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Applied and Environmental Microbiology, November 1998, p. 4513-4521, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Analysis of Subfossil Molecular Remains of Purple
Sulfur Bacteria in a Lake Sediment
Marco J. L.
Coolen and
Jörg
Overmann*
Paleomicrobiology Group, Institute for the
Chemistry and Biology of the Marine Environment, University of
Oldenburg, D-26111 Oldenburg, Germany
Received 22 June 1998/Accepted 1 September 1998
 |
ABSTRACT |
Molecular remains of purple sulfur bacteria
(Chromatiaceae) were detected in Holocene sediment layers
of a meromictic salt lake (Mahoney Lake, British Columbia,
Canada). The carotenoid okenone and bacteriophaeophytin a
were present in sediments up to 11,000 years old. Okenone is specific
for only a few species of Chromatiaceae, including
Amoebobacter purpureus, which presently predominates in the chemocline bacterial community of the lake. With a
primer set specific for Chromatiaceae in combination with denaturing gradient gel electrophoresis, 16S rRNA gene sequences of
four different Chromatiaceae species were retrieved from
different depths of the sediment. One of the sequences, which
originated from a 9,100-year-old sample, was 99.2% identical to the
16S rRNA gene sequence of A. purpureus ML1 isolated
from the chemocline. Employing primers specific for A. purpureus ML1 and dot blot hybridization of the PCR
products, the detection limit for A. purpureus ML1 DNA could be lowered to 0.004% of the total community DNA. With this
approach the DNA of the isolate was detected in 7 of 10 sediment layers, indicating that A. purpureus ML1
constituted at least a part of the ancient purple sulfur bacterial
community. The concentrations of A. purpureus DNA
and okenone in the sediment were not correlated, and the ratio of DNA
to okenone was much lower in the subfossil sediment layers (2.7 · 10
6) than in intact cells (1.4). This indicates
that degradation rates are significantly higher for genomic DNA than
for hydrocarbon cell constituents, even under anoxic conditions and at
the very high sulfide concentrations present in Mahoney Lake.
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INTRODUCTION |
Mahoney Lake is a small saline
meromictic lake in the central south region of British Columbia
with a 6-meter-deep oxic mixolimnion overlying permanently anoxic
bottom layers. An extremely dense accumulation of phototrophic bacteria
has been detected in the chemocline of the lake (28, 30).
Previous cultivation experiments and light microscopic observations
showed the presence of three species of purple sulfur bacteria
(Chromatiaceae) in Mahoney Lake (30, 34). In the
pelagic chemocline, 98% of all phototrophic bacteria were identified
as Amoebobacter purpureus, and 2% belonged to the
species Thiocapsa roseopersicina (30). A third
Chromatiaceae species, Thiorhodovibrio
winogradskyi, was found in high numbers in littoral sediments but
not in the chemocline of the lake (34).
At present, the high rates of CO2 assimilation and
H2S oxidation of the dense population of A. purpureus in the chemocline form the basis of an
intense cycling of carbon and sulfur compounds in Mahoney Lake
(27, 29, 31). A stratigraphic analysis of the Holocene
sediments revealed that the lake became meromictic more than
9,000 years ago (20). This raises the question of whether
purple sulfur bacteria have been of ecological significance ever since.
In previous paleolimnological studies, fossil carotenoids have served
as a sensitive measure of past communities of phototrophic bacteria
(4, 50, 51). Accordingly, the carotenoid okenone, which is specific for A. purpureus and
only nine other species of the Chromatiaceae (6, 9,
39), has been detected in sediment layers up to 11,000 years old
(36).
The aims of the present study were (i) to examine whether and to what
extent intact ancient DNA is preserved in a sulfide-rich sediment and
(ii) to search specifically for 16S rRNA gene sequences of
Chromatiaceae present in the subfossil sediment layers. This analysis would provide a more solid basis for the reconstruction of
past microbial communities and environmental conditions in Mahoney Lake.
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MATERIALS AND METHODS |
Sediment core.
A sediment core (length, 6 m; diameter,
5.5 cm) was obtained with a piston corer from the deepest part in the
center of Mahoney Lake in May 1985 (36). Sections of 1-m
length were stored at 
20°C. Slices of the core were cut out with a
sterile knife. Depending on the age of the sediment, 16 to 170 g
of sediment was used for the extraction of genomic DNA. A total of 10 samples were analyzed. For the depths and ages of the different samples
see Table 1.
Precautions to prevent contamination and PCR controls.
Because our study relied on the analysis of 16S rRNA gene sequences by
PCR amplification, it was of utmost importance to prevent any
contamination of the sediment samples by foreign DNA.
A fresh surface of the sediment slices was generated by lifting the
upper 0.5-cm-thick layer of the cohesive sediment at one edge with a
sterile scalpel, thereby creating a sterile crack parallel to the
surface of the core. This generated an uncontaminated area through
which subsamples were obtained immediately with a sterile scalpel and
forceps. As a control for contamination during DNA extraction, a
parallel sample without sediment was subjected to the whole extraction
and purification procedure. All glassware was sterilized by dry heat (5 h at 160°C). Centrifuge bottles were cleaned with detergent, rinsed
with sterile filtered (0.2-µm pore size) and autoclaved water, and
cleaned with ethanol before they were autoclaved twice. All
amplification reactions were carried out in a laminar flow hood which
was exclusively used for PCR and cleaned with sodium hypochlorite
solution (0.6%, wt/vol) before each use. A special set of pipettes was
employed exclusively for the PCR work. Pipette tips with sterile
sealing filters (Biozym SafeSeal-Tips; Biozym Diagnostik, Hessisch,
Oldendorf, Germany) were used to prevent contamination by aerosols.
Disposable gloves were always worn. Each PCR amplification series
included one reaction without DNA template, which served as a control
for contaminations during the pipetting of the reaction mixture
components. The second control, containing 1 µl of the extraction
control, was similarly subjected to PCR amplification.
Extraction of total DNA.
An extraction protocol modified
after Ogram et al. (26) and Steffan et al. (47)
was employed for the sediment samples. The sediment was resuspended in
50 to 100 ml of lysis buffer (100 mM Tris-HCl, 500 mM EDTA, 2%
[wt/vol] glucose [pH 8.0]) and treated with lysozyme (final
concentration, 2 mg · ml1) for 1 h at room
temperature. Sodium dodecyl sulfate (SDS) was added to a final
concentration of 0.5% (wt/vol), and the samples were incubated for 5 min at 60°C. Proteinase K was added (final concentration of 0.5 mg
ml1), and the samples were incubated for 60 min at
37°C. Following centrifugation of the samples (6,048 × g, 30 min), the supernatant was transferred to a new sterile
centrifuge tube. The pellet was resuspended in 50 to 100 ml of 0.12 M
Na-phosphate buffer (pH 8.0), incubated for 45 min at 70°C, and then
centrifuged. This step was performed a second time.
The three supernatants were pooled and centrifuged again (30 min at
16,300 ×
g). The DNA in the supernatant was
precipitated
by addition of NaCl (0.5 M final concentration) and
polyethylene
glycol (PEG-8000, 25%) and incubation at 4°C for
24 h. Polyethylene
glycol and proteins were removed from the
pellet by extraction
with equal volumes of phenol, phenol-chloroform,
and chloroform
(
43). Humic compounds remaining in the
samples were removed
by adsorption onto acid-washed
polyvinylpolypyrrolidone (0.1 g
per ml of sediment
[
50]). A standard Na-acetate-ethanol precipitation
of
the liquid phase followed (
43), and the resulting
precipitate
was collected by centrifugation (20 min at 4,300 ×
g). The pellet
was redissolved in TE buffer (10 mM Tris-Cl, 1 mM EDTA [pH 8.0])
and purified by standard CsCl density
centrifugation (
43). After
a final standard precipitation
with Na-acetate-ethanol, the purified
DNA was dissolved in TE buffer
and quantified by binding of the
fluorescent dye Hoechst
33258.
Amplification of 16S rRNA gene sequences from the DNA extracts was
possible only after an additional purification step, which
was carried
out in the laminar flow hood. The samples were run
on sterile 1.5%
agarose gels in 1× TAE buffer (40 mM Tris-acetate,
1 mM EDTA [pH
8.0]), and the DNA was visualized by ethidium bromide
staining. The
portion of the gel containing DNA was cut out with
a sterile scalpel
and transferred to the elution chamber of a
Biotrap electroelution
system (Schleicher & Schuell, Dassel, Germany).
Electroelution
proceeded at 100 V for 12 h in accordance with
the instructions of
the manufacturer. The extraction control was
treated in an identical
manner. In this case, a gel slice which
corresponded in size to the
largest slice generated for the DNA
samples was cut
out.
Genomic DNA of the pure cultures and of samples from the chemocline of
Mahoney Lake were extracted by hot phenol as described
previously
(
37).
Specific amplification of Chromatiaceae 16S rRNA
genes.
Bacterial 16S rRNA genes of members of
Chromatiaceae present in the chemocline and in 10 different
Holocene sediment layers of Mahoney Lake were amplified in an UNO II
thermal cycler (Biometra, Göttingen, Germany). Three
Chromatiaceae species isolated from Mahoney Lake were used
as reference strains. A. purpureus ML1 and T. roseopersicina ML2 had been isolated from the pelagic chemocline, whereas T. winogradskyi DSMZ 6702T originated
from the littoral sediment. The specificity of the amplification
protocol was checked against a reaction mixture containing 50 ng of
Escherichia coli B12-H105 DNA as a template.
The forward primer used, Chr986f (
32), complements a target
sequence which is exclusively found in 16S rRNA sequences of
the
members of
Chromatiaceae that are presently available in the
RDP, EMBL, and GenBank databases (
2,
21,
48). The primer
sequence was 5'-AGCCCTTGACATCCTCGGAA-3'; its target region
extends
from
E. coli 16S rRNA positions 986 through 1005. The reverse
primer (GC1392r) binds to a universally conserved region at
E. coli 16S rRNA positions 1392 to 1406, shown underlined in
the
sequence
5'-CGCCCGCCGC GCCCCGCGCCCGGCCCGCCGCCCCCGCCCC
ACGGGCGGTGTGTAC-3'
(
1), and includes a 40-base GC clamp (
24).
Twenty-five nanograms of genomic DNA was amplified with 0.2 pmol of
each of the primers Chr986f and GC1392r, resulting in
a 461-bp-long
amplification product. Each amplification reaction
mixture contained 10 µmol of each deoxyribonucleoside triphosphate
and 5 µl of 10× PCR
buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3],
15 mM
MgCl
2, 0.01% [wt/vol] gelatin; Perkin-Elmer,
Weiterstadt,
Germany) and was adjusted to a final volume of 50 µl
with sterile
filtered (0.2 µm) and autoclaved double-distilled water.
A hot
start (denaturation at 96°C for 4 min, followed by the addition
of 1 U of Ampli
Taq DNA polymerase [Perkin-Elmer] at
80°C) was
performed. The melting temperature was set at 94°C for
30 s, and
the extension was carried out at 72°C for 40 s.
The temperature
ramp was set at 4°C s
1. Our step-down
PCR protocol included 15 cycles at an annealing
temperature of 65°C
(1.5 min) and 25 cycles at 58°C (1 min). A
final extension step was
performed for 10 min at 72°C. The sensitivity
of the approach was
increased by reamplification of 1 µl of each
PCR product for 30 cycles at a constant annealing temperature
of 58°C for 1
min.
PCR amplification specific for A. purpureus.
We designed a second forward primer, Ap454f
(5'-AGCGCAGGGTTAATACCCCTG-3', E. coli 16S rRNA
positions 454 to 474), which complements a target sequence of the 16S
rRNA gene which is specific to A. purpureus ML1. DNA
fragments to be analyzed by denaturing gradient gel electrophoresis
(DGGE) were amplified with the same primer (underlined in the sequence
shown below) but combined with the GC clamp (primer ApGC454f,
5'-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCAGCGCAGGGTTAATACCCCTG-3'). The specific primer was combined with the universal primer 907r (5'-CCGTCAATTCCTTTGAGTTT-3') (17). Amplification
was carried out for 25 cycles at a constant annealing temperature of
58°C (40 s). All other PCR conditions were identical to those of the protocol described above. The resulting 473-bp-long DNA fragments were
used as a target for DNA-DNA hybridization in dot blots.
Quantification of A. purpureus DNA by dot
blot hybridization.
Amplification products of the PCR specific for
A. purpureus ML1 were purified with QIAquick spin
columns, and the volume of the eluate was adjusted to 500 µl with
sterile deionized water. Samples were denatured for 7 min at 100°C
and vacuum blotted onto positively charged nylon membranes (Boehringer,
Mannheim, Germany). The membrane was baked (25 min at 120°C) and
prehybridized in 10 ml of DIG Easy Hyb buffer (Boehringer) at 40°C.
For the calibration of the dot blot, different amounts of genomic DNA
of A. purpureus ML1 were amplified in parallel and
the purified PCR products were blotted adjacent to those of the
sediment samples.
An
A. purpureus-specific probe was generated by
random labelling with digoxigenin (DIG)-11-dUTP using the PCR DIG probe
synthesis
kit (Boehringer) and the primers Ap454f and 907r. Primers and
other PCR components were removed from the probe (QIAquick purification
spin kit; Qiagen, Hilden, Germany). Hybridization was carried
out for
12 h at 40°C in 10 ml of prewarmed Easy Hyb buffer containing
1 pmol of the denatured probe. After hybridization, the blot was
washed
twice for 5 min in 2× SSC (150 mM NaCl, 15 mM Na-citrate
[pH 7.0])
plus 0.1% SDS at room temperature, followed by two stringent
washing
steps (15 min in 0.1× SSC-0.1% SDS at 68°C). The hybridization
signal was detected with the DIG luminescence detection kit. Lumi-Film
(Boehringer) was exposed for 6 min and developed, and the image
was
digitized with a flatbed scanner. For quantification of the
individual
dots, the ZERO-Dscan software (Scanalytics, Billerica,
Mass.) was
employed.
DGGE.
The 461-bp-long 16S rRNA gene sequences generated with
the Chromatiaceae-specific primer set were separated by DGGE
(23, 24). DGGE was carried out in a Bio-Rad D gene system.
PCR samples were applied directly onto 6% (wt/vol) polyacrylamide gels
(acrylamide/N,N'-methylene bisacrylamide ratio, 37:1
[wt/wt]) in 1× TAE buffer (pH 7.4) which had been prepared from
sterile solutions and casted between sterilized glass plates. The gels
contained a linear gradient of 30 to 70% denaturant (100%
denaturant = 7 M urea plus 40% [vol/vol] formamide). Electrophoresis proceeded for 5 h at 200 V and 60°C. Afterwards, gels were stained for 20 min with sterile ethidium bromide solution and photographed.
Sequencing of DGGE fragments.
DGGE fragments were cut out
with a sterile scalpel. The DNA of each fragment was eluted in sterile
1× TAE (pH 7.4) by electrophoresis (3 h, 200 V) in Centricon 50 concentrators inserted into a Centrilutor Micro electroelutor (Amicon,
Witten, Germany). One microliter of the purified and concentrated DNA
of each DGGE band was reamplified with 0.2 pmol of the primers Chr986f
and 1392r (this time without GC clamp). Primers and deoxyribonucleoside
triphosphates were removed with the QIAquick PCR purification spin kit
(Qiagen), and the amount of DNA was quantified with the fluorescent dye Picogreen (MoBiTec, Göttingen, Germany).
Sequences of the 16S rRNA gene fragments were determined by cycle
sequencing based on the dideoxy method (
44) and with the
SequiTherm EXCEL Long-Read Sequencing Kit-LC (Biozym). Each reaction
contained 300 fmol of template DNA and 2 pmol of primer labelled
with
the infrared fluorescent dye IRD-41 (MWG Biotech, Ebersberg,
Germany).
The 16S rRNA gene fragments from members of
Chromatiaceae were sequenced with the primer Chr986f at an annealing temperature
of
49°C. Fragments obtained with the primer pair specific for
A. purpureus ML1 were sequenced with the primer 907r
(
17) at
an annealing temperature of 53°C. The full 16S
rRNA gene sequences
of the three strains isolated from Mahoney Lake
were determined
as described previously (
37). Sequence data
were collected with
a LiCor-4000 automated sequencer (LiCor, Lincoln,
Nebr.).
Phylogenetic analysis.
Each sequence was checked for
chimeras by employing the CHECK_CHIMERA option of the ribosomal
database project (RDP). The program CLUSTAL W (49) was used
to align the sequences recovered from subfossil sediment layers with
those of the three Mahoney Lake isolates, ML1, ML2, and DSMZ
6702T, and all the sequences of Chromatiaceae
presently available in the RDP (21) and GenBank databases
(2). Distance matrices were calculated according to the
algorithm of Jukes and Cantor (14) with the DNADIST program
of the PHYLIP 3.57c program package (10). Only the base
positions that were identical in more than 50% of the aligned
sequences were included in the analysis. The phylogenetic trees were
inferred from evolutionary distances calculated with the FITCH program
of PHYLIP by using the least-squares algorithm of Fitch and Margoliash
(11).
Photosynthetic pigments.
One-centimeter sections of the
sediment core at 25 different depths were freeze-dried and ground in a
mortar. Subsamples (100 mg) were extracted twice with acetone (99.5%,
at 4°C for 24 h) in the dark. After centrifugation, absorbance
of the supernatants was determined in a Perkin-Elmer Lambda 2S
UV-visible-light spectrophotometer. Within the long-wavelength region,
we detected only one absorption peak, at 749 nm, which corresponds
exactly to the absorption maximum of bacteriophaeophytin a
(BPh a) (the Mg2+-free degradation product of
bacteriochlorophyll a). Because no other known bacterial
pigment absorbs at this wavelength, concentrations could be directly
determined in the acetone extracts without further chromatographic
separation. A specific absorption coefficient of 72.21 (g of BPh
a cm1) (calculated from the data given in
reference 25) was employed. The concentrations of
okenone have been determined by high-performance liquid chromatography
in a previous study (36) and are used here for comparative purposes.
Nucleotide sequence accession numbers.
The 16S rRNA gene
sequences of A. purpureus ML1, T. roseopersicina ML2, and T. winogradskyi DSMZ
6702T have been deposited at the EMBL database under
accession nos. AJ006212 to AJ006214, and the environmental sequences
have been deposited under nos. AJ006193 to AJ006197.
| |
RESULTS |
Extraction of total DNA.
The amounts of DNA recovered from the
sediment were highly variable among the different layers (Table
1). Analysis of the sizes of the DNA
fragments by agarose gel electrophoresis revealed that samples
extracted from younger sediment layers were up to 23 kb long, while the
maximum length decreased abruptly between the 3,720- and 5,580-year-old
sediment layers (Table 1). No DNA was present in the extraction control
as judged by the fluorometric assay. In no case did we detect
amplification signals after either amplification or even
reamplification of the extraction control (Fig.
1). These results provide strong evidence
that the extracted and purified DNA indeed originated from the sediment
layers and was not contaminated with foreign DNA.
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FIG. 1.
16S rRNA gene fragments from three strains of
Chromatiaceae from the extant bacterial community in the
chemocline and from samples from 10 different sediment layers of
Mahoney Lake were amplified with primers Chr986f and GC1392r. An
additional reaction mixture containing 1 µl of the DNA extraction
control (see text) was included in the PCR. In two of the reactions,
primer dimers formed during PCR (i.e., the 100-bp-long fragments in
5,580- and 8,220-year-old samples).
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The DNA content of intact cells of
A. purpureus ML1
determined for pure cultures was 7.85 µg · mg (dry
weight)
1.
Specific detection of Chromatiaceae.
Chr986f is the
first primer described for the specific amplification of 16S rRNA
genes of members of Chromatiaceae. With this primer,
67% of all Chromatiaceae 16S rRNA gene sequences which are
presently available in the RDP, EMBL, and GenBank databases can be
detected (32).
The phylogenetic tree constructed for all 16S rRNA gene sequences of
Chromatiaceae revealed that the strain of
A. purpureus isolated from Mahoney Lake (ML1) is not closely
related to the
type strain (DSMZ 4197
T; see Fig.
3). Strain
ML1 therefore represents a new species,
but its description has to be
postponed until additional 16S rRNA
gene sequences of
Chromatiaceae become available. Provisionally,
we therefore
use the former designation,
A. purpureus ML1,
in
the present
communication.
Four of the 10 sediment DNA samples yielded products during
Chromatiaceae-specific PCR (Fig.
1). The 16S rRNA gene
fragments
amplified with primers Chr986f and GC1392r from genomic
DNA of
the three Mahoney Lake reference strains could all be separated
on DGGE gels based on their different melting behaviors (Fig.
2). These fingerprints were used for
comparison with the signals
obtained from sediment DNA samples.
The fragments amplified from
the 9,100-year-old layer, and that from
the extant population
in the chemocline of the lake, had a melting
position identical
to that of
A. purpureus ML1
(Fig.
2). The 383-bp-long and 382-bp-long
sequences determined for both
fragments showed only three gaps
when aligned with the sequence of
A. purpureus ML1. The gaps fell
into a region
of band compression on the sequencing gel and therefore
most likely
represent a sequencing artifact. It appears unlikely
that the gaps were
the result of a chemical alteration of the
template DNA, as no mismatch
was found when the second primer
pair, Ap454f and 907r, was used
for amplification of the old DNA
samples. Based on the results of the
above sequence comparison,
a maximum error of 0.5% can be assumed for
the sequencing of 16S
rRNA genes from subfossil sediment layers.
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FIG. 2.
Separation of the 16S rRNA gene fragments depicted in
Fig. 1 on a DGGE gel. A negative image of an ethidium bromide-stained
gel is shown. Percentages on the left denote concentrations of
denaturant in the gel. Arrows point to the melting position of 16S rRNA
gene fragment of A. purpureus ML1.
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Based on its position on the DGGE gel, the amplification product which
originated from the 1,970-year-old sediment sample
was affiliated with
T. winogradskyi DSMZ 6702
T (Fig.
1). Its partial
sequence differed from that of the pure
culture by two gaps and a
mismatch at
E. coli position 1332. The
latter consisted of a
uracil present in strain DSMZ 6702
T which was replaced by
an adenine in the environmental sequence.
The uracil is not located in
a double-stranded portion of the
16S rRNA molecule (
13) and
might therefore represent a real
difference between the
sequences.
Two additional sequences in the 660- and 3,720-year-old sediment layers
were detected (Fig.
2). The amplification products
of both samples
exhibited a distinct melting behavior and had
a 16S rRNA gene sequence
which clearly differed from those of
all other
Chromatiaceae
species sequenced so far (Fig.
3). The
greatest sequence homology was found with
Chromatium gracile
DSMZ
203
T and amounted to 94.7% (for the 660-year-old
sample) and 93.4%
(for the 3,720-year-old sample).
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FIG. 3.
Phylogenetic tree containing all available sequences of
Chromatiaceae and the sequences obtained from the pelagial
zone and sediment of Mahoney Lake. Shaded boxes indicate strains which
contain the carotenoid okenone. y., years.
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Specific amplification and quantification of A. purpureus ML1 16S rRNA genes.
When
Chromatiaceae-specific amplification conditions were used,
the 16S rRNA gene of A. purpureus ML1 was
detected in only a single sediment layer. One of the additional
sequences which were retrieved from the other layers belongs to a
species (T. winogradskyi) which does not contain the
carotenoid okenone. However, this sequence was recovered from the same
sediment sample in which okenone reached a comparably high
concentration, 1.52 mg · (g of sediment)1 (see Fig.
6). This discrepancy could be due to either a skewed representation of
the 16S rRNA gene sequences of different Chromatiaceae species as a result of amplification bias or, alternatively, an actual
absence of DNA of A. purpureus ML1. This
prompted us to develop our highly sensitive and specific method for the
quantification of A. purpureus ML1 16S rRNA
gene sequences in the DNA extracts.
At the outset, it was mandatory to confirm the specificity of the
primer pair Ap454f and 907r and of the amplification protocol
used for the generation of template DNA for the dot blot hybridization.
Following PCR, all amplification products were analyzed by DGGE
(Fig.
4). The melting behavior of each fragment
was identical
to that of PCR products of
A. purpureus ML1, indicating a high
specificity of the
primer pair Ap454f and 907r. The specificity
was further confirmed
by sequencing of the DGGE bands generated
from the extant
chemocline population (not shown in Fig.
4) and
from the 660-year-old
sample. The sequence homology of both fragments
with the 16S rRNA gene
of the reference strain
A. purpureus ML1
was
100%. Evidently, our method for the detection of 16S rRNA
gene
sequences of
A. purpureus ML1 had the high
specificity required
for a quantitative analysis.
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FIG. 4.
Analysis of the melting behavior of 16S rRNA gene
fragments generated by PCR with primers Ap454f and 907r. To obtain
a visible amount of the DNA fragment and to introduce the GC clamp, the
PCR products were reamplified with primers ApGC454f and 907r. A
negative image of an ethidium bromide-stained DGGE gel is shown.
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For the quantification of
A. purpureus ML1 DNA
in the sediment samples, it was assumed that the number of 16S
rRNA genes in
strain ML1 is equal to that in cells of the natural
population.
A usable calibration curve between the amount of pure
culture
template DNA and the hybridization signal of its amplification
product was achieved only when the number of thermal cycles of
the PCR
was limited to a maximum of 25 and when a constant annealing
temperature was chosen (i.e., omitting a step down; compare with
the
Materials and Methods
section).
Hybridization with the
A. purpureus ML1 probe
could be detected for 7 of the 10 sediment extracts (Fig.
5; the very faint
signal in the
1,970-year-old sample could be visualized by increasing
the exposure
time). The oldest sediment layer containing detectable
amounts of
A. purpureus ML1 DNA was deposited 8,220 years
ago.
Contrary to the results of the amplification with
Chromatiaceae-specific
primers, no signal was obtained
for the 9,100-year-old sample.
Conversely, a significant amount of
A. purpureus ML1 gene sequences
was detected in
the 660-year-old sample which had not yielded
a corresponding signal
with
Chromatiaceae-specific primers. This
discrepancy
most likely results from a much larger amplification
bias when
Chromatiaceae-specific primers are used than when the
primer pair specific for
A. purpureus ML1
is used.
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FIG. 5.
Quantification of A. purpureus
ML1 DNA in the Holocene sediment layers by amplification with
primers Ap454f and 907r and dot blot hybridization. A digitized
image of an exposed Lumi-Film is shown. Twenty-five nanograms of
genomic DNA was added to the PCRs for T. winogradskyi DSMZ
6702T (Trv.), T. roseopersicina
ML2 (Tca.), and E. coli. For amplification of the
chemocline sample, only 10 pg was used. Control denotes the
amplification product of 1 µl of the extraction control (see text).
For the detection of samples from subfossil sediments, 25 ng of
extracted DNA was used. Sample numbering corresponds to that in Table
1. The different amounts of genomic DNA from A. purpureus ML1 used for calibration of the dot blot are
given in picograms.
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Slight cross-hybridization of the probe occurred with the blotted DNA
of
T. winogradskyi DSMZ 6702
T. However, in our
case this cross-hybridization does not interfere
with the
quantification of
A. purpureus ML1, for the
following
reasons. Firstly, DGGE and sequence analysis of the
amplification
products used for the dot blot did not show any 16S rRNA
gene
fragments other than those of
A. purpureus
ML1 (see above). Secondly,
the intensity of the signal
generated with 25 ng of genomic DNA
from
T. winogradskyi
DSMZ 6702
T is comparable to that obtained with 1.2 pg of
genomic DNA from
the
A. purpureus ML1 standard
(Fig.
5). Therefore, the PCR conditions
are approximately 20,800 times
more specific for the amplification
of
A. purpureus ML1 DNA than for the phylogenetically closely
related
T. winogradskyi DSMZ 6702
T.
Comparison of the vertical concentration profiles of okenone and
A. purpureus ML1 DNA revealed that the two
parameters are
not correlated in the Mahoney Lake sediment (Fig.
6). In addition,
the fraction of
A. purpureus ML1 DNA among the total
community
DNA was unexpectedly low given the high concentrations of
okenone
present in the sediment. Interestingly, the concentrations of
total DNA and
A. purpureus DNA were
tightly correlated (Fig.
7;
r2 = 0.968;
P < 0.001).
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 6.
Vertical distribution of BPh a (Bph a) and
okenone expressed in milligrams per gram (dry weight) [mg (g
d.w.)1] (A) and total DNA and DNA of A. purpureus ML1 (Amb. purpureus) in the
sediment of Mahoney Lake. For direct comparison, the concentrations of
okenone (dotted line) are also depicted in panel B. Total DNA is
expressed in micrograms per gram (dry weight) [µg (g
d.w.)1], and A. purpureus DNA is
expressed in nanograms per gram (dry weight) [ng (g
d.w.)1].
|
|
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 7.
Correlation between amounts of total DNA and DNA of
A. purpureus ML1. Dotted lines delineate 95%
confidence interval of second-order nonlinear regression. d.w., dry
weight.
|
|
The maximum fragment size of the extracted DNA decreased
with the age of the sediment layer and reached about 400 bp in the
older layers. Our quantification of DNA from
A. purpureus ML1
critically depended on the amplification of
a 450-bp-long DNA
fragment. Therefore it appeared possible that the
size of the
community DNA, rather than the percentage of
A. purpureus ML1
DNA within the community DNA, might limit
the amplification reaction.
If this was the case,
A. purpureus ML1 DNA should be overrepresented
in sediment
samples with long fragment lengths but represent a
significantly lower
percentage in samples containing small DNA
fragments. In contrast, our
actual results demonstrated that there
was no correlation between the
amount of
A. purpureus ML1 DNA
detected and the
maximum fragment length of the extracted total
DNA (Fig.
8).
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 8.
Relative proportion of A. purpureus ML1 DNA of the total subfossil DNA compared to
the range of fragment lengths of the subfossil DNA.
|
|
All partial 16S rRNA gene sequences obtained in the present study
were checked with the CHECK_CHIMERA option of RDP. According
to this
analysis none of the sequences represented
chimeras.
Photosynthetic pigments.
BPh a was the only
bacterial tetrapyrrole pigment detected in acetone extracts of the
subfossil sediment. The position of the long-wavelength absorption
maximum of the extracts coincided with that of BPh a,
indicating that no bacteriochlorophyll a was present.
Compared to the concentrations of okenone determined in a
previous study (36), the concentrations of BPh
a were significantly lower in all sediment layers. No close
correlation between the two pigments of purple sulfur bacteria was
apparent (Fig. 6). Intact cells of A. purpureus
ML1 in pure cultures contained 8.0 µg of bacteriochlorophyll
a · mg (dry weight)1 (corresponding to 7.8 µg of BPh a · mg [dry weight]1) and
5.6 µg of okenone · mg [dry weight]1). The
ratio of BPh a to okenone in the Mahoney Lake sediment was 0.228 ± 0.179 (n = 25) and thus was much
lower than that of intact cells (1.38).
| |
DISCUSSION |
Evidence for a subfossil origin of the isolated DNA.
In
previous studies, DNA has been isolated from up to 3-million-year-old
layers of a deep marine sediment, and 16S rRNA gene fragments could be
amplified from the extracted DNA (41) and sequenced
(38). However, within such marine sediment layers, substantial populations of metabolically active bacteria have been
detected by epifluorescence microscopy, cultivation methods, and
measurements of sulfate reduction rates (38). In contrast, a
study of 500- to 8,900-year-old profundal sediment layers of the deep
mesotrophic lake Lake Constance revealed the presence of only
metabolically inactive endospores of heterotrophic bacteria. Rates of
sulfate reduction and methanogenesis were below the detection limit
(42). The predominance of metabolically inactive endospores in these limnic sediments has been attributed to their high clay content, which effectively prevents pore water seepage and thus exchange of carbon substrates and electron acceptors. At the same time
vertical transport of bacterial cells is prevented (42).
In the sediments studied so far, it is very difficult to distinguish
between the DNA derived from physiologically active bacteria
and that
of ancient origin. By comparison, the bottom sediment
of Mahoney Lake
is well suited for the study of a past microbial
community; the 16S
rRNA gene sequences investigated in the present
study are very unlikely
to have originated from metabolically
active cells, for three reasons.
First, concentrations of sulfide
and polysulfide measured in the
monimolimnion of Mahoney Lake
do not increase between a water depth of
12 m and the sediment
surface at 14.5-m depth (
31). It
is thus evident that terminal
degradation of organic matter presently
does not occur in the
profundal sediment and that a considerable
part of the chemoheterotrophic
bacterial community, at least
sulfate- and sulfur-reducing bacteria,
must be
inactive.
Second, the predominance of short fragments in the DNA extracts (Table
1) is difficult to reconcile with the presence of
a large number of
intact bacterial cells. We omitted the bead-beating
step of the
original method of Ogram et al. (
26) in order to
minimize
the shearing of the DNA during extraction of the sediment
samples. By a
method similar to ours, very large fragments of
genomic DNA with
lengths of 20 to 25 kb could be isolated from
503-meter-deep marine
sediments (
41). In contrast, the considerable
fragmentation
of the DNA isolated during the present study indicates
that the
majority of the extracted DNA did not originate from
intact bacterial
genomes.
Third, further support for a subfossil origin of the isolated DNA comes
from the well-studied physiology of purple sulfur
bacteria. In the
present, meromictic state of Mahoney Lake, underwater
irradiance does not penetrate the chemocline and thus does not
reach
the surface of the profundal sediment. However, the water
level of
Mahoney Lake fluctuated considerably in the past, and
holomixis
occurred during dry periods with low water levels (
20).
Temporarily, light might therefore have reached bottom sediments
but
would have been rapidly attenuated within the first millimeter
of the
thickness of the sediment (
16). Several species of the
Chromatiaceae are able to grow chemolithotrophically in the
dark
with reduced sulfur compounds and in the presence of oxygen as
an
electron donor (
8,
12,
15,
35). However, growth under
these
conditions is strictly dependent on the presence of molecular
oxygen,
which is highly unlikely to have penetrated far into the
organic-matter-rich Mahoney Lake sediment. Even more significant
is the
fact that
A. purpureus ML1 is not capable of
growing chemolithotrophically
in the dark (
35).
Taken together, all results of our molecular biological work, of
physicochemical measurements, and of paleoclimatological
studies
provide strong evidence that the 16S rRNA gene sequences
of
Chromatiaceae isolated from the profundal sediment layers of
Mahoney Lake are indeed of subfossil origin and do not belong
to a
metabolically active extant
population.
Comparison of the different subfossil molecular remains of purple
sulfur bacteria.
Similar to other limnic sediments (4)
the analysis of bacteriochlorophylls in the Mahoney Lake sediment
revealed that bacteriochlorophyll a is completely converted
to BPh a during deposition. In contrast to other lakes,
(3, 4, 51), however, okenone concentrations exceeded by
far those of all other carotenoids in Mahoney Lake (36). It
was therefore totally unexpected that A. purpureus ML1 DNA represented only a minute fraction of
the total community DNA. As discussed below, these data indicate a
preferential degradation of the DNA of A. purpureus ML1 as compared to its photosynthetic pigments.
For intact
A. purpureus cells we determined the
ratios of DNA to okenone and of DNA to BPh
a to be 1.4 and 0.98, respectively.
By contrast, these ratios were much decreased
in the subfossil
sediment layers (mean values, 2.7 · 10
6 and 2.1 · 10
5, respectively),
indicating that the DNA of
A. purpureus ML1 is
degraded significantly faster than its specific carotenoid,
okenone.
An alternative explanation would be that about 99.999% of
the
DNA present in the Mahoney Lake sediment cannot be extracted
by
following established methods. The DNA yield of the Mahoney
Lake
sediments (up to 19.1 µg · g [dry weight] of
sediment
1; Table
1) falls well into the range reported
for other sediments
(11.8 to 26 µg · g [dry weight] of
sediment
1;
22,
26,
47). It is
therefore very unlikely that a major
fraction of the genomic DNA was
missed in our molecular biological
analysis. Because the ratios
of
A. purpureus DNA to okenone
were
equally low in the uppermost and lowermost sediment layers
(2.9
· 10
6 and 1.2 · 10
6,
respectively), the preferential degradation of DNA must have
occurred
already during sedimentation of the cells or very soon
after their
burial in the
sediment.
Theoretically, the low ratio of DNA to okenone or DNA to BPh
a could also be caused by a predominance of
Chromatiaceae species
which contain the carotenoid
okenone but are different from
A. purpureus
ML1, as okenone has been found in nine other species
of the
Chromatiaceae (
6,
9,
39). By means of specific
amplification of 16S rRNA gene sequences of
Chromatiaceae,
A. purpureus ML1 was the only
okenone-containing strain of this family
which could be positively
identified. As a limitation of our approach,
the identity of the
carotenoid belonging to the
Chromatiaceae species with the
two new 16S rRNA gene sequences (from 660- and
3,720-year-old
sediments) cannot be deduced from their phylogenetic
affiliation as
long as the corresponding strains have not been
isolated in pure
culture. When DNA samples were amplified with
the nonspecific
eubacterial primer pair 341f and 907r (
17) and
the
resulting fragments were analyzed by DGGE, five different
16S rRNA gene
sequences were detected in the DNA samples, but
none was affiliated
with the purple sulfur bacteria (
33).
A. purpureus is the only okenone-containing bacterium
presently found
in the pelagial and littoral zones of the lake by
cultivation
experiments, light microscopical observations
(
30,
34), and
PCR or DGGE (Fig.
2). This combined evidence
renders it rather
unlikely that an additional 16S rRNA sequence
type from member(s)
of
Chromatiaceae was missed by our
approach. Finally,
A. purpureus ML1 could
account for only 0.0002% of the total
Chromatiaceae biomass
if the degradation rates for its genomic DNA were not
different from
that of
okenone.
The amount of DNA from
A. purpureus ML1 did not
correlate with the amount of okenone. It has to be concluded not
only that
the degradation rate of DNA from
A. purpureus ML1 is much higher
than that of okenone but
also that the mechanisms of degradation
and preservation must differ
markedly between the two cellular
constituents. The concentrations of
total DNA correlated with
those measured for
A. purpureus ML1, which indicates that DNA
from this strain
must have represented a rather constant fraction
of the total input of
DNA during the history of Mahoney
Lake.
Relevance of subfossil 16S rRNA gene sequences for
paleomicrobiological studies.
Fully hydrated DNA spontaneously
degrades into short fragments within several thousands of years, the
most important route for decay being depurination (19). High
ionic strength and adsorption to hydroxyapatite result in a retardation
of the rate of depurination by one order of magnitude
(19). As a first approximation, the extent of chemical
modifications of the subfossil DNA from Mahoney Lake was assessed by
sequence analysis of the amplified fragments. We found that for
stretches of 461 and 473 bp (A. purpureus-specific amplification), the sequencing error
was 
0.5% and thus not significantly increased in comparison to that
of DNA extracted from the extant bacterial community. Our findings
corroborate previous findings that amplification of 16S rRNA gene
fragments is reliable for samples up to several tens of thousands of
years old (19, 40). The molecular biological analysis of
ancient DNA obviously can also be applied to genomic DNA of
subfossil microbial communities in aquatic sediments, provided that
their physiological activity in situ has ceased shortly after deposition.
Based on a stratigraphic analysis of the sediment core, the
paleoenvironmental conditions of Mahoney Lake have been reconstructed
(
20). The lake became saline early after its formation.
Changes
of climate resulted in frequent and dramatic changes in water
levels, which in turn triggered the transition from stably stratified
(meromictic) through shallow nonmeromictic to transiently
approaching
dryness and then the reverse sequence. A total of at least
eleven
such cycles has been detected by stratigraphic analysis of the
sediment core. Laminated sediments are particularly
characteristic
of meromictic lakes and comprise about
one-third of the length
of the sediment core from Mahoney Lake
(
20). The vertical position
of the laminated sediment layers
indicates that meromixis developed
ca. 9,150 years ago and repeatedly
occurred throughout the whole
lake history. Some meromictic
periods lasted for 1,100
years.
However, even during the remaining two-thirds of the lake history,
which presumably encompassed holomictic periods, okenone
remained
the predominant carotenoid in the sediment (
36), indicating
that okenone-containing purple sulfur bacteria dominated. The
coexistence of okenone and nucleic acids of
A. purpureus ML1 found
in the present study now indicates
that this strain was part of
the purple sulfur bacterium layer and must
have colonized the
lake soon after the formation of Mahoney Lake, after
the retreat
of the Wisconsin ice shield. It still remains unclear
whether
A. purpureus ML1 was the dominant
Chromatiaceae species in the
past chemocline bacterial
community.
So far, mostly photosynthetic pigments deposited in the sediment of
aquatic systems have been used as indicators for past
changes in the
trophic status and the development of freshwater
lakes (
3,
4,
5,
18,
36,
45,
51). Proteins and
aldoses are other biochemical
indicators that were studied for
their utility as records of
depositional history (
7), and they
provide information on
the relative diagenetic stage and reaction
potential of the deposited
organic matter. As shown in the present
study, the analysis of
nucleotide sequences has the potential
to provide information on the
species composition of past microbial
communities, but it may be
significantly impaired by the variability
in degradation rates of the
genomic DNAs of different bacterial
species.
| |
ACKNOWLEDGMENTS |
We are indebted to K. J. Hall for providing the samples of
the sediment core and J. T. Beatty for support during the initial phase of the project. J. Glaeser and K. Nauhaus helped with the DNA
extraction. We thank H. Cypionka for support and stimulating discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Paleomicrobiology Group, Institute for the Chemistry and Biology
of the Marine Environment, University of Oldenburg, P.O. Box 2503, D-26111 Oldenburg, Germany. Phone: 49-441-970-6376. Fax:
49-441-798-3583. E-mail:
j.overmann{at}palmikro.icbm.uni-oldenburg.de.
| |
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Applied and Environmental Microbiology, November 1998, p. 4513-4521, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
