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Applied and Environmental Microbiology, September 1999, p. 3843-3849, Vol. 65, No. 9
Molecular Ecology
Group1 and Department of
Microbiology,2 Max-Planck-Institute for
Marine Microbiology, D-28359 Bremen, Germany
Received 21 December 1998/Accepted 9 June 1999
A shallow-water hydrothermal vent system in the Aegean Sea close to
the island of Milos (Greece) was chosen to study the diversity and
distribution of the chemolithoautotrophic sulfur-oxidizing bacterium
Thiomicrospira. Cell numbers in samples from different regions around a solitary vent decreased toward the center of the vent
(horizontal distribution), as well as with depth (vertical distribution), corresponding to an increase in temperature (from ca. 25 to 60°C) and a decrease in pH (from ca. pH 7 to 5).
Thiomicrospira was one of the most abundant culturable
sulfur oxidizers and was even dominant in one region. Phylogenetic
analysis of Thiomicrospira spp. present in the highest
most-probable-number (MPN) dilutions revealed that most of the obtained
sequences grouped in two new closely related clusters within the
Thiomicrospira branch. Two different new isolates, i.e.,
Milos-T1 and Milos-T2, were obtained from high-dilution
(10 Hydrothermal vents are located at
tectonically active regions all over the world; the most spectacular
are those at the deep-sea floor, where very unusual light-independent
ecosystems based on chemolithoautotrophic sulfur-oxidizing bacteria
exist (see, e.g., references 16 and
17). However, hydrothermal vents have also been
found in several shallow-water areas (see, e.g., references 15, 26, 28, 30, and 31). These
shallow-water vents differ from their deep-sea counterparts mainly by
the presence of light. Accordingly, the biology of these ecosystems is
different too; e.g., at shallow-water vents, photosynthetic organisms,
such as benthic microalgae and cyanobacteria, can be present (see,
e.g., references 9 and 26).
Off the Greek island of Milos in the Aegean Sea, shallow-water vents
were found at depths ranging from the littoral zone down to 115 m
(9). A rich macrofauna depending on endosymbiotic bacteria
is absent at the Milos vents (9, 29). Until now, the only
sulfur-oxidizing bacteria (SOB) described for the hydrothermal vents
around Milos were thiobacilli and Achromatium volutans at vents less than 30 m deep (9) and Thioploca
at a vent 46 m deep (11). These identifications were
based on microscopic observations. For Achromatium volutans
and Thioploca, this is appropriate because of the
particular morphological features of these organisms. For thiobacilli, however, microscopic identification is more problematic, because the rod-shaped morphology might easily be confused with, e.g.,
the recently described rod-shaped isolates of Thiomicrospira (5, 6).
Members of the genus Thiomicrospira are
chemolithoautotrophic bacteria, which use reduced sulfur compounds as
electron donors and obtain carbon from CO2 (7,
19). Although Thiomicrospira spp. were detected in and
isolated from several different environments (see, e.g., references
3 and 34), their ecological
importance remained obscure for most habitats. Several isolates were
obtained from intertidal mud flats (3, 18, 35, 36). However,
in a recent study it was found that Thiomicrospira
constituted only a minor fraction of the total sulfur-oxidizing
bacterial community in this habitat, pointing to a minor role of these
microorganisms (4). On the other hand, an analysis by
molecular methods, i.e., denaturing gradient gel electrophoresis (DGGE)
and sequencing of PCR amplified 16S rDNA fragments, demonstrated that
Thiomicrospira spp. were dominant community members of
hydrothermal vent sites at the Mid-Atlantic Ridge (21).
Here, we present the results of an investigation of the diversity and
distribution of Thiomicrospira populations at a
shallow-water hydrothermal vent off Milos. For this study, a
combination of molecular and microbiological methods was used in an
attempt to understand the ecological importance of these microorganisms.
Sampling.
Samples were taken in June and September 1996 and
in June 1997 at a solitary gaseous hydrothermal vent, located at a
water depth of 8 m in Palaeochori Bay (24°31.220'E,
36°40.391'N), a sandy bay in the southeastern part of the Greek
island of Milos in the Aegean Sea. The mean composition of the
discharged gases from the different seeps was 80.5% CO2,
1.2% H2S, 0.8% CH4, and 0.4% H2
(10). The reduced hydrothermal fluid has an increased salinity of up to 58
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Distribution and Diversity of Sulfur-Oxidizing
Thiomicrospira spp. at a Shallow-Water Hydrothermal Vent in
the Aegean Sea (Milos, Greece)
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
5) enrichments. Phylogenetic analysis indicated that
isolate Milos-T1 is related to the recently described
Thiomicrospira kuenenii and Hydrogenovibrio
marinus, whereas isolate Milos-T2 grouped with the MPN sequences
of cluster 2. The predominance of strain Milos-T2 was indicated by its
identification in several environmental samples by hybridization
analysis of denaturing gradient gel electrophoresis (DGGE) patterns and
by sequencing of one of the corresponding bands, i.e., ML-1, from the
DGGE gel. The results shown in this paper support earlier indications
that Thiomicrospira species are important members of
hydrothermal vent communities.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, compared to 39 for the ambient seawater (29).
Subsampling of sediment cores. For all samples taken in June 1996, the following layers were investigated: water directly from above the sediment surface and sediment samples from depths of 0 to 10, 10 to 20, and 20 to 30 mm. For samples taken in September 1996, cores were sliced at a finer resolution as follows: water directly from above the sediment surface and sediment from depths of 0 to 5, 8 to 13, and 16 to 26 mm. Water from just above the surface was sampled with a syringe. Sediment cores were subsampled by extruding the sediment from the polycarbonate tubes and slicing each core. Dilutions for the MPN series were performed directly after slicing. Subsamples of the same layers for DNA extraction were frozen immediately in liquid N2 until used in further processing.
MPN counts of sulfur-oxidizing bacteria.
The MPN technique
was used to estimate the abundance of SOB in the different subsamples
taken in September 1996. Sediment cores were subsampled as described
above and serially diluted (1:10 steps) with mineral medium without a
substrate. Between every dilution step, the samples were vigorously
shaken on a vortex apparatus to dislodge the bacteria from the sediment
particles. From each dilution, three replicate tubes containing growth
medium were inoculated and incubated at their approximate in situ
temperature. Mineral medium with 20 mM thiosulfate (added by sterile
filtration to the autoclaved medium) as the sole electron donor was
used. The composition of the mineral medium was (per 1,000 ml of
H2O) 29 g of NaCl, 1 g of
(NH4)2SO4, 1.5 g of
MgSO4 · 7H2O, 0.42 g of
CaCl2 · 2H2O, 0.5 g of
K2HPO4, 0.7 g of KCl, 0.05 g of
vitamin B12, and 1 ml trace element solution with EDTA
(32). Bromthymol blue was added as a pH indicator at a
concentration of 4 mg liter1.
K2HPO4 was autoclaved separately and added to
the medium after autoclaving.
by adding NaCl,
was performed. This salinity was similar to the salinity of the
outflowing brine (29). This parallel MPN series was carried
out to investigate whether there were SOB specifically adapted to the
higher salt concentration.
The cultures were incubated in the dark, to avoid growth of
phototrophic bacteria, and at their approximate in situ temperatures (see Fig. 1). Growth was monitored by observing a color change of the
pH indicator and by microscopic observation. The presence of
Thiomicrospira cells in the MPN cultures was determined by using a Thiomicrospira-specific primer set in a PCR with
cells taken directly from the cultures (3). The numbers of
SOB and Thiomicrospira cells were determined by using the
MPN index of the American Public Health Association (1) in
three parallel determinations.
Isolation and cultivation of bacteria. High-dilution enrichments were performed with samples taken in June 1996 and June 1997, with the intention of obtaining the most abundant SOB. The medium and the culture conditions were the same as for the MPN cultures. Aliquots from the tubes were transferred on solid agar plates, containing the same culture medium and 1% (wt/vol) agar (Difco). All known Thiomicrospira species and strains isolated so far form intensely yellow colonies due to sulfur precipitation. Additionally, they all produce acid. Colonies with these characteristics were chosen for identification by a Thiomicrospira-specific PCR (3). The positive colonies obtained were transferred at least three times before being considered pure.
Nucleic acid extraction. DNA was extracted from sediment and water samples by the method described by Zhou et al. (37) and modified as described by Sievert et al. (25).
Oligonucleotides used for PCR. Oligonucleotides TMS128F and TMS849R are specific for the 16S rDNA of bacteria belonging to the genus Thiomicrospira (3). With these primers, about 700-bp 16S rDNA fragments are obtained. The primers have been used for the identification of Thiomicrospira isolates and for the detection of bacteria belonging to this genus in the MPN cultures. The PCR products obtained from the highest MPN dilutions in which Thiomicrospira could be detected were sequenced to determine the phylogenetic affiliation of the respective organisms.
Oligonucleotides GM3F and GM4R are specific for the 16S rDNA of members of the domain Bacteria and were used as primers in a PCR to amplify the nearly complete (1,500-bp) genes of the new isolates. The PCR products obtained with these primers were used for sequencing. The primer pair GM5F and 907R amplifies the 16S rDNA of members belonging to the domain Bacteria and was used to obtain 550-bp fragments for DGGE analysis. The sequences of both primer pairs (GM3F plus GM4R and GM5F plus 907R) have been published by Muyzer et al. (21).PCR amplification of 16S rDNA fragments. PCR amplifications were performed as described by Muyzer et al. (21). A touchdown PCR (12) was performed for primer pair GM5F plus 907R (annealing temperature from 65 to 55°C in 20 cycles). For primer pairs TMS128F plus TMS849R (annealing temperature of 44°C), and GM3F plus GM4R (annealing temperature of 40°C), no touchdown PCR was used. The amplification products were analyzed as described by Muyzer et al. (21) before being subjected to further characterization by DGGE analysis or DNA sequencing.
DGGE analysis of PCR products. DGGE was performed with the D-Gene system (Bio-Rad Laboratories, Inc.). The protocol as described by Brinkhoff and Muyzer (3) was used: 1-mm-thick, 6% (wt/vol) polyacrylamide gels, 1× TAE electrophoresis buffer (40 mM Tris-HCl [pH 8.3], 20 mM acetic acid, 1 mM EDTA), 20 to 70% denaturant, and an electrophoresis time of 20 h at a constant voltage of 100 V. After electrophoresis, the gels were stained with ethidium bromide and photographed as described previously (22).
Hybridization analysis of blotted DGGE patterns. DGGE patterns were transferred to nylon membranes (Hybond-N+; Amersham, Little Chalfont, United Kingdom) by electroblotting and hybridized with the digoxigenin-labeled Thiomicrospira-specific probe TMS849R, as previously described (3).
Sequencing of PCR products. PCR products were purified by using the Qiaquick Spin PCR purification kit (Qiagen Inc., Chatsworth, Calif.). The Taq Dyedeoxy Terminator cycle-sequencing kit (Applied Biosystems, Foster City, Calif.) was used to sequence the 16S rDNA fragments. The sequencing primers for the nearly complete 16S rDNA of bacterial isolates were GM3F, GM1F, and GM4R (8). The sequencing primers for Thiomicrospira 16S rDNA fragments obtained from MPN cultures with the specific primer pair were the same Thiomicrospira-specific oligonucleotides, TMS128F and TMS849R (3). One band in the DGGE gel, which hybridized with the Thiomicrospira-specific probe TMS849R, was excised from the gel, reamplified with the primer pair GM5F and 907R, and sequenced with the Thiomicrospira-specific primer TMS849R. The sequence reaction mixtures were electrophoresed on an Applied Biosystems 373S DNA sequencer.
Comparative analysis of 16S rRNA sequences. The 16S rRNA sequences were aligned with those obtained from the Ribosomal Database Project (20) and GenBank (2). Sequence alignments were prepared with the sequence editor SEQAPP (14). Phylogenetic trees were created by using the neighbor joining algorithm with maximum-likelihood correction as implemented in the test version of PAUP 4 developed by Swofford.
Nucleotide sequence accession numbers. The sequences obtained in this study are available from the EMBL nucleotide sequence database under accession nos. AJ237757 to AJ237769.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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MPN counts of SOB and Thiomicrospira cells.
Numbers of cultivable aerobic chemolithoautotrophic SOB and
Thiomicrospira cells counted by MPN serial dilution were
variable for the different zones and layers (Fig.
1). Where Thiomicrospira spp.
were present, they accounted for between 2 and 100% of the total
numbers of cultivable SOB, which indicates that
Thiomicrospira spp. belonged to the dominant SOB in this
habitat. Generally, the numbers of SOB varied between not detectable
and 1.4 × 106 cells g (wet weight) of
sediment1. Thiomicrospira cell numbers varied
between not detectable and 2.7 × 105 cells g (wet
weight) of sediment1.
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1, could be obtained.
Thiomicrospira cells were not detectable in the MPN cultures
of this zone (Fig. 1A).
In the second zone, at 117 cm from the vent center, the numbers of SOB
and Thiomicrospira cells were different for the two cores
examined. The surface of the first core (core I, Fig. 1B) contained the
white precipitate mentioned above, which was absent at the surface of
the second core (core II, Fig. 1C). Core II was used for the MPN counts
with the increased salinity. Whereas the cell numbers of SOB obtained
for the three sediment layers for core II were only slightly smaller
than those obtained for core I, the numbers of
Thiomicrospira spp. were appreciable smaller in the sediment
samples of core II than of core I. This is also indicated by the
finding that Thiomicrospira spp. constituted a much smaller
fraction of the total numbers of SOB in core II than in core I. The
largest numbers of SOB and Thiomicrospira at 117 cm were
obtained with the sample from the water above the sediment surface of
core I, which contained the white precipitate (4.1 × 104 and 1.9 × 104 cells ml of
water1, respectively [Fig. 1B]).
For the third zone, at 200 cm from the vent center, the smallest
numbers of SOB and Thiomicrospira cells were obtained for the water above the sediment surface (Fig. 1D). The largest numbers of
SOB (1.4 × 106 cells g1) were found in
the second sediment layer (8 to 13 mm deep), while the largest numbers
of Thiomicrospira cells (2.7 × 105 cells
g1) were found in the first sediment layer (0 to 5 mm
deep). Here Thiomicrospira species were also one of the
dominant SOB, since they accounted for between 2 and 100% of the total
numbers of SOB. Comparison of the different zones revealed generally
the largest numbers of SOB and Thiomicrospira cells in the
sediment layers of the third zone.
Detection of Thiomicrospira by DGGE and hybridization analysis. DGGE profiles of 16S rDNA fragments obtained from sediment and water samples from June and September 1996, respectively (Fig. 2A and C), were hybridized with a probe specific for the genus Thiomicrospira (Fig. 2B and D). Positive signals were obtained with the bands from two isolates and with bands from samples of different zones and layers. The results for the samples from June 1996 are described first. For the first zone, at 10 cm from the vent center, a signal was obtained for the water above the sediment (Fig. 2B, lane 3). For the second zone (123 cm from the vent center), signals were obtained for the water above the sediment and the first sediment layer (Fig. 2B, lanes 7 and 8). No signals were obtained for the third zone (Fig. 2B, 165 cm from the vent center). For the fourth zone (235 cm) a signal was obtained for the water above the sediment (Fig. 2B, lane 14) and a weak signal was obtained for the first sediment layer (Fig. 2B, lane 15).
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Phylogenetic analysis of Thiomicrospira from MPN
cultures.
Although the Thiomicrospira species from the
highest dilutions of the MPN counts performed in September 1996 were
not isolated, partial sequences of their 16S rDNA could be determined
by using the Thiomicrospira-specific primer pair for
amplification and sequencing. With the exception of the sequence of
Tms-MPN/Milos-CIV1, all the sequences from the MPN cultures were very
similar (ca. 99%) and grouped in two clusters, i.e., clusters 2 and 3 (Fig. 3). The sequence of
Tms-MPN/Milos-CIV1, obtained from the MPN culture from the deepest
sediment layer (16 to 26 mm deep) of the second zone (117 cm from the
vent center) and cultured at 55 salinity, was very different from
all others and was shown to be the deepest-branching
Thiomicrospira sequence known so far. For one MPN culture
(Tms-MPN/Milos-DIV5), a mixed sequence was obtained, indicating that
more than one Thiomicrospira sequence type was present in
this culture.
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Characterization of Thiomicrospira isolates.
Two
isolates, which were both obtained from 105 dilutions of
the enrichments, gave a positive signal with the
Thiomicrospira-specific PCR. Strain Milos T-1 was received
from a sample taken in June 1996 from the upper sediment layer,
underneath the white precipitate, at 123 cm from the vent center.
Strain Milos T-2 was isolated directly from the white precipitate in
June 1997. While isolate Milos T-1 is vibrio shaped, like most other
Thiomicrospira spp., strain Milos T-2 is rod-shaped, like
two recently described new Thiomicrospira species, T. frisia (5) and T. chilensis (6).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The high abundance of Thiomicrospira cells in some zones of the shallow-water hydrothermal vent off the isle of Milos can probably be attributed to the presence of high concentrations of reduced sulfur compounds and CO2 (10), which both favor the growth of chemolithoautotrophic sulfur-oxidizing bacteria.
The distribution pattern of Thiomicrospira as determined by the MPN counts probably reflects the influence of environmental parameters, such as temperature, pH, and oxygen concentration. These environmental parameters were determined concurrently along the same transect (25). Temperatures of about 30 to 60°C and pH values between 5.7 and 5.0 were measured for the layers of the first zone (30 cm from the center of the vent). This might explain the small numbers of SOB in general and the absence of Thiomicrospira.
At 117 cm from the center of the vent, the numbers of SOB and
Thiomicrospira cells were decreasing with depth (Fig. 1B).
While the environmental conditions of this zone are moderate at the sediment surface (25°C and pH 6.5), temperatures of about 40°C and
pH values of 5.5 and lower at the deeper layers, as well as a
decreasing oxygen concentration with depth, seemed to inhibit the
growth of SOB and Thiomicrospira. The numbers of SOB as
determined in the MPN count with an increased salinity of 55 (Fig.
1C) were slightly smaller for the sediment layers than were those
obtained with 30 salinity, even though the higher value reflected
the in situ concentration of the outflowing brine. This indicates that
SOB adapted to a higher salinity were not dominant. The finding that
Thiomicrospira spp. constituted a smaller fraction of the total numbers of SOB in the sediment samples from core II than in those
from core I might be an indication that Thiomicrospira spp.
were more strongly affected by the increased salinity. However, it
could also be related to natural variability. The second possibility is
supported by the observation that bands hybridizing with the Thiomicrospira-specific probe were less abundant and weaker
for the samples from core II than for those from core I (Fig. 2B and D). The significantly smaller numbers of SOB and
Thiomicrospira cells determined for the surface layer of the
MPN counts with the increased salinity might be due to the absence of
the white precipitate of the respective sample (25).
At 200 cm from the center of the vent, moderate conditions (25°C and pH 7.0) and a deeper oxygen penetration seemed to allow the presence of larger numbers of SOB and Thiomicrospira cells in deeper layers (Fig. 1D). The predominance of Thiomicrospira cells in the first sediment layer of this zone indicates that the environmental parameters favored the growth of members of this genus. It is worth noting, however, that Thiomicrospira spp. were not the only SOB present in the highest positive dilutions (24). At 200 cm, Thiomicrospira cells were also found in deeper layers at nearly the same order of magnitude as in the upper layers, although oxygen was not present (25). This phenomenon was described previously for this genus in sediment of an intertidal mud flat (4). However, in the latter habitat, Thiomicrospira populations were found to be metabolically active only in the oxic part of the sediment.
Comparison of the results of the hybridization patterns with the MPN
results showed differences for the distribution of
Thiomicrospira, especially for the first zone, i.e., at 10 and 30 cm from the vent center. No Thiomicrospira cells were
detected here by the MPN counts. On the other hand, hybridization
analysis of both DGGE patterns gave positive signals for this zone
(Fig. 2B and D). The physicochemical parameters should actually allow
the growth of Thiomicrospira spp. at the surface of this
region, as indicated by the positive band from the samples taken in
June 1996 (Fig. 2B, lane 3). However, the presence of
Thiomicrospira in the sediment samples taken in September
1996 (Fig. 2D, lanes 4 to 6) is in contrast to the environmental
parameters. It is unlikely that Thiomicrospira spp. grew in
these layers, since the in situ temperature (40 to 60°C) was at or
above the upper growth limit of known species (7) and since
oxygen, which is needed as electron donor (7), was not
present (25). That these Thiomicrospira
populations were not detected by the MPN determination is probably a
result of their special adaptation to the extreme environmental
conditions that were not imitated in the MPN culture. This possibility
would be supported by the finding of the sequence Tms-MPN/Milos-CIV1, which was obtained from the MPN cultures with a salinity of 55. Its
phylogenetic position and the extreme environment where the sequence
was found (temperature around 40°C, pH 5.0 to 5.5, and no oxygen)
might mean that the corresponding organism has a different physiology
from known Thiomicrospira species. However, the position of
the Thiomicrospira-positive bands for the samples obtained at 30 cm from the vent center were at the same position in the gel as
was the band for the isolate Milos-T2, which argues against a unique
population. Most probably, resuspension of the upper sediment layers
due to a storm, before to the sampling in September 1996, was
responsible for the occurrence of Thiomicrospira populations in deeper sediment layers (25). This would also account for the observation that bands belonging to Thiomicrospira spp.
were confined to the upper sediment layers and the water above the sediment in June 1996 (Fig. 2B, lanes 3, 7, 8, 14, and 15), which is in
agreement with the known physiological capabilities of this genus
(7, 19), whereas they were more homogeneously distributed with no clear trend in September 1996 (Fig. 2D).
By using a PCR-DGGE-hybridization assay, it was found that a particular bacterial population representing 0.1% of the total community could still be detected (27). In the present study, we could detect Thiomicrospira populations, even though their contribution to the total cell numbers (determined by acridine orange direct counts [25]) was well below 0.1%. Different explanations, which are not mutually exclusive, could account for this. First, we do not know the extent to which Archaea contributed to the total cell counts. Thus, the contribution of Thiomicrospira cells to the total bacterial cells might be higher. Second, the association of cells in clumps or with particles, as well as the insensitivity of the detection method (33), might have led to an underestimation of Thiomicrospira cell numbers. In addition, it is conceivable that different and highly related Thiomicrospira populations were present in the highest dilutions. Third, a bias toward Thiomicrospira might have been caused by the DNA extraction and/or the PCR; i.e., Thiomicrospira cells were more efficiently lysed and/or their 16S rRNA might have been specifically overamplified compared to other sequences. Furthermore, not all cells stained with acridine orange might have contained enough DNA for amplification. The hybridization analysis of DGGE patterns with the environmental samples obtained in June and September 1996 showed that all bands, which gave positive signals, ran at the same position in the denaturing gel as those from the new isolated Thiomicrospira strain Milos T-2. Based on these data, it can be seen that this is apparently the dominant Thiomicrospira sequence type of this ecosystem. This was confirmed by the isolation of this organism from a high-dilution enrichment of a sample taken in June 1997 and additionally by the sequence similarity of a band excised from a DGGE gel, having 99.8% sequence homology to strain Milos T-2. No bands corresponding to the band of strain Milos T-1 were found in the DGGE patterns, and no similar sequence was obtained from the MPN cultures, even though strain Milos T-1 was also isolated from a high-dilution enrichment, like strain Milos T-2. This finding cannot be explained by the present data.
Almost all the sequences presented in this study show differences among
each other of less than 2%. Therefore, physiological adaptations to
the different zones of the studied vent system might be possible
without greater differences in the 16S rDNA. An indication for this is
that all sequences obtained from the MPN cultures of the second zone
(117 cm from the vent center), grown at a salinity of 30, are
identical. On the other hand, most sequences obtained from the MPN
counts of the same zone, grown at a salinity of 55, formed another
cluster. This might reflect an adaptation of specific
Thiomicrospira populations to a higher salinity. This would
be similar to the finding that highly related cyanobacterial
populations are adapted to different temperatures according to their
occurrence in a thermal gradient along an outflow of a hot spring
(13). It was generally observed that the more moderate
conditions in the outer zone of the vent correlated with a higher
phylogenetic diversity of the Thiomicrospira sequences obtained from this zone. This reflects a trend that is also apparent for the total bacterial community (25).
Due to the presence of light, chemolithoautotrophic SOB were found not to be the only primary producers of the shallow-water vent system off Milos (9, 29). However, results from this study clearly indicate that even though differences between the deep-sea and shallow-water hydrothermal vent systems exist, Thiomicrospira is in both cases an important member of the sulfur-oxidizing community and must be taken into account in further ecological and microbiological investigations of these ecosystems.
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ACKNOWLEDGMENTS |
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We thank Wiebke Ziebis, Susanne Menger, and Guido Lützenkirchen for SCUBA diving, sampling, and help with field work, and we thank the mechanical workshop of the MPI for building the sampling devices. We are grateful for support by other participants of the EU-funded project Hydrothermal Fluxes and Biological Production in the Aegean Sea. We acknowledge the Greek authorities for permission to undertake SCUBA diving and field work.
This research was financially supported by the Max-Planck-Society, Munich, Germany, and by the European Union under MAST CT-95-0021.
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FOOTNOTES |
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* Corresponding author. Present address: Netherlands Institute for Sea Research, P.O. Box 59, NL-1790 AB Den Burg, The Netherlands. Phone: 31-222-369-521. Fax: 31-222-319-674. E-mail: gmuyzer{at}nioz.nl.
Present address: Institute for Chemistry and Biology of the Marine
Environment, Carl von Ossietzky Universität, D-26111 Oldenburg, Germany.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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