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Applied and Environmental Microbiology, December 2001, p. 5392-5402, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5392-5402.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Microbial Communities in the Chemocline of a
Hypersaline Deep-Sea Basin (Urania Basin, Mediterranean Sea)
Andrea M.
Sass,
Henrik
Sass,
Marco J. L.
Coolen,
Heribert
Cypionka, and
Jörg
Overmann*
Paleomicrobiology Group, Institute for the
Chemistry and Biology of the Marine Environment, University of
Oldenburg, D-26111 Oldenburg, Germany
Received 5 April 2001/Accepted 23 August 2001
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ABSTRACT |
The Urania basin is a hypersaline sulfidic brine lake at the bottom
of the eastern Mediterranean Sea. Since this basin is located at a
depth of ~3,500 m below the sea surface, it receives only a small
amount of phytoplankton organic carbon. In the present study, the
bacterial assemblages at the interface between the hypersaline brine
and the overlaying seawater were investigated. The sulfide
concentration increased from 0 to 10 mM within a vertical interval of
5 m across the interface. Within this chemocline, the total
bacterial cell counts and the exoenzyme activities were elevated.
Employing 11 cultivation methods, we isolated a total of 70 bacterial
strains. The 16S ribosomal DNA sequences of 32 of the strains were
identical to environmental sequences detected in the chemocline by
culture-independent molecular methods. These strains were identified as
flavobacteria, Alteromonas macleodii, and Halomonas
aquamarina. All 70 strains could grow
chemoorganoheterotrophically under oxic conditions. Sixty-six strains
grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the
isolates could grow both chemoorganotrophically and
chemolithotrophically. While the most probable numbers in most cases
ranged between 0.006 and 4.3% of the total cell counts, an unsually
high value of 54% was determined above the chemocline with media
containing amino acids as the carbon and energy source. Our results
indicate that culturable bacteria thriving at the oxic-anoxic interface
of the Urania basin differ considerably from the chemolithoautotrophic
bacteria typical of other chemocline habitats.
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INTRODUCTION |
In pelagial lakes and certain
marine environments, strong salinity or temperature gradients can
prevent mixing and hence lead to stratification of the water column.
Steep vertical gradients of oxygen, sulfide, ammonium, and sometimes
methane concentrations occur across the interface layer (22, 30,
41). In most cases, these chemical gradients across the
chemocline originate from microbial activities. Compared to the
microbial assemblages in the upper and lower water layers, the
microbial assemblages at chemoclines are characterized not only by
higher levels of bacteria, protozoans, and zooplankters (23,
41) but also by higher microbial activities, like
CO2 fixation and exoenzyme activities (31, 41,
43). In many stratified ecosystems, the chemocline is characterized by an intense cycling of sulfur compounds (30, 43,
54). The chemocline thus offers a variety of ecological niches
on a small spatial scale and consequently may also harbor a highly
diverse microbial community.
In the pelagic marine environment, oxic-anoxic interfaces are mostly
limited to coastal lagoons or fjords; the euxinic Black Sea is a
notable exception. However, several hypersaline basins have been
discovered on the seafloor in different oceanographic provinces, like
the Gulf of Mexico (51), the Red Sea (46), and the Mediterranean Sea (29). In the Mediterranean Sea,
these so-called brine lakes most likely developed by the dissolution of
5- to 8-million year-old Messinian evaporites (9) which became exposed to seawater during the collision of the African and
Eurasian tectonic plates. Alternatively, the depressions may have been
formed by accrecionary processes and may have subsequently been filled
with fossils and highly concentrated relics of Messinian seawater
trapped in the interstitial areas of the deep-sea sediments (58).
The deep-sea anoxic hypersaline basins of the eastern Mediterranean Sea
(Fig. 1) are located far below the photic
zone. In addition, the primary productivity in the surface waters is
extraordinarily low due to the antiestuarine circulation pattern of the
Mediterranean Sea (61). Because of the low production and
long sedimentation path, only a small flux of organic carbon reaches
the deep-sea community at the chemoclines of the brine lakes. At the
same time, the occurrence of sulfide in the Bannock and Tyro basins
(20; W. Ziebis, M. E. Böttcher, A. Weber, J.-C.
Miquel, S. Sievert, and P. Linke, J. Conf. Abstr. Goldschmidt Conf.
2000 5:1134, 2000), which are depleted in 34S
(33; Ziebis et al., J. Conf. Abstr. Goldschmidt Conf.
2000), may indicate that active sulfate-reducing bacteria are present. So far, little is known about the abundance, diversity, and
physiological capabilities of microorganisms that thrive in these
unusual chemocline systems. Only recently, microbial sulfate reduction
in the water column of the deep Urania basin was directly shown by
radiotracer techniques (Ziebis et al., J. Conf. Abstr. Goldschmidt
Conf. 2000).
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FIG. 1.
Locations of the Urania basin and other hypersaline
brine lakes in the eastern Mediterranean Sea.
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In this paper we describe the environmental conditions in the
chemocline of the Urania basin in the eastern Mediterranean Sea and an
analysis of the indigenous microbial community and present evidence
that at least some of the numerically significant bacteria can be
cultivated by using a set of different cultivation conditions.
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MATERIALS AND METHODS |
Sampling site.
The Urania basin (Fig. 1) is a
horseshoe-shaped brine lake located west of Crete in the eastern
Mediterranean Sea. The surface of this brine lake lies 3,470 m below
sea level (35), and the maximum brine thickness is
200 m at its southwestern end. The salinity of the brine is five
times higher than that of standard seawater (58).
Sampling was performed during cruise M40/4 of the R/V
Meteor
at station 76 (35°13,83'N, 21°28,29'E) in January 1998. Water
samples were taken at 5- or 10-m intervals from 16 different water
depths by using a rosette sampler (Hydro-Bios, Kiel, Germany)
equipped
with Niskin bottles (General Oceanics, Miami, Fl.). Sampling
through
the chemocline was performed at 5-m intervals. Three depths
were chosen
for microbiological analyses: 3,455 m (above the chemocline),
3,475 m
(within the chemocline), and 3,500 m (below the chemocline).
The
vertical position of the chemocline was located by means of
the drop in
oxygen concentration recorded with a CTD probe (see
below).
Chemical and physical parameters.
Temperatures and oxygen
concentrations in the water column were measured online during sampling
by using a CTD probe (type SBE 19 CTD; Seabird Electronics, Bellevue,
Wash.) connected to the rosette sampler. Depth was determined with the
pressure sensor of the CTD probe.
Chloride and sulfate concentrations were measured by ion chromatography
(Sykam, Gilching, Germany) (
3). Because of their
high salt
concentrations, samples of seawater and the brine were
diluted 100- to
1,000-fold in eluent prior to analysis. Standards
were prepared as
mixed salt solutions that had a composition corresponding
to that of
the brine and were diluted as well. Parallel analyses
of several
standards demonstrated that the standard errors due
to dilution were
between 0.1 and 3%. The
ortho-phosphate content
was
determined photometrically by the molybdenum blue method
(
50).
For determination of sulfide concentrations, water
samples were
immediately mixed with a 1 M zinc acetate solution so that
the
final zinc concentration was 100 mM. The samples were stored in
glass ampoules which were flushed with N
2 and sealed with
butyl
rubber stoppers. Sulfide was assayed spectrophotometrically by
using the methylene blue method of Cline (
11).
Sulfur isotope values were determined by separate precipitation of
sulfide and sulfate as Ag
2S and BaSO
4 and
subsequent isotope
analysis by combustion isotope ratio monitoring mass
spectrometry
(
7). Because of the high concentrations of
chloride in the
brine samples, sulfide was first precipitated as ZnS;
the latter
was collected and acidified, and the liberated sulfide was
flushed
out by a stream of N
2 and reprecipitated as
Ag
2S in a second trap.
Isotope fractionation was calculated
from ratios of
34S to
32S for samples
(
Rsample) and for the V-CDT sulfur standard
(
Rstd),
as follows:
34S = [(
Rsample/R
std)
1] × 1,000.
Microbiological parameters.
The potential activities of the
hydrolytic exoenzymes alkaline phosphatase (EC 3.1.3.1),
-glucosidase (EC 3.1.21), and leucine aminopeptidase (EC 3.4.1.1)
were measured on board immediately after sampling by using
fluorescently labeled substrate analogues (25, 26). The
measurements for brine samples were obtained under anoxic conditions.
Total cell counts were determined by epifluorescence microscopy.
Samples were preserved with filtered (pore size, 0.1 µm)
glutardialdehyde at a final concentration of 2%. Bacterial cells
were
stained with DAPI (4',6-diamidino-2-phenylindole) (
48).
Source of organisms.
In this study a total of 70 bacterial
strains were investigated (Table 1). The
strains were isolated from the three water depths of the Urania basin
by employing 11 isolation procedures (Table 1). Besides agar plates
containing peptone, hydrolyzed casein, and soluble starch (CPSm medium)
(13), liquid anaerobic cultures with different substrates
and sulfide-oxygen gradient tubes were employed. The basal liquid
medium consisted of artificial seawater (13). One
milliliter of trace element solution SL 10 (60) and 1 ml
of a solution of selenite and tungstate (60) were added,
and the pH was adjusted to 7.5. After autoclaving, 10 ml of a sterile
filtered vitamin solution (4) per liter and 10 ml of a
sterile sodium bicarbonate solution (20 g · liter
1) per liter were added. For anoxic incubation
experiments the medium was reduced with 400 µM sterile sodium
sulfide. The salt concentrations of the medium used for samples from
the brine and the sediment surface were elevated to twice those of
seawater by adding a concentrated salt solution (290 g of NaCl per
liter, 40 g of MgCl2 · 6H2O per
liter).
In order to generate a variety of incubation conditions, the basal
medium was supplemented with either acetate (15 mM), lactate
(10 mM), a
mixture of volatile fatty acids (containing 3.75 mM
acetate, 1.5 mM
propionate, 1.5 mM butyrate, 0.75 mM isobutyrate,
0.75 mM valerate,
0.75 mM 2-methylbutyrate, and 0.75 mM 3-methylbutyrate),
or a mixture
of amino acids (containing 2 mM alanine, 1 mM arginine,
1 mM
asparagine, and 1 mM cysteine). Thiosulfate-disproportionating
bacteria
were grown with 15 mM thiosulfate and 2 mM acetate. Sulfur-oxidizing
bacteria were obtained in media containing thiosulfate (10 mM)
or
polysulfide (15 mM S
0 and 6 mM sulfide) and either
supplemented with nitrate (15 mM)
or incubated under oxic conditions.
All the media described above
were employed in most-probable-number
(MPN) dilution series. After
6 weeks, growth was monitored, viable cell
counts were calculated,
and strains were isolated from different
parallel preparations
of the highest positive dilutions by repeated
application of the
deep agar dilution method (
60).
Gradient tubes were established by adding polysulfide to anoxic
artificial seawater containing sterile molten agarose (1%).
After
solidification, the tubes were incubated under
air.
DNA extraction.
Immediately after sampling, 8-liter portions
of water from the three selected depths were filtered through sterile
polycarbonate filters (diameter, 45 mm; pore size, 0.2 µm; Millipore,
Bedford, Mass.). DNA was extracted with buffered phenol at 55°C,
phenol-chloroform, and chloroform (44). Subsequent
purification was performed with an EasyPure kit (Biozym, Hessisch
Oldendorf, Germany).
Genomic DNA from bacterial cultures was extracted by a freeze-thaw
protocol (
28). One microliter of a cell pellet was added
to 50 µl of filter-sterilized 50 mM Tris buffer (pH 7.4). The
suspension was heated to 100°C for 3 min and then incubated at
80°C for 3 min. This procedure was repeated five times, and 1
µl
of the extract was added to 50 µl of the PCR
mixture.
Amplification, separation, and sequencing of 16S rDNA
fragments.
DNA fragments (length, 630 bp) of the natural community
and of pure cultures were amplified by using eubacterial primers GC357f containing a 40-bp GC clamp (5'-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC CCC TAC GGG AGG CAG CAG-3') (37)
and 907r (5'-CCG TCA ATT CCT TTG AGT TT-3'). A hot start at
96°C (4 min) was applied; during cycling, the melting temperature was
set at 94°C (30 s), and the extension temperature was 72°C (1 min).
A step-down procedure was applied by using an annealing temperature of
61°C for the first 10 cycles and of 56°C for the subsequent 20 cycles.
The 16S ribosomal DNA (rDNA) fragments generated were separated on the
basis of their melting behavior by denaturing gradient
gel
electrophoresis (DGGE) (
37). DGGE was carried out with the
Bio-Rad D Gene system (Bio-Rad, Munich, Germany) as described
previously (
44). Individual DNA bands from the natural
community
and from strains with multiple rRNA operons were excised with
a sterile scalpel and then electroeluted (Centrilutor
Micro-Electroelutor;
Amicon, Witten, Germany) and purified
(
12). 16S rRNA genes of
other pure cultures which did not
display multiple bands during
DGGE were amplified and sequenced
directly.
Sequencing was performed with a SequiTherm Long-Read kit (Epicentre,
Madison, Wis.) and an automated infrared laser fluorescence
sequencer
(Li-Cor model 4000 DNA sequencer) (
44). The primers
used
for sequencing were the same as those used for PCR amplification,
except that the GC clamp of primer GC357f was
omitted.
Phylogenetic analysis.
For each 16S rDNA sequence, the most
closely related sequence and the sequence of the most closely related
cultured bacterial strain were retrieved from the GenBank database by
using BLASTN 2.0 (1) and from the Ribosomal Database
Project by using the SIMILARITY_CHECK option (34) (Table
2).
The sequences were aligned with the CLUSTAL W program
(
57). Nucleotide positions that differed in more than 50%
of all sequences
were excluded from the analysis. This resulted in a
total of 452
informative nucleotide positions. Phylogenetic trees were
calculated
with the maximum-likelihood program (DNAML) and the
parsimony
program (DNAPARS) of the PHYLIP 3.57c package
(
19).
ERIC PCR.
In order to investigate the genomic heterogeneity
among isolates of the same phylotype, enterobacterial repetitive
intergenic consensus (ERIC) fingerprinting (59) was
carried out. The primers used were ERIC1R (5'-ATG TAA GCT CCT GGG
GAT TCA C-3') and ERIC2 (5'-AAG TAA GTG ACT GGG GTG AGC
G-3'). The PCR conditions used and the method used for
visualization of products have been outlined earlier (44).
The resulting ERIC band patterns were subjected to a cluster analysis.
A binary 0/1 matrix was created based on the absence
or presence of DNA
bands. Pairwise distances were calculated with
the SimQual option of
the NTSYSpc 2.02j computer program (Exeter
Software, Setauket, N.Y.) by
employing the Dice coefficient for
two-state data. For the cluster
analysis we employed the SAHN
option of the package using the
unweighted pair group method with
arithmetric average for
clustering.
Nucleotide sequence accession numbers.
Sequences
representing each of the 12 different cultured phylotypes, five
environmental sequences, and four sequences of multiple operons of
isolate UM4b have been deposited in the GenBank database under
accession numbers AF321058 through AF321078.
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RESULTS |
Chemical and physical characterization of the habitat.
On our
sampling date, the chemocline of the Urania basin was located at a
depth of 3,470 m below the sea surface. Within a 5-m depth interval,
the oxygen concentration decreased below the detection limit, whereas
the sulfide concentration reached 10 mM. Similarly, the concentrations
of chloride, sulfate, and phosphate increased markedly (to 2.8 M, 85 mM, and 41 µM, respectively) (Fig. 2A and
B; Table
3). Across this chemocline the
temperature increased slightly (by 2.5°C), and there was a
significant decrease in pH from 8.6 to 6.8. The 34S
values for sulfate were 20.6 ± 0.14
above the chemocline and 25.4 in the brine. The 34S value of sulfide was found
to be 15.2 ± 0.2 in samples from the chemocline, as well as
in brine samples. This confirmed earlier measurements (Ziebis et al.,
J. Conf. Abstr. Goldschmidt Conf. 2000).
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FIG. 2.
Physicochemical parameters, total cell counts, and MPN
across the oxic-anoxic interface of the Urania basin. (A) Oxygen and
sulfide concentrations; (B) sulfate, ortho-phosphate, and
chloride concentrations; (C) total cell counts; (D) viable cell counts
in anoxic media containing the amino acid mixture (solid bars) and in
oxic media containing thiosulfate (cross-hatched bars). Only values for
the media which yielded maximum MPN are shown. The percentages are
percentages relative to the total cell count. The error bars indicate
standard deviations.
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Total and viable cell counts.
The total concentration of
bacterial cells reached 1.3 × 105 cells · ml1 in the chemocline and increased to a maximum of
2.2 × 105 cells · ml1 at 10 m below the chemocline (Fig. 2C). The concentrations within and just
below the chemocline were thus slightly, but significantly (P < 0.005), elevated compared to the concentrations
in most water layers above the chemocline and to the concentrations in
the brine below the chemocline.
The maximum viable cell counts were obtained for samples from 3,455 m
when anoxic media with alanine, arginine, asparagine,
and cysteine as
the sole carbon and energy sources were used.
In these media up to 54%
of all bacterial cells could be cultivated
by the MPN technique (Fig.
2D). In contrast, the MPN values for
the chemocline were highest in
media supplemented only with thiosulfate
and incubated under oxic
conditions; a smaller fraction of the
cells (4.3%) grew in these
media. The culturability was substantially
lower in samples from the
anoxic brine, reaching 0.03% of total
cell counts with
thiosulfate as the substrate and 0.006% with
amino acids as the
substrates.
Exoenzyme activity.
Within the chemocline, the activities of
the exoenzymes -glucosidase, aminopeptidase, and alkaline
phosphatase were significantly increased compared to the activities in
the water layers above and below (Fig.
3). The highest activity values were
those observed for alkaline phosphatase, which reached 20.5 ± 1.5 nM · h1. The cell-specific exoenzyme activities in
the chemocline were 15.8 × 1017 mol · cell1 · h1 for alkaline phosphatase
and 4.58 × 1017 and 1.57 × 1017
mol · cell1 · h1 for
aminopeptidase and -glucosidase, respectively (Fig. 3).
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FIG. 3.
Extracellular -glucosidase, leucine aminopeptidase,
and alkaline phosphatase activities in the Urania basin. The error bars
indicate standard deviations.
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16S rDNA fingerprinting.
After amplification of environmental
DNA with the eubacterial primer set, 12 distinct bands were separated
by DGGE (Fig. 4A). DNA samples from the
three different depths examined yielded similar DGGE fingerprints;
therefore, only the chemocline sample was analyzed further. After
excision and sequencing, five of the bands (A1, A2, A4, A7, and A8)
contained a single 16S rDNA, as judged from the absence of ambiguous
base positions on sequencing gels. The five sequences were included in
the phylogenetic analyses. In all cases, these molecular isolates
clustered with sequences of typical marine bacteria (Fig.
5). Phylotype A4 was distantly related to
the sequence of an uncultured member of the 
subclass of the class
Proteobacteria (-proteobacteria) recovered from Pacific deep-sea sediments; its closest cultured relative was the marine organism Roseobacter algicola (Fig. 5). The partial
sequences of bands A1 and A7 were identical or almost identical
(99.8%) to sequences of the marine species Halomonas
aquamarina and Alteromonas macleodii, respectively. The
closest relative of environmental sequence A8 was an uncultured
bacterium from a hydrocarbon seep. Finally, pronounced band A2
contained a 16S rDNA sequence which fell in the
Cytophaga-Flavobacterium group and was closely related to an
uncultured marine bacterium detected in the phycosphere of a marine
diatom (Fig. 5). An analysis by the parsimony method fully supported
the phylogenetic tree generated by the maximum-likelihood method.
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FIG. 4.
Comparison of 16S rDNA fragments of the strains isolated
with fingerprints of the natural bacterial community in the chemocline
by DGGE fingerprinting. Negative images of ethidium bromide-stained
gels are shown. (A) Anaerobically isolated strains; (B) aerobically
isolated strains. Strains were assigned to species and to genera if
sequence identities were >97 and >93%, respectively. Strains marked
by shading had 16S rDNA sequences identical to those of band A1, A2, or
A7. CFB-group, Cytophaga-Flexibacter-Bacteroides
group.
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FIG. 5.
Phylogenetic affiliations of the strains isolated and
the molecular isolates from the chemocline bacterial community.
The tree is based on 452 informative nucleotide positions of the 16S
rRNA gene. Chlorobium limicola was used as the outgroup.
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Of the 70 bacterial isolates obtained in the present study, 27% were
affiliated with the
-proteobacteria, 62% were affiliated
with the
-proteobacteria, 8% were affiliated with the
Cytophaga-Flavobacterium group, and 3% were affiliated with
the high-G+C-content gram-positive
bacteria (Fig.
4 and
5). A
comparison of the 16S rDNA fingerprints
of the natural bacterial
community with those of the 70 cellular
isolates revealed that about
one-half of our strains (32 strains)
potentially represent bacteria
which are also detectable by culture-independent
methods in the natural
bacterial community of the chemocline (Fig.
4). Sequencing confirmed
that strains with 16S rDNA fingerprints
corresponding to those of bands
A1, A2, and A7 indeed also contained
the same sequences (Fig.
5).
Eighteen strains of
H. aquamarina represent phylotype A1 of
the natural community. Six cellular
isolates had partial 16S rDNA
sequences identical to the band
A2 sequence and grouped with the
Cytophaga-Flavobacterium division.
Eight of the strains isolated (U4, U7, U8, U10, U12, UM4b, UM7, UM8)
showed multiple DGGE bands (Fig.
4B) which were indicative
of the
presence of multiple 16S rRNA operons (
39). Sequencing
the
four most frequently detected bands (lowermost bands
rrnA through
rrnD in Fig.
4B) of two of the strains (U4, UM4b)
revealed
that the bands with different melting behaviors differed in
only
one or two bases. All sequences clustered closely with the
A. macleodii sequence (Fig.
5); one of the bands
(
rrnD) corresponded
to band A7 of the natural bacterial
community.
In order to estimate the abundance of phylotypes A1, A2, and A7 in the
natural community, we performed MPN-PCR (
40) at fivefold
dilution steps using three replicates. For each dilution, the
presence
of phylotypes A1, A2, and A7 among the amplification
products was
checked by DGGE. Amplicons of phylotypes A1 and A7
were detected in PCR
assay mixtures that contained 2 ng of genomic
DNA, whereas amplicons of
A2 were found also with the next dilution
step mixtures (containing 0.4 ng of DNA). Based on the MPN values
obtained (1.6 × 10
10 and 8.2 × 10
10 targets per g of
genomic DNA, respectively), phylotypes A1 and
A7 contribute about 3.6%
of the total eubacterial template in
the chemocline (confidence
interval, 0.003 to 17.3%) and phylotype
A2 contributes about 18.3%
(confidence interval, 1.4 to 88.6%).
Genetic and physiological diversity within the same phylotype.
The three groups of bacterial strains which represented phylotypes of
the natural community consisted of 6 to 18 identical partial 16S rDNA
sequences and hence phylogenetically were very closely related or even
identical. Therefore, we investigated whether the different bacterial
strains of the same phylotype were genetically and physiologically
different or whether members of one homogeneous population had been
obtained. The genomic heterogeneity as analyzed by ERIC PCR was
considerable in all three groups (Fig. 6). Only a few strains yielded identical
ERIC fingerprint patterns. Even strains from the same water sample and
isolated in the same medium were genetically different. Genetic
diversity was highest among members of the A. macleodii
cluster. The diversity among strains of the same phylotype was also
assessed by physiological characterization of the 70 strains using 67 growth substrates and different incubation conditions (A. Sass, J. Overmann, M. Coolen, H. Cypionka, and H. Sass, submitted for
publication). These tests revealed considerable physiological
differences between strains of the same phylotype isolated from the
same depth, and the results support the view that different bacterial
ecotypes had been isolated from the chemocline of the Urania basin.
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FIG. 6.
Genetic diversity of the three groups of bacterial
isolates which represent numerically significant phylotypes. The
dendrograms are based on ERIC PCR band patterns. CF-group,
Cytophaga-Flavobacterium group.
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Culturability.
Most notably, the success of cultivation of
bacteria belonging to phylotypes A1, A2, and A7 was found to depend on
the type of medium employed and also on the sampling depth (Table 1). All members of the Cytophaga-Flavobacterium group were
obtained by cultivation on complex solid media under oxic conditions
when the sample obtained above the chemocline was used. No isolate belonging to this group was obtained from the chemocline, although a
corresponding molecular fingerprint was clearly detected in this sample
by the culture-independent method (Fig. 4A, band A2). This result may
reflect vertical differences in the viability of the bacteria. Strains
of A. macleodii were isolated on the complex solid media, as
well as in media containing only thiosulfate and O2 (Table
1). The numerous strains of H. aquamarina were isolated
either as aerobic organoheterotrophs on complex solid medium or as
anaerobic organoheterotrophs with fatty acids or lactate and in media
designed for chemolithotrophs containing polysulfides or supplemented
with thiosulfate.
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DISCUSSION |
Representative character of the culture collection.
Different
statistical analyses showed that the culture collection investigated in
the present study is large enough to represent the majority of the
bacteria which are culturable by our methods. First, the cumulative
plot of the different phylotypes reached saturation (Fig.
7); a total of 13 phylotypes could be
distinguished. Rarefaction analysis (Fig. 7) also revealed that
continued isolation of strains would have yielded very few new
phylotypes. Second, a high sample coverage value was obtained in the
present study. The coverage (C) [C = 1 (n1/N), where
n1 is the number of phylotypes which occurred
only once in our culture collection and N is the total
number of strains (36)] was 94.4%. This very high value indicates that our culture collection included almost all of the bacterial phylotypes of the natural community which grew in the 11 media used.
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FIG. 7.
Diversity of phylotypes in the collection of 70 bacterial isolates. A total of 13 phylotypes were detected. After the
order of strains was randomized, sequential detection of phylotypes was
plotted in a cumulative manner ( ). For comparison, a rarefaction
curve (solid line) was produced from the data by employing the freeware
program Analytical Rarefaction 1.2 (compiled by Steven M. Holland; available at http://www.uga.edu/~strata/Software.html). The
dotted lines indicate 95% confidence intervals.
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Bacterial culturability.
One of the most pressing problems in
bacterioplankton ecology is the small fraction of the total bacterial
cells which can be cultivated (2, 18). Especially low
values have been reported for seawater samples (0.001 to 0.1%)
(2), and it has been suggested that the vast majority of
the numerically important bacteria have not been cultured so far
(55). In the present study, three 16S rDNA sequences
detected in the natural community were also present in almost one-half
of our bacterial isolates. Commonly, only those 16S rDNA sequences
which constitute more than 1% of all template molecules in a natural
bacterial community are detectable by the PCR-DGGE approach
(38). Based on the results of our MPN-PCR analysis,
phylotypes A1, A2, and A7 contributed approximately 4 to 18% of all
eubacterial template molecules. The isolates of the
Cytophaga-Flavobacterium group are especially important
numerically. Our results support the view that at least some of the
abundant marine bacteria are readily culturable (21, 47).
Only a single sampling was performed, and limited amounts of sample
material from the deep sea were available to us. Therefore, more
precise quantification of phylotypes A1, A2, and A7 has to await future molecular investigations of the Urania basin chemocline.
Eight of the isolates which corresponded to a molecular isolate were
identified as
A. macleodii. This species has been shown
to
constitute a major fraction of bacterioplankton in eutrophied
Mediterranean seawater (
49), and it has been speculated
that
microheterogeneity occurs at the
rrn operon level of
this species.
Interestingly, 16S rDNA fingerprinting revealed that all
eight
strains of
A. macleodii isolated in the present study
contained
the same multiple
rrn operons which differed at
one or two nucleotide
positions over the 452 nucleotide positions
analyzed.
A. macleodii obviously is a widely distributed
marine species which, in contrast
to many other marine bacteria, is
characterized by the existence
of multiple, heterogeneous
rrn operons.
Environmental conditions in the Urania basin chemocline.
In
the chemoclines of deep-sea hypersaline brines, the concentrations of
particulate organic carbon and dissolved organic carbon are
significantly increased. It has been proposed that these conditions may
stimulate bacterial activity (24).
Compared to other deep-sea brine lakes, the outstanding feature of the
Urania basin is its high sulfide content (Table
3).
The sulfide
concentration in this basin is up to 10 mM (mean concentration
in the
brine, 8.6 mM) and is higher than the sulfide concentration
in any
other marine water body investigated to date. The sulfur
isotope values
for sulfide and sulfate agree well with earlier
measurements (Ziebis et
al., J. Conf. Abstr. Goldschmidt Conf.
2000) and indicate that sulfide
is formed from microbial reduction
of sulfate. The apparent isotope
fractionation in the brine, about
41
, is within the range observed
in pure cultures of sulfate-reducing
bacteria (up to
46
)
(
32). Corresponding to the high sulfide
concentrations,
high rates of sulfate reduction (6 to 14 µmol
· liter
1 · day
1) have been determined
in the brine (Ziebis et al., J. Conf. Abstr.
Goldschmidt Conf. 2000).
These values are high compared to those
in other anoxic pelagic
systems, like the Black Sea (3 to 36 nmol
· liter
1 · day
1) (
31).
The primary production in the oligotrophic eastern Mediterranean Sea is
about 16 g of C · m
2 · year
1 (
61). Based on the
stoichiometry of sulfate reduction, which
can be calculated by
2<CH
2O> + SO
42 + 2H
+ 
H
2S + 2H
2O + 2CO
2, the primary production (if all of it reaches
the
chemocline) could supply a maximum of 0.24% of the carbon
demand of
the sulfate-reducing bacteria beneath 1 m
2 of the brine
lake. Obviously, sedimentation of phytoplankton
from the photic water
layers is far from sufficient to explain
the sulfate reduction rates
observed. Hence, organic carbon must
reach the Urania basin by other
routes, such as lateral advection
of suspended organic matter from the
continental shelf or erosion
of sediment layers rich in organic carbon
(so-called green mud
containing >2% organic carbon) (
20)
which become exposed to
the brine water. Another possible substrate for
sulfate reduction
may be methane (
6,
27), which has been
detected at high concentrations
in the brine (2.6 to 3.8 mM)
(
20).
Since the vertical sedimentation flux of organic carbon appears to
contribute little to the overall carbon budget,
chemoorganoheterotrophic
bacteria are expected to have little
biogeochemical significance
in the chemocline of the Urania basin. In
contrast, it is expected
that chemolithotrophic sulfur-oxidizing
bacteria constitute a
major fraction of the microbial community in the
chemocline of
the Urania basin since a pronounced sulfide gradient
exists at
the interface with oxic water layers. Both our
culture-independent
analysis and an analysis of the culturable fraction
of the bacterioplankton
yielded a different result,
however.
Biogeochemical implications.
The activity of leucine
aminopeptidase is associated exclusively with heterotrophic bacteria
(10). This enzyme, unlike alkaline phosphatase and
-glucosidase, is not induced by its natural substrates (polypeptides
and proteins) in marine sediments; however, it is inhibited by glycine
and other amino acids (5). Since our cell-specific values
are comparable to maximum values determined in other environments (up
to 2.97 × 1017 mol · cell1 · h1 [13, 25]),
a large fraction of the bacterial cells present in the chemocline must
be physiologically active.
The exoenzymes alkaline phosphatase and
-glucosidase are inducible
by their substrates (organic phosphoesters and cellobiose,
respectively) and are subject to catabolite repression by their
products (glucose and phosphate, respectively) (
10,
52).
Cell-specific
activities of these two enzymes may thus be used as
indicators
of the presence of degradable biopolymers (
10,
14,
15,
42).
Since the cell-specific phosphatase activities determined
in the
present study were much lower than those of phosphate-deficient
bacterial communities (3 × 10
15 to 7.7 × 10
15 mol · cell
1 · h
1 [
42]), bacteria in the chemocline of
the Urania basin appear
not to be limited by inorganic phosphate.
Obviously, the supply
of phosphate from the anoxic brine is sufficient
to repress the
synthesis of alkaline phosphatase in the cells. In
contrast to
the specific activities of alkaline phosphatase, the
specific
activities of
-glucosidase exceeded values determined for
other
pelagic water samples (50 × 10
20 mol · cell
1 · h
1 [
25]) by a
factor of >30. It can be concluded that degradable
carbohydrate
biopolymers are present and are degraded by bacteria
in the chemocline
despite the supposedly small amount of organic
matter which reaches the
Urania deep-sea
environment.
The strains affiliated with the
Cytophaga-Flavobacterium
group were obligate chemoorganoheterotrophs and corresponded to a
major
DGGE band of the natural bacterial community (band A2).
All 70 strains,
including the 21 strains isolated in media designed
for
chemolithotrophs, (Table
1), could grow chemoorganoheterotrophically
under oxic conditions (Sass et al., submitted). Similar results
have
been obtained for marine sediments and hydrothermal vents,
where liquid
MPN series containing thiosulfate yielded heterotrophic
isolates with
the highest dilutions (
56). Conversely, 15 of
the strains
isolated as chemoorganoheterotrophs could not grow
chemolithotrophically with reduced sulfur compounds. Sixty-six
strains
could grow on peptone, casein hydrolysate, and yeast extract,
whereas
only 15 strains did not utilize polymeric carbohydrates.
Together with
the high specific activity of
-glucosidase, our
results indicate
that chemoorganotrophic bacteria constitute a
significant fraction of
the natural bacterial
community.
The 16S rDNA sequences retrieved by the culture-independent PCR-DGGE
approach did not match sequences of typical chemolithoautotrophic
chemocline bacteria expected to thrive in a chemocline with oxygen
and
sulfide countergradients (
45). However, 55 of our isolates
were facultatively chemolithoautotrophic and used reduced sulfur
compounds (Sass et al., submitted). Molecular isolate A4 is affiliated
with the genus
Roseobacter, which comprises many
facultatively
chemolithotrophic sulfide oxidizers (
56).
Members of the
Pseudomonas stutzeri group (Fig.
5, compare
isolates U32 and U65) have been
shown to utilize thiosulfate under oxic
conditions as well as
under anoxic conditions in the presence of
organic carbon substrates
(
53). Possibly, oxidation of
sulfide in the chemocline of the
Urania basin is mediated to a large
extent by such mixotrophic
bacteria. Our findings are in line with
those of other studies
of marine waters and sediments, in which
heterotrophic sulfur-oxidizing
bacteria are common (
56).
The bacterial isolates obtained in the present study resemble those
which are frequently obtained from the marine water column
and
represent a subset of the standard pelagic bacterial groups
that is
frequently found with molecular methods (
18,
36,
47,
49,
55). It therefore appears that a major fraction of the
chemocline bacteria may originate in upper water layers and reach
the
pycnocline by sedimentation. Even if the typical heterotrophic
bacteria
are of allochthonous origin, our results indicate that
these organisms
must still be capable of resuming active metabolism
at the oxic-anoxic
interface and growing to higher cell
densities.
Hypersaline sediments from the Kebrit Deep at the bottom of the Red Sea
harbor unknown
Euryarchaeota, as well as
Bacteria which are distantly related to the
Aquificales and
Thermotogales (
17). The salt concentration in
hypersaline brine sediments
of the Red Sea is 4.3 M and thus exceeds
the chlorinity in sediments
of the Urania basin. Most of our isolates
originated from significantly
less saline (1 M chloride) chemocline
waters of the Urania basin
and were identified as proteobacteria,
high-G+C-content gram-positive
bacteria, or members of the
Cytophaga-Flavobacterium group. Future
research should
reveal whether the composition of microbial communities
changes along
the salinity gradient in the Urania basin and whether
Archaea are abundant in the hypersaline
brine.
In conclusion, the present polyphasic study provided new insights into
the ecophysiology of bacteria in the chemocline of
the Urania basin.
Physiologically active chemoorganotrophic bacteria
are present and
appear to be more dominant than chemolithoautotrophs.
Our repeated
isolation of bacteria that had the same partial 16S
rDNA sequence but
produced different ERIC band patterns and had
distinct physiological
properties could merely reflect genetic
variation within one species.
Alternatively, the microdiversity
may indicate that there are different
ecotypes which occupy different
ecological microniches in the
chemocline of the Urania
basin.
| |
ACKNOWLEDGMENTS |
We thank the master and the crew of the R/V Meteor for
their help during collection of the water samples and sediment cores and Michael Böttcher for help with determination of sulfur
isotope data.
This work was supported by grants Cy 1/8-1 and Cy 1/10-1 from the
Deutsche Forschungsgemeinschaft to H.C. and J.O.
| |
FOOTNOTES |
*
Corresponding author. Present address: Institut
für Genetik und Mikrobiologie, Universität München,
Maria-Ward-Str. 1a, D-80638 Munich, Germany. Phone: 49-89-2180-6123. Fax: 49-89-2180-6125. E-mail:
j.overmann{at}lrz.uni-muenchen.de.
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Applied and Environmental Microbiology, December 2001, p. 5392-5402, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5392-5402.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
