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Applied and Environmental Microbiology, March 1999, p. 1325-1330, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Situ Analysis of Phototrophic Sulfur Bacteria in
the Chemocline of Meromictic Lake Cadagno (Switzerland)
Mauro
Tonolla,*
Antonella
Demarta,1
Raffaele
Peduzzi,1 and
Dittmar
Hahn2,3
Cantonal Institute of Bacteriology, Microbial
Ecology (University of Geneva), CH-6904 Lugano,1
and Swiss Federal Institute of Technology (ETH), Institute of
Terrestrial Ecology, Soil Biology, CH-8952
Schlieren,2 Switzerland, and
Department of Biology, Rutgers University, Newark, New Jersey
07102-18113
Received 5 August 1998/Accepted 8 December 1998
 |
ABSTRACT |
Comparative sequence analysis of a 16S rRNA gene clone library from
the chemocline of the meromictic Lake Cadagno (Switzerland) revealed
the presence of a diverse number of phototrophic sulfur bacteria.
Sequences resembled those of rRNA of type strains Chromatium okenii DSM169 and Amoebobacter purpureus DSM4197, as
well as those of four bacteria forming a tight cluster with A. purpureus DSM4197 and Lamprocystis roseopersicina
DSM229. In situ hybridization with fluorescent (Cy3 labeled)
oligonucleotide probes indicated that all large-celled phototrophic
sulfur bacteria in the chemocline of Lake Cadagno were represented by
C. okenii DSM169, while small-celled phototrophic sulfur
bacteria consisted of four major populations with different
distribution profiles in the chemocline indicating different
ecophysiological adaptations.
| |
TEXT |
Lake Cadagno is an alpine lake
situated 1,923 m above sea level in the Piora valley in the south of
Switzerland (46°33'N, 8°43'E). The lake has a surface area of 26 by
105 m2 and a maximum depth of 21 m. Due to
the infiltration of water through dolomite rich in gypsum, Lake Cadagno
is a meromictic lake characterized by a high salinity of the
monimolimnion and a permanent chemocline at a depth of between 9 and
14 m separating the aerobic epilimnion from the anaerobic,
sulfidogenic hypolimnion (22, 33). A turbidity maximum in
this chemocline suggests that a bacterial community making use of
energy gradients is present. It is assumed that such a community may
mainly consist of sulfate-reducing bacteria (10) and
lithotrophic sulfur bacteria (14, 17), as well as
phototrophic sulfur bacteria if light reaches the anoxic part of the
lake (11, 18, 21).
Since many of these organisms require complex gradients of, e.g.,
light, sulfide, oxygen, and pH for optimal growth (24), studies of their ecology are often impeded by the fact that they are
difficult to isolate or even resist cultivation, which is an essential
prelude to characterization by traditional laboratory methods.
Therefore, studies often focus on bacteria with distinctive morphological features which can be analyzed by light or
epifluorescence microscopy after staining with fluorochromes such
as acridine orange (13) or 4'-6-diamidino-2-phenylindole
(DAPI) (12, 23). This approach, however, is restricted to a
few organisms with highly distinctive morphologies. Based on a
combination of cell size and autofluorescence as distinctive parameters
for different populations of phototrophic sulfur bacteria, the major
populations in the chemocline of Lake Cadagno, for example, are
identified as Chromatium okenii and Amoebobacter
purpureus (7, 22).
The aim of our study was to analyze populations of phototrophic sulfur
bacteria in the chemocline of Lake Cadagno by molecular methods. These
studies were based on comparative sequence analysis of a 16S rRNA gene
clone library (19, 29) from the chemocline and 16S ribosomal
DNA (rDNA) of phototrophic sulfur bacteria, including C. okenii DSM169, Lamprocystis roseopersicina DSM229, A. purpureus DSM4197, and Amoebobacter roseus
DSM235. Specific oligonucleotide probes were subsequently designed and
used to enumerate specific populations of phototrophic sulfur bacteria in the chemocline.
Sequence analysis.
Nucleic acids from C. okenii
DSM169, L. roseopersicina DSM229, A. purpureus
DSM4197, and A. roseus DSM235, as well as from bacterioplankton in water samples from the chemocline, were isolated by
the method described by Ausubel et al. (4). 16S rRNA genes were amplified by PCR on a Perkin-Elmer (Rotkreuz, Switzerland) GeneAmp
2400 thermal cycler with primers UNI16SRNA-3F (5' ATT CTA GAG TTT GAT
CAT GGC TCA) and UNI16SRNA-1419R (5' ATG GTA CCG TGT GAC GGG CGG TGT
GTA), respectively. Thirty-five rounds of temperature cycling (94°C
for 30 s, 52°C for 30 s, and 72°C for 1 min) were
followed by a final 7-min incubation at 72°C. Amplified fragments
were cloned into Phagemidvector Bluescript and transformed into
Escherichia coli TOP-10 (Stratagene, Heidelberg, Germany). Plasmid DNA was extracted and purified with the QIAprep-spin plasmid purification procedure kit (Qiagen, Inc., Chatsworth, Calif.). Three
hundred fifty clones of the rDNA clone library were screened by
restriction analysis with the endonucleases HaeIII and
MboI (Promega, Wallisellen, Switzerland), respectively, to
determine phylotype distribution (16). rDNA of
representative clones of the phylotypes as well as of C. okenii DSM169, L. roseopersicina DSM229, A. purpureus DSM 4197, and A. roseus DSM235 was
reamplified, and the amplification products were purified by using
Centri-Sep columns (Princeton Separations, Inc., Adelphia, USA) and
sequenced with an ABI PRISM Ready Reaction dye deoxy terminator cycle
sequencing kit and an ABI Prism 310 automated sequencer (Perkin-Elmer).
High similarity values were obtained for sequences of clones compared
to sequences of representative cultures of phototrophic sulfur bacteria
(Table 1) when the phylogenetic positions
of C. okenii DSM169, L. roseopersicina DSM229,
A. purpureus DSM4197, A. roseus DSM235 and
selected clones after EMBL/GenBank database searches with FASTA
(20) were preceded by alignment of sequences of selected
bacteria by using the CLUSTAL W program (version 1.6) (30).
Clone 359 showed a nearly identical sequence to C. okenii DSM169, differing in just one base, whereas clone 345 showed a sequence
identical to that of A. purpureus DSM4197. A high similarity value of 99.2% was also obtained for rRNA sequences of A. purpureus DSM4197 and L. roseopersicina DSM229.
Sequences of representative clones of four phylotypes, clones 136, 261, 335, and 371, respectively, displayed similarity values of between 98.3 and 99.9% to each other and to sequences obtained from both A. purpureus DSM4197 and L. roseopersicina DSM229. This
indicated the occurrence of a phylogenetically very tight cluster of
different phototrophic sulfur bacteria in the chemocline of Lake
Cadagno. Similarity values of all clones to other phototrophic sulfur
bacteria such as, e.g., A. roseus, Chromatium
vinosum, Thiorhodococcus minus, or
Thiorhodococcus gelatinosa were generally below 95% (Table 1).
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TABLE 1.
Percent similarity between the 16S rRNAs of clones from
the chemocline of Lake Cadagno and those of closely
related organismsa
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The formation of a tight cluster of clones 136, 261, and 345 with
sequences from A. purpureus and L. roseopersicina, with clones 371 and 335 still closely related to
this cluster, was further shown in a phylogenetic tree which was
calculated by the neighbor-joining method (26) in CLUSTAL W
(Fig. 1). These results suggested that
small-celled phototrophic sulfur bacteria in the chemocline of Lake
Cadagno consisted of at least four major populations, while
large-celled phototrophic sulfur bacteria were represented only by
C. okenii.
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FIG. 1.
Neighbor-joining tree based on the aligned sequences of
selected clones from the 16S rRNA gene library of the chemocline of
Lake Cadagno and of selected bacteria searched from the EMBL/GenBank
databases by FASTA through the GCG package. The distance scale
indicates the expected number of changes per sequence position. Bars
and probe designations indicate target groups of phototrophic sulfur
bacteria for specific oligonucleotide probes.
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Probe design.
In order to investigate the significance of the
sequence information obtained from the environment, specific
oligonucleotide probes were designed that allowed us to detect all
sequences within the cluster representing the small-celled phototrophic
sulfur bacteria (Fig. 1). All probes were targeting 16S rRNA at the
position 453 to 478 according to the E. coli numbering
(5). Sequence comparison in this highly variable region
revealed at least five differences on a small stretch of 26 bases
between sequences of the phototrophic sulfur bacteria investigated
which allowed us to design highly specific probes (Table
2). Probe Apur453 targeted A. purpureus, but it also targeted the rRNA of bacteria harboring the
sequence of clone 345. Probe Laro453 targeted L. roseopersicina, as well as the rRNA of bacteria harboring the
sequence of clone 136, while probe S453D targeted the 16S rRNA of
bacteria harboring the sequence of clone 261, and probe S453F targeted
the rRNA of bacteria harboring the sequences of clones 335 and 371.
Large-celled phototrophic sulfur bacteria were analyzed with probe
Cmvi453 targeting
C. vinosum and with probe Cmok453
targeting
C. okenii and the rRNA of bacteria harboring the
sequence in clone
359, although it had a mismatch to probe Cmok453 at
position 474
(Table
2). Probe specificity with reference to the
available
16S rRNA sequences was checked with the ARB program
(
28) and
in the EMBL/GenBank databases by using FASTA
(
20) through the
GCG package (Genetics Computer Group,
University of Wisconsin,
Madison). Pure cultures of phototrophic sulfur
bacteria, such
as
C. okenii DSM169,
C. vinosum
DSM180,
L. roseopersicina DSM229,
A. purpureus
DSM4197, and
A. roseus DSM235, as well as those of
bacteria
from other phyla, like
Desulfotomaculum orientis DSM765,
Desulfovibrio desulfuricans DSM642,
Burkholderia
cepacia DSM50181,
Brevundimonas diminuta DSM1635, and
Campylobacter jejuni DSM4688
were used to test probe
specificity and to establish appropriate
in situ hybridization
conditions for the specific detection. The
specificity of the
hybridization was then adjusted by the addition
of 40% formamide to
the hybridization buffer (except for probe
Cmok453, for which the
formamide concentration was 35%) and by
a reduction of NaCl in the
washing buffer to 80 or 56 mM, depending
on the formamide concentration
during hybridization (35 and 40%,
respectively) (
35).
Population analysis of phototrophic sulfur bacteria in the
chemocline.
Bacteria were analyzed in water samples from the
chemocline obtained with a thin-layer pneumatic multisyringe sampler
(University of Zürich, Institute of Microbiology, Zurich,
Switzerland) (31). During sampling, the physicochemical
parameters (temperature, conductivity, pH, dissolved oxygen, and
turbidity) were measured with a Hydropolytester HPT-C profiler
(Züllig AG, Rheineck, Switzerland) and showed the characteristic
stratification profile of Lake Cadagno (22, 33). The rapid
decrease of oxygen (data not shown) and the increase in sulfide
concentrations (Fig. 2a) determined
colorimetrically in water samples (31) indicated the
formation of a condensed chemocline at a depth of between 11.5 and
14 m. The maximum turbidity was found at a depth of 11.7 m
(Fig. 2a), suggesting the presence of bacterioplankton taking advantage
of the downward O2 or light fluxes and the upward fluxes of
reduced compounds (11).
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FIG. 2.
Vertical distribution of physicochemical parameters and
bacteria in the chemocline of Lake Cadagno at a depth of between 11 and
14 m. (a) Sulfide ( ) and turbidity ( ). (b) Cells detectable
after in situ hybridization with probes Cmok453 ( ) and Laro453
( ). (c) Cells detectable after in situ hybridization with probes
S453D ( ) and Apur453 ( ). (d) Cells detectable after in situ
hybridization with probe S453F ( ); the sum of cells detectable after
in situ hybridization with probes Apur453, Laro453, S453D, and S453F
( ); and the number of small-celled phototrophic sulfur bacteria
determined by using autofluorescence and cell size as distinctive
criteria ( ). The data, determined from 40 microscopic fields of
three samples, are expressed as means ± standard errors.
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In situ hybridization with fluorescent (Cy3-labeled) oligonucleotide
probes was performed with aliquots (3 µl) of paraformaldehyde-fixed
water samples (
n = 3) spotted onto gelatin-coated
slides [0.1%
gelatin, 0.01% KCr(SO
4)
2]
(
9) and hybridized and concomitantly
stained with DAPI
according to the method of Zarda et al. (
35).
The analysis
was performed in a top-to-bottom approach, initially
detecting members
of the domain
Bacteria (probe EUB338) (
3);
subsequently detecting bacteria of the
(ALF1b) (
15),
(BET42a)
(
15),
(GAM42a) (
15), and
(SRB385) (
2) subdivisions
of
Proteobacteria; and
finally detecting different populations
of phototrophic sulfur bacteria
(Table
2). The slides were examined
by epifluorescence microscopy with
filter sets F31 (AHF Analysentechnik,
Tübingen, Germany
[D360/40, 400DCLP, D460/50]) and F41 (AHF Analysentechnik
[HQ535/50,
Q565LP, HQ610/75]). Microorganisms were counted at
×1,000
magnification in 40 fields covering an area of 0.01 mm
2
each (
8). Numbers were expressed as means ± standard
errors.
The sum of all bacteria detected with probes targeting
phototrophic
sulfur bacteria was finally compared to the numbers of
autofluorescent
bacteria determined by epifluorescence microscopy with
filter
set F41. Autofluorescence was taken as a distinctive parameter
characteristic of all phototrophic sulfur
bacteria.
In situ hybridization with probe EUB338 allowed us to detect between 38 and 90% of the DAPI-stained cells in the chemocline
(
31),
which was comparable to other aquatic environments, such
as, e.g.,
anoxic water of a stratified fjord (20 to 50%) (
25),
lake
snow (55 to 100%) (
34), the winter cover and pelagic zone
of a high mountain lake (40 to 81%) (
1), and water from two
artificial ponds (35 to 67%) (
12). Averaged over the whole
chemocline,
cells hybridizing with probes ALF1b, BET42a, GAM42a, and
SRB385
accounted for 23, 17, 45, and 15% of the DAPI-stained bacteria,
respectively (
31). On average, 33% of the DAPI-stained
bacteria
were represented by autofluorescent cells which hybridized to
probe GAM42a (
31) and morphologically resembled the
large-celled
phototrophic sulfur bacterium
C. okenii, with a
cell size of 4.5
to 6 by 8 to 15 µm (
32), and the
smaller-celled species
A. purpureus,
with a cell size of 3.3 to 3.8 by 3.5 to 4.5 µm (
6).
All large-celled phototrophic bacteria enumerated by using a
combination of cell size and autofluorescence as a distinctive
parameter hybridized to probe Cmok453 targeting the
C. okenii type strain, DSM169 (Fig.
3a). None of these cells showed
hybridization
signals with probe Cmvi453 targeting
C. vinosum (data not shown).
These results demonstrated that type
strain DSM169, which was
isolated from a pond, really represented the
large-celled species
C. okenii in Lake Cadagno.
C. okenii occurred in numbers of between
(3 ± 4) × 10
3 and (24 ± 7) × 10
3 cells
ml
1 in the chemocline, with a maximum occurrence at a
depth of 12.5
m (Fig.
2b). Maximum numbers were comparable to
those of earlier
studies which were (48 ± 13) × 10
3
cells ml
1 (
22) or to those in other lakes,
such as Lake Belovod, with
approximately 50 × 10
3
cells ml
1 (
27). The occurrence of
C. okenii correlated with environmental
parameters, including anoxic
conditions and high sulfate and low
sulfide concentrations (Fig.
2a).
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FIG. 3.
In situ detection of phototrophic sulfur bacteria with
Cy3-labeled probes Cmok453, targeting C. okenii (a);
Apur453, targeting A. purpureus (b); S453D, targeting clone
261 (c); and S453F, targeting clones 335 and 371 (d). Bar, 10 µm.
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Compared to the vertical distribution of
C. okenii,
A. purpureus-like cells showed a broader distribution, with high cell
densities in the lower part of the chemocline (Fig.
2b to d) which
might be due to their high sulfide tolerance (
6). In situ
hybridization
with probes Apur453, Laro453, S453D, and S453F targeting
a phylogenetically
and morphologically very tight cluster of different
small-celled
phototrophic sulfur bacteria resulted in the detection of
distinct
populations of phototrophic sulfur bacteria in the chemocline
of Lake Cadagno (Fig.
3b to d). Probe Apur453 detected up to (7
± 2) × 10
5 cells ml
1 at a depth of 12.5 m, whereas probes Laro453, S453D, and S453F
detected (1 ± 0) × 10
5, (7 ± 3) × 10
5, and (4 ± 1) × 10
5 cells ml
1 at their maximum occurrence at
a depth of 12.5 m, respectively
(Fig.
2b to
d).
These results showed that the type strain of
A. purpureus,
DSM4197, did not represent the major population of
Amoebobacter-like
cells in the chemocline of Lake Cadagno
and that different populations
were present. This result was not
surprising, because earlier
studies with an isolate obtained from Lake
Cadagno (LcCAD1) had
already indicated some size differences from the
type strain that
was isolated from Lake Schleinsee in Germany
(
6). Populations
of small-celled phototrophic sulfur
bacteria not only differed
with respect to total population sizes but
also differed with
respect to population profiles in depth. While
populations detected
with probe S453D only revealed a distinct maximum
occurrence at
a depth of 12.5 m, populations detected with probe
Apur453 showed
a second maximum occurrence at a depth of 13.1 m,
and those detected
with probe S453F showed an evenly high distribution
at a depth
of between 12 and 13.1 m (Fig.
2b and
c).
In the overall depth profile, the sum of the individual numbers
detected after hybridization with probes Apur453, Laro453,
S453D, and
S453F was comparable to the cell numbers determined
by using a
combination of cell size and autofluorescence as a
distinctive
parameter for small-celled phototrophic sulfur bacteria
(Fig.
2d).
Between 96.4 and 100% of autofluorescent cells were
further detected
throughout the whole chemocline when a combination
of all probes was
used. These results suggested that sequence
information for the major
populations of small-celled phototrophic
sulfur bacteria was obtained
and retrieved in the 16S rRNA gene
clone library from bacterioplankton
in the chemocline of Lake
Cadagno. Small-celled phototrophic sulfur
bacteria in the chemocline
of Lake Cadagno comprised four major
populations with different
distribution profiles indicating different
ecophysiological adaptations
and requirements (
32). Future
studies of populations of small-celled
phototrophic sulfur bacteria in
the chemocline of Lake Cadagno
will therefore include an analysis of
temporal and spatial distributions
of specific populations in relation
to environmental factors such
as light, electron donors, and oxygen and
carbon sources (
21,
32).
Nucleotide sequence accession number.
The 16S rDNA sequences
determined in this study have been deposited in the EMBL/GenBank
databases under accession no. AJ223234, AJ223235, AJ006221, and
AJ006057 to AJ006063, respectively.
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ACKNOWLEDGMENTS |
This work was supported by grants from the Swiss National Science
Foundation (NF31-46855.96), the Swiss Federal Office of Environment,
Forests and Landscape (BUWAL) and the canton of Ticino (Switzerland).
We are indebted to N. Ruggeri and A. Caminada for technical support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Cantonal
Institute of Bacteriology, Microbial Ecology, Via Ospedale 6, CH-6904
Lugano, Switzerland. Phone: 41-91-923 25 22. Fax: 41-91-922 09 93. E-mail: mauro.tonolla{at}ti.ch.
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Applied and Environmental Microbiology, March 1999, p. 1325-1330, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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