic archaeal community. Our latest insights into their distribution in hypersaline ecosystems are
presented as well. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Halobacteriaceae ; Saltern crystallizer; Dead Sea; 16S rRNA; Polar lipid ; Salinibacter
1. Introduction
Environments with NaCl concentrations approaching
saturation are often populated by dense microbial communities. As a result of the lack of predation and the often
high nutrient levels, densities of 107 ^108 cells ml31 and
higher are not unusual. Many halophilic microorganisms
have a high content of carotenoid pigments, and as a
result the waters of the Great Salt Lake (Utah), crystallizer ponds of solar salterns, and hypersaline soda lakes
such as Lake Magadi (Kenya) are often bright red. Red
waters are even sometimes found in the Dead Sea.
Red halophilic Archaea of the family Halobacteriaceae
[1] dominate in these environments. At the time of writing
(August 2001), the family Halobacteriaceae consisted of 15
genera with 40 species. Most are pigmented red due to a
high content of C-50 carotenoid pigments (K-bacterioruberin and derivatives) in their membrane, in some cases
accompanied by the purple retinal pigment bacteriorhodopsin.
* Tel. : +972 (2) 658 4951; Fax: +972 (2) 652 8008.
E-mail address : [email protected] (A. Oren).
Most ecological studies in the past have been restricted
to isolation and characterization of microorganisms from
the environment. This approach has yielded valuable information on the biodiversity present [2]. However, the
percentage of the microorganisms recovered as colonies
on agar plates is generally low, and therefore this type
of data provides little information on the true community
structure.
The application of molecular biological techniques to
microbial ecology, in particular the use of approaches
based on the characterization of ribosomal RNA sequences, has shown that the cultured organisms are generally
di¡erent from those that dominate in the natural environment. In addition, we know little about the growth rates
of halophilic Archaea in situ and about the factors that
lead to their death.
This review intends to provide an overview of the experimental approaches used in recent years to increase our
understanding of the ecology of halophilic Archaea in
ecosystems with salt concentrations approaching saturation. Most of these investigations have been performed
in saltern crystallizer ponds, and therefore the insights
obtained from these studies are presented ¢rst. Then follows a discussion of a number of additional hypersaline
environments in which molecular techniques have been
0168-6496 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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applied to obtain information on the nature of the halophilic archaeal communities.
It was recently discovered that red heterotrophic Bacteria, no less salt-dependent and salt-tolerant than the most
halophilic among the Archaea, may coexist with the halophilic archaeal community [3]. Information about their
distribution in hypersaline ecosystems is presented as well.
2. Ecology of extremely halophilic Archaea
2.1. Saltern crystallizer ponds
Multi-pond solar salterns present a gradient of salinities,
from seawater salinity to halite saturation. The salt concentration in each pond is kept relatively constant, and
microbial community densities are generally high.
Although salterns are super¢cially similar all over the
world, they do di¡er with respect to nutrient status and
retention time of the water, depending on climatic conditions [4].
Many species of halophilic Archaea have been isolated
from crystallizer ponds, the NaCl-saturated ponds in
which halite is deposited. Isolates include the type strains
of Haloferax mediterranei, Haloferax gibbonsii, Haloferax
denitri¢cans, Halogeometricum borinquense, Halococcus
saccharolyticus, Haloterrigena thermotolerans, Halorubrum
saccharovorum, Halorubrum coriense, Haloarcula hispanica
and Haloarcula japonica [1]. Halobacterium salinarum
could be grown from brine samples from crystallizer
ponds in Eilat, Israel and Newark, CA, USA in anaerobic
enrichment cultures in the presence of L-arginine [5]. From
saltern ponds near Alicante, Spain, species of Haloarcula,
Haloferax, Halorubrum and Halobacterium have been recovered at a high frequency [6,7]. The signi¢cance of such
results is limited as the number of colonies obtained is
only a small fraction of the total numbers of prokaryotes
present.
Microscopic examination of saltern crystallizer brines
worldwide generally shows that £at, square or rectangular,
gas-vacuolated cells dominate in the community [8^10]
(Fig. 1). Such square halophiles were ¢rst described by
Walsby [11] from a brine pool on the coast of the Sinai
peninsula, Egypt. They were reported to contain bacteriorhodopsin [12]. Unfortunately, this type of square £at gasvacuolated cells has not yet been brought into culture.
Molecular, culture-independent rRNA-based studies
have been performed to characterize the archaeal communities in saltern crystallizer ponds. These studies indicated that neither of the species recovered on agar plates
represents a major part of the archaeal community in
those ponds. In a study of Spanish salterns, 5S rRNA
was extracted from the microbial assemblages without prior ampli¢cation, and electrophoretically compared with 5S
rRNAs from cultured halophilic Archaea. The crystallizer
ponds yielded two bands, neither of which matched with
that of any of the cultured halophilic Archaea [13]. Another approach used was based on comparison of restriction digests of 16S rDNA ampli¢ed from the DNA extracted from the biomass. Restriction fragment length
polymorphism was determined by ampli¢cation with Archaea- or Bacteria-speci¢c primers, followed by digestion
with AluI, HinfI and MboI. Bacterial diversity was found
to decrease with salinity, while archaeal diversity increased
[14].
More detailed information on the nature of the Archaea
present in crystallizer ponds was obtained by sequencing
16S rDNA genes ampli¢ed from DNA isolated from the
biomass. Most of these studies were performed in the Santa Pola salterns near Alicante, Spain. From the crystallizer
ponds, one archaeal phylotype was recovered almost exclusively. It is only distantly related to Haloferax, its clos-
Fig. 1. Mixed community of halophilic microorganisms from a saltern crystallizer pond near Alicante, Spain, collected by ¢ltration and viewed by scanning electron microscopy. The picture shows square £at Archaea and rod-shaped cells, probably belonging to the genus Salinibacter. Bar, 1 Wm. Courtesy of F. Rodr|¨guez-Valera, Universidad Miguel Hernändez, Alicante.
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A. Oren / FEMS Microbiology Ecology 39 (2002) 1^7
est cultured relative [7,15^17]. The same phylotype also
dominated the archaeal community in the crystallizers of
the Eilat salterns [17]. Similar techniques have been employed in a study of the microbial diversity in the microbial mats covering the sediments of saltern ponds on the
Mediterranean coast of France (Salin-de-Giraud). Two
concentrator ponds were sampled, one with a salinity
that £uctuated between 90 and 158 g l31 , and a second
in which the salt concentration varied between 164 and
228 g l31 (i.e. salinities much below those of the crystallizer ponds). Fourteen and 23 sequences of Halobacteriaceae from the respective sites were characterized. These
sequences were very diverse, and spread all over the phylogenetic tree of the family [18].
The technique of £uorescent in situ hybridization
(FISH) now enables a direct characterization of the
archaeal communities in saltern ponds, while exploiting
16S rRNA sequence information derived from cultured
halophiles or from environmental samples. A probe designed to react with the dominant phylotype obtained
from the Alicante crystallizers reacted with the yet
uncultured, £at, square, gas-vacuolated cells, again con¢rming their abundance in the ecosystem [19]. No cultures
of this organism exist as yet; more or less square Archaea
obtained in culture such as the motile strain 801030/1
isolated from the Sinai brine pool [20] and described
as Haloarcula quadrata [21] are phylogenetically unrelated.
Polar lipids are excellent biomarkers that can be exploited to obtain information on the nature of the microbial communities inhabiting hypersaline environments.
The lipids of the halophilic Archaea are easy to di¡erentiate from bacterial or eukaryal lipids. The structure of the
archaeal lipids is based on phytanyl groups bound by
ether linkages to the glycerol backbone, with various types
of substituents on the third carbon of the glycerol. A considerable diversity in polar lipid structure exists among the
genera and species of the Halobacteriaceae [1]. As polar
lipids can easily be characterized by thin-layer chromatography, they can conveniently be used to characterize not
only pure cultures of halophilic Archaea, but natural communities as well.
In saltern crystallizer ponds, lipid patterns were found
to be relatively simple and to be quite similar in di¡erent
geographic locations. Four main polar lipid fractions were
detected in the Eilat salterns: the diphytanyl derivatives of
phosphatidylglycerol (PG), and of the methyl ester of
phosphatidylglycerophosphate (Me-PGP), the diphytanyl
derivative of phosphatidylglycerosulfate (PGS), and a single glycolipid, chromatographically identical to S-DGD-1
(1-O-[K-D -mannose-(2-SO3
4 )-(1PC4P)-K-D -glucose]-2,3-diO-phytanyl-sn-glycerol) [22]. As this lipid pattern was
found in a community dominated by square, £at, gas-vacuolated cells, it may be assumed that this is also the polar
lipid composition of this organism [10]. Lipid extracts of
biomass collected from crystallizers of the more nutrient-
3
enriched Newark, CA salterns showed a higher complexity
than the oligotrophic salterns of Eilat [23].
[methyl-3 H]Thymidine was used to estimate the growth
rates of prokaryote communities in the saltern of Eilat
[24]. Calculated doubling times of the heterotrophic community in the crystallizer ponds were between 6 and 22.6
days. Similar values were reported in a study of saltern
ponds in Spain, with estimated doubling times between 2.5
and 5 days at salinities between 250 and 372 g l31 [8].
Incorporation of [3 H]leucine has also been used to assess
the growth rate of Bacteria and Archaea in Spanish salterns. Doubling times thus estimated in high-salinity ponds
(250^380 g l31 ) were generally between 2 and 5 days, but
occasionally as long as 70 days [25].
We still know little about the factors responsible for the
death of halophilic Archaea in their natural environment.
Protozoa have never been encountered in large numbers, if
at all, in saltern crystallizer ponds. Death by lysis due to
bacteriophages, however, may occur, as appeared from
studies in Spanish salterns [8,25]. Numbers of presumed
viruses as high as 109 ml31 were observed in NaCl-saturated brines examined in the electron microscope. Between
1% and 10% of the £at square Archaea had visible phages
inside; the estimated burst size was more than 200 viruses
per cell. However, calculations showed that viruses did not
exert a strong control over the prokaryotic abundance and
growth rate: at the highest salinities the percentage of cells
lost daily by viral lysis was calculated to be lower than 5%
[8].
Attempts have been made to assess whether halophilic
archaeocins (halocins) excreted by halophilic Archaea may
inhibit the growth of other archaeal species in the saltern
crystallizers, and thus regulate the archaeal community
size and composition in these ponds. No indication was
obtained that halocins are an important ecological factor
in this ecosystem [26].
2.2. The Dead Sea
The Dead Sea presents unique challenges to the halophilic microorganisms inhabiting it because of its peculiar
ionic composition. Divalent cations dominate over monovalent cations (presently about 1.9 M Mg2‡ and 0.4 M
Ca2‡ , with in addition 1.7 M Na‡ and 0.2 M K‡ ). The
pH is relatively low (about 6.0). Microbial blooms occur
in the lake only after winter rain £oods cause the formation of a diluted upper water layer. A dilution of 10^20%
is required to trigger the development of a bloom of the
unicellular green alga Dunaliella, accompanied by large
numbers of halophilic Archaea. Thus, a prokaryotic community of up to 1.9U107 cells ml31 was observed in 1980,
and even higher numbers (3.5U107 ml31 ) were measured
in 1992. In both those years the lake was colored red due
to the archaeal carotenoids. At other times algae are absent from the water column, and hardly any prokaryotic
microorganisms can be detected [27].
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Bacteriorhodopsin may also be present in the Dead Sea
biota. Biomass collected from the lake in 1981 had the
characteristic purple color of bacteriorhodopsin. The report of its abundance in the community (up to 0.6 nmol
l31 or 0.4 nmol mg protein31 ) [28] was the ¢rst account of
the occurrence of this pigment in any natural community
of halophilic Archaea. For comparison, Javor [4] found
2.2 nmol l31 bacteriorhodopsin in the archaeal community
of an oligotrophic saltern crystallizer pond in Baja California (Mexico), while in a more eutrophic saltern in California the retinal pigment could not be detected. At least
one Dead Sea isolate, Halorubrum sodomense, can synthesize purple membrane. In the autumn of 1981, a time at
which halophilic Archaea were still abundant but very few
Dunaliella cells were found, the low level of light-dependent CO2 ¢xation was probably driven by bacteriorhodopsin rather than by chlorophyll. Evidence for this was obtained from the action spectrum of the process and by the
use of speci¢c inhibitors [29]. Di¡erent mechanisms have
been suggested to explain the nature of the bacteriorhodopsin-driven CO2 photoassimilation, such as carboxylation of propionyl-CoA to yield K-ketobutyrate or reactions leading to the biosynthesis of glycine.
Studies with speci¢c inhibitors such as bile acids and
antibiotics a¡ecting protein synthesis (see also Section
3.1) showed that in 1988 (a period in which only low
numbers of microorganisms were present in the lake's
water column) all heterotrophic activity could be attributed to halophilic Archaea [30,31].
A variety of archaeal halophiles have been isolated from
the Dead Sea, including Haloferax volcanii, Haloarcula
marismortui, Hrr. sodomense, and Halobaculum gomorrense. Little is known about the importance of these and
possibly other, as yet uncultured species. Analyses of polar
lipids extracted from the community that formed the
bloom in 1992 showed a simple pattern. Only three polar
lipid fractions were detected: PG, Me-PGP, and a single
glycolipid, chromatographically identical to S-DGD-1;
PGS was absent [32]. Such a lipid composition is characteristic of the genus Haloferax and also of Hbl. gomorrense, a species isolated from the bloom.
We know little about the factors responsible for the
decline in bacterial numbers following the occasional massive blooms. The ¢nding of large numbers of virus-like
particles in the lake [33] suggests that bacteriophages
may be involved in controlling the community size of prokaryotes in the Dead Sea.
2.3. Solar Lake
Solar Lake is a small lake on the Sinai peninsula
(Egypt) on the shore of the Gulf of Aqaba. In summer
the water column of the pond is hypersaline (about 200 g
l31 ) and aerobic down to the bottom (maximum depth
4.5^5 m). In winter the lake is strati¢ed, with a layer of
less saline (about 60 g l31 ) water £oating on top of the
heavier bottom waters (180^200 g l31 ). The hypolimnion
rapidly turns anaerobic, and heats up to temperatures of
55^60³C and higher due to heliothermal heating.
The archaeal biodiversity of the water column of Solar
Lake was recently studied throughout the annual cycle.
Archaeal 16S rDNA was ampli¢ed from the biomass, separated by denaturing gradient gel electrophoresis, and sequenced. Archaea were abundantly detected in the water
column, both during summer mixing and during winter
strati¢cation, including in the hot anaerobic, sul¢de-rich
hypolimnion [34]. Of the 165 archaeal clones analyzed, 144
belonged to the Halobacteriaceae, including 92 out of the
104 clones obtained from the anaerobic layer during strati¢cation. Two clusters of clones of Halobacteriaceae sequences recovered shared 94% sequence identity. Their
closest cultivated relative is Haloferax (89% identity), but
an even closer relationship was found with the phylotype
most abundant in saltern crystallizers [15^17], now assigned to the gas-vacuolated £at square Archaea [19]. Representatives of this cluster were found both in the aerobic
and in the anaerobic parts of the water column, and at
temperatures ranging from 15 to 55³C [34].
2.4. Antarctic hypersaline lakes
The hypersaline lakes of the Vestfold Hills lake system
of Eastern Antarctica have been the subject of a number
of studies on microbial distribution. Recently, 16S rDNA
sequencing techniques have been included in these studies.
One of these lakes is Deep Lake, a monomictic, 36 m deep
lake with a salinity of 320 g l31 and temperatures between
314 and 318³C. The biodiversity in Deep Lake is low,
and is dominated by Archaea of the family Halobacteriaceae. The predominant phylotype was closely related to
Halorubrum lacusprofundi, a cold-tolerant halophilic Archaeon originally isolated from this lake. Furthermore,
three deep-branching clusters of novel types of Archaea
were detected [35]. Analysis of polar lipids recovered
from the lake's sediments yielded a lipid ¢ngerprint very
similar to that of Hrr. lacusprofundi [36].
2.5. African alkaline hypersaline lakes
Lake Magadi (Kenya), an alkaline lake in the East African Rift Valley, is salt-saturated, and contains a precipitate of trona (sodium sesquicarbonate). The pH of the
brine is about 10. Alkaliphilic members of the Halobacteriaceae dominate the microbial community.
A number of halophilic alkaliphilic Archaea have been
isolated from the lake and characterized (Natronobacterium gregoryi, Natrialba magadii, Halorubrum vacuolatum,
Natronococcus occultus). The archaeal community populating the crystallizer ponds of the alkaline (pH about
12) solar salterns on the shore of the lake has recently
been characterized by 16S rDNA sequencing of clones
obtained after PCR ampli¢cation from DNA extracted
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A. Oren / FEMS Microbiology Ecology 39 (2002) 1^7
from the biomass [37,38]. Most sequences recovered
shared more than 95% identity to each other, but only
88^90% to Natronomonas pharaonis, their closest relative
among the cultivated haloalkaliphilic Archaea. Two other
clones retrieved were only 76% similar to any known
archaeal sequence, showing that also in this extreme environment the microorganisms that dominate the community are still awaiting isolation.
3. Ecology of extremely halophilic Bacteria
3.1. Activity of halophilic Bacteria at the highest salt
concentrations
Until recently it was assumed that at salt concentrations
at or near NaCl saturation, Archaea of the family Halobacteriaceae are the only active aerobic heterotrophs. This
assumption was based in part on culture experiments: red
colonies of organisms that did not grow below 100^150 g
l31 salt were generally the only colony type recovered.
Secondly, activity measurements in the presence of inhibitors speci¢c for either archaeal or bacterial activities suggested that above about 250 g l31 salt essentially all heterotrophic activity could be attributed to Archaea.
Already in 1956 it was proposed that bile acids (BactoOxgall) may be a useful agent to di¡erentiate between red
archaeal halophiles (not yet recognized as such at the time)
and other types of halophilic microorganisms [39]. The
ability of bile acids (deoxycholate, taurocholate) at low
concentrations to lyse halophilic Archaea was exploited
in studies of the uptake of radioactively labeled amino
acids in saltern brines in Eilat. Above 300 g l31 salt, 50
mg l31 taurocholate caused complete inhibition [40]. Similarly, in Spanish salterns of the Ebro delta and near Alicante, taurocholate completely inhibited incorporation of
[3 H]leucine at the highest salinities, while below 200 g/l
relatively little inhibition was observed [25].
Antibiotics speci¢cally a¡ecting protein synthesis in Archaea or in Bacteria have also been employed in ecological
studies. Specially useful are anisomycin (inhibiting Archaea and Eucarya) and chloramphenicol or erythromycin, inhibiting the bacterial protein synthesis machinery.
Incorporation of labeled amino acids by samples collected
from crystallizer ponds in Eilat was inhibited more than
95% by anisomycin [30,31]. Erythromycin has been employed in similar experiments in saltern ponds in Spain.
While as expected [3 H]leucine incorporation was fully inhibited by erythromycin in the lower salt concentration
range, only half of the activity was inhibited in the Archaea-dominated crystallizer ponds [25]. In the Eilat salterns most of the amino acid uptake at the highest salinities
was resistant to chloramphenicol [30,31].
Also DNA synthesis can be targeted by speci¢c antibiotics. Aphidicolin, a potent inhibitor of halobacterial
DNA polymerase, completely abolished incorporation of
5
[methyl-3 H]thymidine in saltern crystallizer ponds in Eilat
[24].
3.2. Salinibacter ruber, an extremely halophilic Bacterium
In spite of the above, it is now becoming clear that
Bacteria may also contribute to the aerobic heterotrophic
prokaryotic community at the highest salt concentrations.
PCR ampli¢cation of 16S rDNA from biomass collected
from saltern crystallizers in Spain, using Bacteria-speci¢c
primers, yielded sequences distantly related to Rhodothermus marinus (Cytophaga/Flavobacterium/Bacteroides phylum) [41]. Similar sequences have also been recovered
from salterns in the south of France [18]. Using £uorescent
oligonucleotide probes designed to detect this phylotype,
the organism was shown to be rod-shaped (Fig. 1), and to
be very abundant: in the crystallizer ponds on Ibiza and
Mallorca between 18 and 27% of all prokaryotes belonged
to this type, in crystallizers on the Canary Islands they
were less abundant (5^8%) [41]. It may be noted that the
¢nding of a speci¢c type of Bacteria adapted to life at the
highest salt concentrations was already predicted by an
earlier study of restriction fragment length polymorphism
of 16S rDNA ampli¢ed from the Spanish salterns using
Bacteria-speci¢c primers [14]. Bacterial restriction fragment patterns were obtained from the crystallizer ponds,
and these were very di¡erent from those retrieved from
lower salinity ponds. The statement by Martinez-Murcia
et al. that the crystallizer environment `probably represents an extremely specialized niche for Bacteria' [14] predates the isolation of such bacteria by 5 years.
The organism harboring this novel phylotype has recently been isolated and described as a new genus and
species : S. ruber. Recognition of bacterial colonies was
based either on their polar lipid pattern or on hybridization with a speci¢c £uorescent 16S rRNA-targeted probe
[3]. Salinibacter is a motile rod, pigmented red by a pigment (probably a carotenoid) with an absorption maximum at 482 nm and a shoulder at 506^510 nm. Because
of the red pigmentation of the colonies this type of Bacterium has probably been overlooked in the past, red colonies growing at 250 g l31 salt having been considered to be
archaeal. The organism is no less halophilic than the
archaeal halophiles: no growth was obtained below 100
g l31 NaCl, and for optimal growth concentrations between 150 and 230 g l31 are required. The physiological
properties of Salinibacter are unusual: the organism apparently uses KCl to provide osmotic balance, while lacking high concentrations of organic osmotic solutes [3].
Thus, its physiology resembles that of the halophilic Archaea more than that of other aerobes within the domain
Bacteria.
HPLC analysis of pigments extracted from Spanish saltern crystallizer ponds enabled the identi¢cation and quantitative assessment of the red pigment of Salinibacter. Approximately 5% of the total prokaryotic pigment
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A. Oren / FEMS Microbiology Ecology 39 (2002) 1^7
absorbance could be attributed to the presence of Salinibacter. The pigment was not detected in samples collected
from crystallizers of Eilat, and possibly traces of it were
found in the salterns in San Francisco Bay near Newark,
CA [42].
ery of Salinibacter shows, breakthroughs are possible, so
that it still may be relatively easy to obtain a proper
understanding of the microbial ecology of hypersaline environments in the near future.
References
4. Epilogue
The above survey shows that our understanding of the
community structure of aerobic halophilic microorganisms
in hypersaline lakes is still limited. Application of rRNAbased characterization techniques has demonstrated that
those microorganisms known in culture are not dominant
in the natural environment, and that most of the ecologically important organisms are still awaiting isolation.
Hbt. salinarum, the type strain and the best-known representative of the family Halobacteriaceae, is not a quantitatively important component of the microbial community
in any hypersaline lake investigated, although it can be
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