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Applied and Environmental Microbiology, September 1999, p. 3982-3989, Vol. 65, No. 9
Molecular Ecology Group, Max-Planck-Institute
for Marine Microbiology, D-28359 Bremen, Germany
Received 2 April 1999/Accepted 2 July 1999
A 16S ribosomal DNA (rDNA) clone library from permanently cold
marine sediments was established. Screening 353 clones by dot blot
hybridization with group-specific oligonucleotide probes suggested a
predominance of sequences related to bacteria of the sulfur cycle
(43.4% potential sulfate reducers). Within this fraction, the major
cluster (19.0%) was affiliated with Desulfotalea sp. and
other closely related psychrophilic sulfate reducers isolated from the
same habitat. The cloned sequences showed between 93 and 100%
similarity to these bacteria. Two additional groups were frequently
encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with
Myxobacteria spp. and Bdellovibrio spp. Many
clones (18.1%) belonged to the Coastal and shelf sediments play a
significant role in the remineralization of organic matter. In shelf
areas, an estimated 32 to 46% of the primary production settles to the
sea floor (54). While part of it is permanently buried, the
majority of this detrital material is reoxidized, mainly through the
action of prokaryotes (54). Steep redox gradients provide
niches for a wide variety of metabolically diverse microorganisms, and
O2, NO3 Few cultivation-independent studies of microbial diversity in marine
sediments have been conducted (6, 15, 22, 43). The sequences
recovered in these studies revealed the presence of mainly unknown
organisms only distantly related to known isolates. To further uncover
microbial diversity in marine shelf sediments and to identify
potentially dominant groups in this habitat, we constructed a 16S
ribosomal DNA (rDNA) clone library using general bacterial primers to
amplify the almost complete gene.
The screening process was tested by statistical analysis to evaluate
whether we had covered total diversity in our clone library by
screening 353 clones. Species diversity can be considered to be
composed of two components: species richness (the number of species in
a community) and species evenness (the distribution of levels of
abundance among the species). Two types of analyses have been used to
assess diversity. Rarefaction is a statistical technique for different
applications in an ecological context and gives an estimation of the
decrease in apparent species richness of a community with decreasing
subsample size (50). A second approach to evaluate whether
diversity within a subsample approaches diversity within a sample of
infinite size is to calculate coverage (14). Coverage
(C) values are calculated by the equation C = 1 We chose to study permanently cold sediments because 90% of the sea
floor has temperatures below 4°C (25). During a cruise to
the Arctic Ocean in September and October of 1995, several studies of
different aspects of microbial life in this habitat, such as the
determination of prokaryotic abundance and the profiling of prokaryotic
rRNA (47, 48), were conducted. Temperature dependence was
determined, rates of polysaccharide hydrolysis (2) and
sulfate reduction were measured (46), and psychrophilic sulfate reducers were enriched (23). Furthermore, benthic
exchange and mineralization rates were determined (12, 24).
All the above-described studies indicated an active microbial community with metabolic rates comparable to those of temperate habitats. Forty-two percent of total benthic mineralization was due to sulfate reduction at the station sampled for the clone library (46). Here and in the accompanying paper (48), we describe the
phylogenetic affiliation and diversity of the prokaryotic community and
quantify the contribution of sulfate-reducing bacteria (SRB) to the
total microbial community.
Study site.
Sediment samples were collected at Hornsund off
the coast of Spitsbergen, Arctic Ocean, in September and October of
1995. The bottom water temperature was 2.6°C (47), and
sediments were anoxic below a depth of approximately 8 mm
(12). For a detailed description of the sampling procedure,
see the report of Sahm and Berninger (47).
DNA extraction and purification.
Total community DNA was
extracted directly from the sediment as described by Zhou et al.
(55). The protocol encompassed three cycles of freezing and
thawing, chemical lysis in a high-salt extraction buffer (1.5 M NaCl)
by heating of the suspension in the presence of sodium dodecyl sulfate
(SDS) and hexadecyltrimethylammonium bromide, and a proteinase K step.
It was slightly modified by performing only two SDS extraction steps.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
High Bacterial Diversity in Permanently Cold
Marine Sediments
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
subclass of the class
Proteobacteria and were closest to symbiotic or free-living
sulfur oxidizers. Probe target groups were further characterized by
amplified rDNA restriction analysis to determine diversity within the
groups and within the clone library. Rarefaction analysis suggested
that the total diversity assessed by 16S rDNA analysis was very high in
these permanently cold sediments and was only partially revealed by
screening of 353 clones.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, manganese and iron
oxides, and SO42 have been identified as the
most important electron acceptors in marine sediments (3,
19). The various processes of microbial carbon mineralization can
be quantified by tracer techniques, and their importance for
biogeochemical cycles in the marine environment is recognized; however,
little is known about the microbial community responsible for them.
(n/N) × 100, where n is the number of
unique clones and N is the total number of clones examined.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Cell lysis efficiency. Cell lysis efficiency of the DNA extraction procedure was checked by enumerating the total number of 4',6-diamidino-2-phenylindole (DAPI)-stained cells in aliquots of sediments taken before and after cell lysis. Ninety-three percent ± 3.4% of the microorganisms were lysed.
PCR amplification of 16S rDNAs. Two universal bacterial primers, EUB008 (17) and EUB1492 (20), were used to amplify 16S rDNAs from the extracted and purified chromosomal DNAs. PCR was performed with a model PHC-3 Temperature Cycler (Techne, Cambridge, United Kingdom) as follows: 50 pmol of each primer, 2.5 µmol of each deoxyribonucleoside triphosphate, 300 µg of bovine serum albumin, 1× PCR buffer, and 1 U of Super Taq DNA polymerase (HT Biotechnology, Cambridge, United Kingdom) were adjusted to a final volume of 100 µl with sterile water. Template DNA (80 to 500 ng) was added to the reaction mixture (preheated to 70°C) to avoid nonspecific annealing of the primers to nontarget DNA. The cycles used were as follows: 1 cycle at 70°C for 1 min; 33 cycles at 95°C for 1 min, 40°C for 1 min, and 72°C for 3 min; and 1 final cycle at 95°C for 1 min, 40°C for 1 min, and 72°C for 10 min. The number of amplification cycles during PCR was reduced as much as possible to reduce PCR biases (52), chimera formations (53), and Taq polymerase error rates; however, 34 cycles were needed to yield sufficient product.
Clone library construction. Products of three parallel PCRs were combined and precipitated to concentrate the DNAs for cloning. DNA was ligated in the pGEM-T-Easy vector by using the protocol of the manufacturer (Promega). Ligation reaction mixtures were purified and used for electroporation of Escherichia coli XL1 Blue (Stratagene GmbH, Heidelberg, Germany) or E. coli JM-109 cells (Promega) as described by Flohr (8). Recombinant transformants were selected by blue and white screening.
Dot blot hybridization. Plasmid DNA was prepared from overnight cultures with a WIZARD Mini Prep Purification Kit (Promega) by following the manufacturer's recommendations. Plasmids were checked for insert presence on agarose gels. All plasmids known to contain the correctly sized insert of 1.5 kb were used for dot blot hybridization.
For blotting, plasmid DNA was denatured for 5 min at 95°C and cooled immediately on ice. Aliquots of 100 to 400 ng of DNA were spotted onto a prewetted nylon membrane (Hybond-N+; Amersham, Little Chalfont, Buckinghamshire, United Kingdom) with a Bio-Rad (Munich, Germany) dot blot apparatus. For additional denaturation, blots were placed on filter paper soaked with 0.4 M NaOH-0.6 M NaCl for 15 min. Finally, membranes were equilibrated with 2× SSC (0.3 M NaCl, 0.03 M sodium citrate [pH 7.0]) for 10 min. For immobilization of the DNA, the membrane was baked at 80°C for 2 h. Oligonucleotide probes were 5' end labeled with [-32P]ATP by using T4
polynucleotide kinase according to the recommendation of the
manufacturer (New England Biolabs, Schwalbach, Germany). The
unincorporated [-32P]ATP was removed from the labeled
probes by using Sephadex columns (NAP columns; Pharmacia Biotech,
Freiburg, Germany) according to the manufacturer's protocol.
Membranes were prehybridized for 1 h at 40°C in hybridization
solution (10× Denhardt solution, 4× SSC, 0.1% SDS, 2 mM EDTA [pH
8.0], 50 µg of salmon sperm DNA per ml [32]) before
32P-labeled probes were added. Hybridization was carried
out at 40°C (except with membranes for hybridization with a
gram-positive probe, for which the temperature was 30°C) for 14 to
16 h. Thereafter, the membranes were washed twice for 30 min with
washing buffer (2× SSC, 0.1% SDS) at hybridization temperature. To
eliminate nonspecific binding, the membranes were washed two more times for 15 min at the dissociation temperature (Td),
which had been determined according to the method of Raskin et al.
(39). Probes and Tds used in this
study are given in Table 1. Control 16S rDNAs different in sequence from each particular probe by one nucleotide were also spotted on membranes and hybridized as well to
check the stringency of washing conditions. Hybridization signals were
analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).
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ARDRA and rarefaction analysis. Amplified rDNA restriction analysis (ARDRA) was performed to analyze the diversity of clones within each group defined by dot blot hybridization. Isolated plasmid DNAs of 16S rDNA clones were used as templates for insert amplification. The PCR was performed as described above, except that the primer annealing temperature was higher (44°C). PCR products were purified, and aliquots of 200 to 400 ng of the amplified insert were digested with 7.5 U of the restriction endonuclease HaeIII (Promega) for 3 h at 37°C. The resulting fragments were analyzed on an 8% polyacrylamide gel, and restriction patterns within each group were compared. Diversity of the clone library was further investigated by rarefaction analysis (16, 18, 50). Rarefaction curves were produced by using the analytical approximation algorithm of Hurlbert (18) and 95% confidence intervals estimated as described by Heck et al. (16). Calculations were performed on a personal computer with the freeware program aRarefactWin (17a).
Sequencing and phylogenetic analysis. Representatives of all major ARDRA pattern groups were chosen for sequencing. Plasmid DNAs from selected 16S rDNA clones were sequenced (partially or in full) by Taq Cycle Sequencing with universal rRNA-specific primers with a model ABI377 (Applied Biosystems, Inc.) or a Li-Cor (MWG Biotech, Ebersberg, Germany) sequencer. A total of 116 clones were sequenced partially or fully. All sequences were checked for chimera formation with the CHECK_CHIMERA software of the Ribosomal Database Project (29), and the phylogenetic affiliations of their 5' and 3' ends were compared. By this procedure seven potential chimeras (6.0%) were detected. This figure probably underestimates the real chimera fraction because it is more difficult to detect chimera formation of two closely related sequences (53). Potential chimeras were eliminated before phylogenetic trees were constructed.
Sequence data were analyzed with the ARB software package (51). Phylogenetic trees were calculated by parsimony, neighbor-joining, and maximum-likelihood analysis with different sets of filters. For tree reconstruction, only full-length sequences were considered.Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence databases under the accession no. AJ240966 to AJ241022. Only sequences of more than 1,000 bases in length were submitted.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Initial clone library analysis.
A sample of 30 clones was
initially selected for sequencing and phylogenetic analysis to get a
first overview of the quality of and the diversity in the 16S rDNA
clone library. Within the 30 clones, we detected 21 different
sequences. Two major groups became evident: approximately 50% of the
16S rDNA clone sequences were related to gram-negative SRB and other
members of the 
subclass of the class Proteobacteria, and
approximately 40% were affiliated with the subclass of
Proteobacteria, most closely with sulfur-oxidizing bacteria.
Additional sequences were related to Cytophaga spp. (one
sequence) and gram-positive bacteria (two sequences).
Grouping of clones by dot blot hybridization and ARDRA. On the basis of the initial sequence analysis, we developed a new probe (Gamma598) and used this probe, in addition to others, for dot blot hybridization (for an overview of probes used, see Table 1).
Of the screened clones, 94.3% hybridized with the bacterial probe EUB338 and contained a 16S rDNA insert. The remaining 5.7% had no hybridization signal with EUB338 (Fig. 1), although all of them had a correctly sized insert of 1.5 kb. ARDRA of this group resulted in 20 different patterns, with each pattern being represented by a single clone (see Fig. 2J). Fourteen of these clones were sequenced, and 12 fell into the division Planctomycetes-Verrucomicrobiales. This result is in agreement with the work of Neef et al., who showed that Planctomycetales spp. have at least one nucleotide that is different from the sequence of probe EUB338 (36). The other clones were distantly related to low-G+C-content gram-positive and green nonsulfur bacteria. Of the EUB-positive clones, 71.9% bound one of the group-specific probes used.-Proteobacteria.
The most abundant group of clones
was affiliated with the subclass of Proteobacteria. A
total of 36.8% of the clones hybridized with different probes specific
for SRB. The majority of these clones were targeted by probe Sval428
(Fig. 1). Sixty-seven clones (19.0%)
hybridized with the probe specific for SRB first isolated from the same
habitat. ARDRA of this fraction resulted in eight different restriction
patterns, subsequently referred to as phylotypes (Fig.
2D). Sequence analysis revealed that the
dominant phylotypes were almost all closely related (95 to 100%) to
SRB isolates from the same cruise (strains with the prefix "LSv,"
Desulfotalea [23, 48]). The most abundant
pattern was found in 27 clones (e.g., Sva1036 and Sva1037), their
sequences showing 95% similarity to the 16S rDNA sequence of LSv20 and
Desulforhopalus vacuolatus (Fig.
3). The sequences of the
second-most-dominant pattern, represented by 17 clones, were affiliated
with Desulfotalea sp. (Sva0999) and LSv23 or LSv53
(Sva0632). Twelve of these clones were identical to the 16S rDNA of
LSv53, a psychrophilic SRB isolated by Knoblauch et al. (23)
during the same cruise but from a different sampling station
(Storfjord). A third dominant phylotype in the Sval428-positive fraction (12 clones, e.g., Sva0010) was phylogenetically related to
Desulfocapsa sp. (93% similarity) (Fig. 3), while a fourth phylotype (6 clones, e.g., Sva0113) was affiliated with LSv23 or LSv53
(97% similarity). Remaining patterns were represented by only one or
two clones.
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-Proteobacteria.
In addition to the SRB, there was a
second dominant group in the clone library represented by 16S rDNA
clone sequences which fell in the subclass of
Proteobacteria. They were only distantly related to known
bacteria (between 85.6 and 92.1%), being related most closely to
sulfur-oxidizing bacteria. This group was detected by dot blot
hybridization with probe Gamma598. Sixty-four clones (18.0%)
hybridized with this new probe developed on the basis of preliminary
screening of 30 clones (see above). After restriction endonuclease
digestion, 22 different ARDRA patterns became evident (Fig. 1 and 2E).
Phylogenetically, the clones formed three distinct clusters (Fig.
4). Clones Sva0071 and Sva0864 belonged
to a cluster that was affiliated with sulfur-oxidizing endosymbiotic
bacteria such as the gill symbionts Solemya velum (92.0%)
and Codakia costata (92.1%). The second cluster, containing
clones Sva1046, Sva0115, and Sva0120, was most closely related to other
clone sequences published by Kato and Li (21), derived from
deep-sea sediments (97.9% highest similarity). The third cluster
(containing, e.g., Sva0091, Sva0854, and Sva0318) could not be assigned
stably. Different tree reconstructions affiliated the sequences with
sulfur-oxidizing endosymbionts, the Beggiatoa-Thioploca
group, or with separate groups. To no members of the above-named groups
did they show more than 89.5% 16S rDNA similarity. The phylogenetic
position in Fig. 4 was consequently indicated by a multifurcation.
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Other probe target groups.
Probe ALF968 was designed to target
the 
subclass of Proteobacteria. This probe is known to
also target some members of the subclass of
Proteobacteria (35). In our study, 59 of the 82 clones hybridizing with probe ALF968 could be assigned to SRB by
hybridization with probe Sval428 and sequencing. Consequently, we
investigated only the diversity of the remaining 23 clones, which
displayed 14 different ARDRA patterns (Fig. 1). The three most abundant
patterns were each represented by three clones. The sequences were most
closely related to Bdellovibrio and Nannocystis or Polyangium sp., i.e., genera of the subclass of
Proteobacteria (Fig. 2F). The remaining patterns were
represented by one or two clones only. Sequencing of 11 of 14 phylotypes hybridizing with ALF968 showed that only one phylotype,
represented by two clones, was indeed affiliated with the subclass
of Proteobacteria (Rhodobacter spp.).
-Proteobacteria), Coxiella sp.,
and Marinobacter sp. (91.5 and 89.8%; both
-Proteobacteria). The less frequent patterns had
sequences related to the subclass of Proteobacteria
(e.g., Marinobacter, Methylophaga, and
Coxiella relatives), to the subclass of
Proteobacteria (Desulfobacula, Desulfosarcina, Nitrospina, and
Bdellovibrio relatives), and to the newly described phylum
Holophaga-Acidobacterium (27).
Rarefaction analysis. We applied rarefaction analysis to evaluate whether screening of 353 clones was sufficient to estimate diversity within the clone library. The expected number of different ARDRA patterns was plotted versus the number of 16S rDNA clones in the clone library. ARDRA of 353 clones resulted in 140 different patterns. The calculated rarefaction curves did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity (Fig. 5).
|
) is plotted
versus the number of clones. Rarefaction curves were also calculated
for the fraction of SRB (
). The dotted lines represent 95%
confidence intervals.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Diversity. The sediments investigated in this study are never exposed to temperatures higher than 3°C and might, therefore, be regarded as extreme environments. Studies of the bacterial communities of extreme environments such as a saltern (31) and a low-pH hydrothermal vent system (33) have indicated low bacterial diversity in these habitats. By applying rarefaction analysis to restriction fragment length polymorphism patterns of their 16S rDNA clone library, Moyer et al. (33) could demonstrate that screening of 48 bacterial clones was enough to detect the majority of taxa in the clone library of a hydrothermal vent microbial mat. One aim of our study was to assess bacterial diversity, which was expected to be limited considering the extremely low environmental temperatures; however, rarefaction analysis revealed that by screening 353 clones, the actual diversity in our clone library was only partially covered. It is unlikely that new major groups will be discovered by analyzing additional clone sequences, since the major groups were the same after 30 and 353 clones were screened. Total phylotype richness, i.e., the number of phylotypes present, on the other hand, might reflect the potential within a microbial community to respond to changes in environmental conditions. At a different time point, those phylotypes not detected or represented by only one clone might play an important role in this habitat.
Another approach that has been used to assess completeness of a clone library analysis is to calculate coverage. In our case, coverage was 71.95%, indicating that almost three-quarters of total diversity in the clone library was detected; however, since coverage is based only on the number of unique clones relative to total richness, not taking evenness into account, it should be regarded only as a rough estimate of diversity within a sample of infinite size. The 16S rDNA inserts of the clones were digested with one tetrameric restriction enzyme. Use of a second enzyme resulted in an increased number of patterns (data not shown). In a study of sulfate-reducing isolates, Rooney-Varga et al. (44) demonstrated that use of four enzymes was necessary to differentiate between sequences having more than 95% similarity. However, since our study was aimed towards an overview of diversity, we concentrated on the differences revealed by one enzyme. The results presented here should therefore be regarded as indicating minimal diversity. As can be seen in Fig. 2, diversity within each probe target group varied greatly. In particular, group 687 showed very little evenness (distribution of the number of clones per pattern), with one phylotype making up 85% of the 687 positive clones; however, since different probe target groups represent different phylogenetic depths (probe 660, e.g., is specific for one genus and probe 804 is specific for a group of different genera), we refrain from comparing levels of diversity among the different groups. The data might, however, serve as a basis in future analyses for comparing levels of diversity of the same target group in different environmental samples.Methodological considerations. Clone libraries of 16S rDNAs have been widely used to investigate in a cultivation-independent approach the microbial communities of different, mainly pelagic or terrestrial habitats (4, 7, 9, 11, 33, 37, 38, 49). They have helped to elucidate common features within the microbial communities of specific habitats such as marine pelagic environments (9, 38) and have provided additional sequence information for the design and evaluation of probes. However, this experimental approach suffers from specific limitations that potentially confer selectivity via differential cell lysis, variable nucleic acid extraction efficiencies, or biased amplification in the PCR. The high lysis efficiency (93% ± 3.4%) and the high overall diversity in the clone library presented here suggest that our analysis was based on a substantial fraction of the bacterial community from Hornsund sediments; however, it is difficult to assess the potential bias introduced during amplification of the 16S rDNA. These biases are due to primer selectivity or erroneous product ratios caused by product saturation in the later cycles of amplification (52). Furthermore, oligonucleotides specific for a very general phylogenetic group, such as the bacterial 16S rDNA primers we used in PCR, are ultimately bound to miss some members of the community, which, in turn, leads to an underestimation of diversity.
Despite the caveats that clone abundance in the library does not necessarily reflect bacterial abundance at the site and that diversity might not be fully covered, the correlations between results of the clone library and results of completely different approaches such as 16S rRNA quantification analysis (see the accompanying paper [48]) and most probable number counts (23) are encouraging. The largest group of clones (19%) was detected by a probe designed especially for SRB isolated on the same cruise (23). While some of these isolates came from the same sampling site on the west coast of Spitsbergen (Hornsund), others were obtained from sediments sampled off the east coast (Storfjord). The clones were closely related to these isolates, with one phylotype even showing 100% sequence identity to strain LSv53, which was isolated from the east coast station (Fig. 3). This phylotype was represented by 12 of 353 clones. The same phylotype was also detected in a denaturing gradient gel electrophoresis-Southern blot analysis described in the accompanying paper; however, as expected, quantitative representation of the phylotypes in the clone library corresponds only weakly to the results from 16S rRNA slot blot hybridization (see the accompanying paper [48]).Phylogenetic composition of the clone library.
The clone
library was dominated by sequences related to
-Proteobacteria. Even within the clones targeted by the
general EUB338 probe only, we could detect one additional phylotype
affiliated with the subclass of Proteobacteria, loosely
related to Desulfobulbus. Twenty-three clones (6.5%)
belonged to this phylotype not targeted by any of the specific SRB
probes. Phylogenetic affiliation makes it likely that they are also
sulfate reducers. The design and application of a new probe specific
for this group and its employment in quantitative rRNA slot blot and in
situ hybridizations will show the extent to which it contributes to the
bacterial community.
subclass
of Proteobacteria, with a similarity value of 92 or 86%.
Since no pure culture representatives for this group have been
isolated, we can only speculate that they might indeed be involved in
the oxidative part of the sulfur cycle. Selective cultivation of sulfur
oxidizers from the same habitat is under way.
When investigating Wadden Sea sediments by fluorescence in situ
hybridization, Llobet-Brossa et al. (26) found members of the Cytophaga-Flavobacterium cluster to be even more
abundant than -Proteobacteria. This cluster has also been
found in marine aggregates (4, 40); Cytophagales,
in general, are known for their ability to associate and glide on
surfaces and to degrade a wide variety of polymeric substances
(42). They were also a significant constituent of our clone
library (5.1%), indicating that Cytophagales might be a
common member of marine sediment microbial communities.
Since sedimentation regularly brings in organic matter from the water
column, we expected to find evidence of allochthonous input in the
sediment. Groups that are commonly found in planktonic communities,
like some genera of -Proteobacteria (13, 34), were not abundant in the clone library; only 2 of 353 clones belonged to the -Proteobacteria. Furthermore, we did not detect
any cyanobacterial sequence and detected only one plastid sequence;
however, the presence of allochthonous microorganisms is probably
dependent on the time of sampling, with higher abundances expected
after a phytoplankton bloom.
Comparison with other clone libraries.
Open ocean and coastal
planktonic communities are well-studied ecosystems with regard to clone
libraries (10, 11, 34, 38, 49). Although the bacterial
communities of these habitats are phylogenetically diverse, distinct
phylogenetic clusters are repeatedly detected. These results are in
line with the idea that in similar climate zones, a limited number of
phylotypes account for a substantial fraction of the bacterioplankton
at certain times (34). It is still an open question whether
the same applies to benthic environments, since only limited data are
available on marine sediments. Devereux and Mundfrom (6)
established a clone library from a sandy marine sediment, selectively
amplifying partial 16S rDNAs of SRB. Gray and Herwig (15)
set up a general 16S rDNA clone library, examining 22 clones. Kato and
Li (21) investigated clones from deep-sea sediments off
Japan. A comparison is difficult, in particular because in many cases
only partial sequences are available, but some trends are noteworthy.
Sequences related to the Desulfotalea-Desulforhopalus
cluster were frequently recovered (Fig. 3). The highest similarity
values among clones with almost complete sequences were between 99.4 and 97.2%. Furthermore, all these clone libraries also contained
sequences related to Myxobacteria and
Bdellovibrio (Fig. 3; see below). Myxobacteria have been known mainly as terrestrial organisms (41); their isolation from coastal marine sediments has been attributed to resting
cells because of their low salt tolerance. Bdellovibrio, on
the other hand, has been repeatedly isolated from marine sediments (45). Considering the fact that related known pure cultures are almost all micropredators (41), they might play a role
in the control of bacterial abundance. Within the sequences of the -Proteobacteria, similar congruencies occurred. Both Kato
and Li (21) and Gray and Herwig (15) found
sequences from the symbiont cluster (Fig. 4) (highest similarities
among clones, between 98 and 92%).
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ACKNOWLEDGMENTS |
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We thank Ulrich Nübel for inspiring discussions and Birgit Rattunde for technical assistance. We acknowledge Steven M. Holland for providing the freeware program aRarefactWin.
This work was supported by the Max-Planck-Society.
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FOOTNOTES |
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* Corresponding author. Present address: Biotechnology I, Technical Microbiology, Technical University Hamburg-Harburg, Denickestr. 15, D-21073 Hamburg, Germany. Phone: 49 (0)40 42878 3336. Fax: 49 (0)40 42878 2909. E-mail: ksahm{at}mpi-bremen.de.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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