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Previous Article | Next Article 
Applied and Environmental Microbiology, August 1999, p. 3721-3726, Vol. 65, No. 8
Max-Planck-Institut für Marine
Mikrobiologie, Bremen, Germany
Received 11 March 1999/Accepted 25 April 1999
Fluorescence in situ hybridization (FISH) with rRNA-targeted
oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and
marine samples. An average of about 50% of the cells were detected by
probes for the domains Bacteria and Archaea,
and of these, about half could be identified at the subdomain level
with a set of group-specific probes. Beta subclass proteobacteria
constituted a dominant fraction in freshwater systems, accounting for
16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the
Cytophaga-Flavobacterium cluster were the most abundant
group detected in the marine systems, accounting for 18% (range, 2 to
72%) of the 4',6-diamidino-2-phenylindole (DAPI) counts, and they were
also important in freshwater systems (7%, range 0 to 18%).
Furthermore, members of the alpha and gamma subclasses of
Proteobacteria as well as members of the
Planctomycetales were detected in both freshwater and
marine water in abundances <7%.
In recent years, molecular methods
based on the comparative analysis of 16S rRNA sequences have yielded
new insights into the diversity of marine and freshwater
bacterioplankton communities (see, e.g., references 4, 17, 25,
35, 37, and 49). Numerous new rRNA
sequences have been found, indicating that the vast majority of
bacterioplankton species are not yet represented in the collections of
marine and freshwater strains. It has even been shown that the
"bacterioplankton" contains members of the Archaea
(11, 13, 15, 39). Whereas microbial diversity can be readily
studied by 16S rRNA gene libraries, it is difficult, if not impossible,
to deduce the community composition from them (3),
especially when they are PCR based (59). Quantitative slot
blot hybridization or fluorescence in situ hybridization (FISH) with
rRNA-targeted oligonucleotide probes is better suited for this task
(3). FISH has the potential to supplement the total cell
counts, which are routinely determined in aquatic samples by the
4',6-diamidino-2-phenylindol (DAPI) membrane filter technique (48), with counts on specific phylogenetic groups. With the recently described improved FISH protocol for aquatic samples (18), FISH seems no longer to be limited to systems with
high nutrient concentrations. Consequently, it was the aim of this study to test the general applicability of this improved protocol to
bacterioplankton and to gain the first insights into differences in the
community compositions of marine and freshwater systems with domain-
and group-specific oligonucleotide probes.
Sampling and fixation.
Data on the sampling time and locations
are summarized in Table 1. Important
characteristics like the trophic state (61), P concentration
(in micrograms per liter (38), area (in square kilometers),
and maximum depth (in meters) of the lakes are the following: Lake
Gossenköllesee, oligotrophic, 1 to 7, 0.065, and 9.9; Lake Lago
di Cadagno, mesotrophic, meromictic, 20 to 30, 0.357, 21; Lake Grosser
Ostersee, mesotrophic, 20 to 25, 1.78, 29.7; Lake Baikal, oligotrophic,
2 to 11, 3.1 × 104, 1,741. Fixation was done by the
method of Glöckner et al. (18). Cells were
concentrated from water samples (1 to 100 ml) on white polycarbonate
filters (diameter, 47 mm; pore size, 0.2 µm; type GTTP 4700 [Millipore, Eschborn, Germany]) by applying a vacuum of <25 kPa.
They were subsequently fixed by covering the filter with 3 ml of a
freshly prepared, phosphate-buffered saline (pH 7.2)-4%
paraformaldehyde (Sigma, Deisenhofen, Germany) solution for 30 min at
room temperature. The fixative was removed by applying vacuum, and the
filter was subsequently covered with 3 ml each of phosphate-buffered
saline and distilled water. Both were immediately removed by applying a
vacuum. Air-dried filters are ready for hybridization and can be stored
at
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Bacterioplankton Compositions of Lakes and Oceans:
a First Comparison Based on Fluorescence In Situ
Hybridization
ABSTRACT
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20°C or room temperature for several months without showing
apparent changes.
TABLE 1.
Sampling sites used in this study
FISH and probe-specific cell counts.
CY3-labeled
oligonucleotides were purchased from Interactiva (Ulm, Germany). The
probe sequences, hybridization conditions, and references are given in
Table 2. Each filter was cut into 12 sections. The filter sections were placed on glass slides and covered
with 20 µl of hybridization solution containing 0.9 M NaCl, 20 mM
Tris-HCl (pH 7.4), 35% formamide, 0.01% sodium dodecyl sulfate, and
50 ng of CY3-labeled oligonucleotide and incubated at 46°C for 90 min
in an equilibrated chamber. Probes BET42a, GAM42a, and PLA886 were used
with competitor oligonucleotides as described previously (32,
40). The filters were transferred to a vial containing 50 ml of
prewarmed (48°C) washing solution (70 mM NaCl, 20 mM Tris-HCl [pH
7.4], 5 mM EDTA, 0.01% sodium dodecyl sulfate) and incubated freely
floating without shaking at 48°C for 15 min. The filter sections were
dried on Whatman 3M paper (Whatman Ltd., Maidstone, United Kingdom),
placed back on a glass slide, and covered with 50 µl of DAPI solution
(1 µg/ml in distilled water filtered through at 0.2-µm filter) for
5 min at room temperature in the dark. Subsequently, they were gently washed in 50 ml of 0.2-µm-filtered distilled water, dried on Whatman 3M paper, and mounted on glass slides with Citifluor AF1 (Citifluor Ltd., Canterbury, United Kingdom). Glöckner et al.
(18) have shown previously that due to firm adhesion of the
cells to the polycarbonate filters, cumulative cell losses are below
10%.
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Total cell counts.
The total cell counts found in our samples
(105 to 107 cells ml
1) were in
the normal range reported for oligotrophic and mesotrophic aquatic
systems (28). The more oligotrophic systems usually contained between 3 × 105 and 9 × 105 cells ml1, and the mesotrophic systems
contained between 1.4 × 106 and 7.0 × 106 cells ml1 (Table 3). Exceptions were the
middle basin of Lake Baikal at 1,455 m, where only 8 × 104 cells ml1 were present and samples from
the oligotrophic Antarctic Ocean at 0 and 40 m, with total cell
counts between 1.3 × 106 and 1.6 × 106 cells ml1.
Domain-specific probing. Detection yields relative to DAPI with the EUB338 probe, complementary to a signature site present in most members of the Bacteria, ranged from 39% in the North Sea to 96% in the Antarctic Ocean at 0 m, with a median of 56% (CI, 46 to 61%; n = 26) (Table 3). All samples examined showed bright hybridization signals and a clear distinction between probe-conferred signals and the background. The fraction of autofluorescent and nonspecifically stained cells as determined with the negative control probe NON338 was moderate in all samples, with a median of 5% (CI, 2 to 7%; n = 17) in the freshwater and 1% (CI, 1 to 4%; n = 11) in the marine systems. Results obtained with controls without the addition of CY3-labeled probes showed that the background signals were derived mainly from chlorophyll-containing cells (e.g., algae and cyanobacteria) and inorganic particles and to a much lesser extent from nonspecifically stained cells.
Consequently, with the recently described protocol (18), FISH seems no longer limited to hypereutrophic and eutrophic aquatic ecosystems (1, 3, 24). Around 50% of the cells could be detected. A closer microscopic examination of the exceptional Antarctic Ocean samples revealed a Phaeocystis algal bloom, which coincided with a quite uniform and clearly detectable bloom of members of the Cytophaga-Flavobacterium group. Members of the Bacteria were more abundant in all samples than were members of the Archaea. Although applied to all samples, probe ARCH915, specific for most members of the Archaea, detected cells only in the samples from Lake Gossenköllesee (6%), the North Sea (3%), and the Pacific Ocean (2%). This supports earlier findings on the widespread occurrence of Archaea in the water column (11, 43), even though they did not reach the high abundances described previously (13, 39). Methodological limitations or temporal and spatial variations could have been the reason for not detecting Archaea in the Antarctic Ocean, where they have been found previously (13, 39).Group-specific probing. When the community composition was further analyzed with a set of oligonucleotide probes targeting larger phylogenetic groups within the domain Bacteria, only about 56% (CI, 44 to 62%; n = 26) of the cells detected with probe EUB338 could be assigned (Table 3). This is certainly due to the incompleteness of the probe set used in this study. This set was developed for biotechnological, more eutrophic systems like activated sludge and obviously needs to be supplemented for use in mesotrophic and oligotrophic aquatic systems. New probes must be developed to target sequences found in the rDNA libraries from freshwater and marine systems (see, e.g., references 4, 25, and 37). In particular, new group-specific probes for the epsilon and delta subclasses of the Proteobacteria and the Chlorobiaceae need to be designed (21, 67).
Beta-subclass proteobacteria.
The incomplete set of probes
nevertheless allowed the detection of differences between the
bacterioplankton composition of marine and freshwater systems. These
differences were most pronounced for the beta-subclass proteobacteria.
These accounted for 16% (CI, 11 to 18%; n = 15) of
DAPI-stained cells in the freshwater samples, equivalent to 1.4 × 105 cells ml1 (CI, 9.9 × 104 to 4.2 × 105 cells
ml1). In the deep layers of the middle basin of Lake
Baikal, they made up 32% of the cells stained with DAPI. The median
abundance of beta-subclass proteobacteria in the marine samples
investigated was 0% (CI, 0 to 15%; n = 11). No
beta-subclass proteobacteria could be found in the Antarctic Ocean and
the North Sea. In the sample from the coastal Pacific Ocean, 4% of the
total counts were detected with the BET42a probe. The upper layers of
the Baltic Sea, which are strongly influenced by freshwater (salinity,
7.2 to 8.0%), were exceptions. Here, the counts ranged between 15 and
29% (Fig. 1A). In most of the samples
examined, beta-subclass proteobacteria were straight to curved rods 1.5 to 4.5 µm long and approximately 1 µm wide. Only in Lake
Gossenköllesee were short filaments (8 to 13 µm long) visible.
Our findings corroborate diversity data obtained by cloning of 16S rRNA
gene fragments from freshwater and marine environments. In 16S rDNA
libraries from pelagic freshwater and lake snow aggregates, the
majority of clones was affiliated with the beta subclass of
Proteobacteria (4, 25, 35, 66), whereas
beta-proteobacterial sequences were not found in most marine libraries
(see, e.g., references 6, 8, 16, 17, 20, 37, and
54) or in marine aggregates (50). The
exceptions are a report from Suzuki et al. (60) that
beta-subclass proteobacteria were found at the coast of Oregon and
reports on PCR detection of ammonium-oxidizing beta-subclass proteobacteria in marine samples (27, 62). Interesting in this respect are the results from the Baltic Sea, a heterogeneous system in which low-salt water (7
salinity) overlays a water body
(12 salinity) influenced by the North Sea (51). At the time of sampling, a halocline between 78 m (8 salinity) and
121 m (11 salinity) clearly separated an upper water body with
high relative abundances of beta-subclass proteobacteria (15 to 29%; 4.1 × 105 to 5.4 × 105 cells
ml1) from a water body with lower abundances (6%;
1.8 × 105 cells ml1) (Fig. 1A). The
higher salinity in the deeper layers might prevent the establishment of
this group in large numbers. This idea is supported by recent findings
by Painchaud et al. (44), who reported increased mortality
of freshwater bacteria from the St. Lawrence River in brackish water
with more than 10 salinity.
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Cytophaga-Flavobacterium group.
Members of the
Cytophaga-Flavobacterium group could be found in all marine
and freshwater samples investigated and usually formed the largest
bacterial group in marine water, with a median of 18% (CI, 10 to 40%;
n = 11). The relative abundance ranged from 2% in the
Baltic Sea (depth, 78 m) to 72% in the Antarctic Ocean
(68°50.57'S 06°01.08'E; depth, 0 m). Members of this group were also present in all freshwater systems studied, with a lower median abundance of 6% (CI, 2 to 12%; n = 15). The
average total cell counts were 3.6 × 105 cells
ml1 (CI, 1.5 × 105 to 9.4 × 105 cells ml1) for marine samples and
7.0 × 104 cells ml1 (CI, 2.8 × 104 to 1.6 × 105 cells ml1)
for freshwater samples. This is in good agreement with cultivation studies that report frequent isolation of Cytophaga and
Flavobacterium spp. from freshwater and marine systems
(4, 22, 36, 47, 56, 69), whereas in some 16S rRNA gene
libraries, sequences of the Cytophaga-Flavobacterium group
seem to be underrepresented (4, 17, 21, 37, 57, 60, 67).
Comparative sequence analysis showed that the frequently used reverse
"universal" primers OX2, 1518R, and 1522R at around
Escherichia coli position 1520 and the forward primer 68F at
E. coli positions 48 to 68 (8, 17, 21, 37, 54, 57, 60,
67) discriminate against the amplification of 16S rDNA of members
of the Cytophaga-Flavobacterium group (e.g., E. coli position 67, where most members of the
Cytophaga-Flavobacterium group have a G instead of a C). The
potential of PCR primers to influence the sequence diversity within 16S
rRNA gene libraries has been discussed previously (33).
Clearly, there is no guarantee that modern molecular methods of
biodiversity research are representative, but by using a combination of
various methods, biases should be detected.
Alpha-subclass proteobacteria.
The medians of relative
abundances and absolute cell numbers of alpha-subclass proteobacteria
detected with the probe ALF968 were significantly higher in the marine
samples (6%; CI, 4 to 11%; 1.1 × 105 cells
ml1; CI, 4.2 × 104 to 1.8 × 105 cells ml1 [n = 11])
than in the freshwater samples used (1%; CI, 1 to 2%; 9.0 × 103 cells ml1; CI, 3.0 × 103 to 7.0 × 104 cells ml1
[n = 15]). In lakes the variation was between 0% in
Lake Lago di Cadagno at 3 and 18 m and 10% in Lake
Gossenköllesee. In marine systems, the range of relative
abundance of alpha-subclass proteobacteria was between 1% in the North
Sea and 14% in the Baltic Sea (18 m). The morphology of the alpha
subclass of Proteobacteria was diverse and included rods,
vibrios, and filaments of various size. Clone sequences affiliated with
the alpha and gamma subclasses of Proteobacteria are
commonly retrieved from marine aquatic ecosystems (16, 17, 20, 37,
60). Our FISH results support the widespread occurrence of
members of the alpha subclass of Proteobacteria with larger
numbers in the marine samples than in the freshwater samples. In no
sample were the detectable alpha-subclass proteobacteria more abundant
than members of the Cytophaga-Flavobacterium group. It
remains to be investigated with, e.g., genus-level probes which groups
of alpha-subclass proteobacteria are frequent in aquatic systems.
Gamma-subclass proteobacteria.
There were no clear-cut trends
for gamma-subclass proteobacteria. In Lake Lago di Cadagno, the
distribution of probe GAM42a-positive cells was strongly influenced by
a layer of Chromatium sp. and Amoebobacter sp. in
the metalimnion (13 m; 5%, 2.1 × 105 cells
ml1). Even though these cells showed a red
autofluorescence, they could be unambiguously assigned to the gamma
subclass of Proteobacteria by the yellow-orange signal
conferred by CY3-labeled probe GAM42a. In the marine samples, only
those from the North Sea and the surface layers of the Antarctic Ocean
showed increased abundances of gamma-subclass proteobacteria, with 6%
(4.2 × 105 cells ml1) and 9% (6.3 × 104 cells ml1), respectively. In all other
samples, the cells detected with GAM42a were at or below 4% of the
total counts. Traditional cultivation methods attributed a high
importance to members of the gamma subclass of
Proteobacteria (5, 30, 69). Our current FISH data
reveal relative abundances of <4%, suggesting that members of this
group may, at least numerically, be only a minor part of the
bacterioplankton. The positive selection for gamma-subclass
proteobacteria on agar plates is a well-known phenomenon which has
recently been analyzed by FISH (63). Many members of this
group are typical copiotrophs, adapted to high nutrient concentrations,
and therefore grow well under laboratory conditions (68).
Moreover, it has been reported recently that the 23S rRNA-targeted
probe GAM42a did not detect all deep-branching bacteria in the gamma
subclass of Proteobacteria (10, 19). In a time of
a rapidly increasing rRNA database, the set of rRNA-targeted
oligonucleotide probes needs to be continuously refined, and therefore
it might be necessary to supplement probe GAM42a with some additional probes.
Planctomycetales.
Planctomycetes are known to be typical and
widespread members of freshwater and marine ecosystems (26,
53), and rRNA clones related to Planctomyces spp. have
also been found associated with marine macroaggregates (12,
50). We found them in small numbers in all freshwater samples (1 to 3%; 2.8 × 104 to 9.0 × 104
cells ml1) and also in the North Sea sample (1%;
7.0 × 104 cells ml1). The cells
detected were usually large cocci with diameters of >1 µm; there was
frequently an uneven distribution of the fluorescent signal in the
cell, due to the lack of probe hybridization to the nucleoid
(14). An explanation for their absence in the marine samples
investigated, except for the North Sea, could be the low relative
abundances (ca. 1%), which is close to the present detection limits of FISH.
Limitations. For most regular water samples, we could not use FISH to assign 40 to 50% of the particles stained with DAPI to one of the three domains Archaea, Bacteria, or Eucarya. A comparison of DAPI and EUB338 pictures showed that the particles stained with DAPI but not with EUB338 probe were usually very small, at about the resolution of the light microscope (0.2 µm). Considering this, an affiliation of the undetected DAPI-stained particles with the domain Eucarya can almost certainly be excluded. This leaves us with two other likely explanations. (i) Bacteria or Archaea remained undetected due to the low ribosome content often found in starved cells (29, 52), to limited probe penetration caused by, e.g., thick cell walls, or to absence of the bacterial and archaeal signatures targeted by the probes. (ii) The DAPI-positive particles which cannot be stained with rRNA-targeted probes are viruses. Viruses are abundant in freshwater and marine aquatic ecosystems (23); in particular, the large ones with sizes up to 150 nm (34, 65) might be detected by sensitive epifluorescence microscopy. Attempts to increase the detection yields by, e.g., the application of highly sensitive enzyme-based signal amplification systems (55) or changes in the cell fixation to increase cell permeability (3) failed (data not shown). Other authors suggested recently that short-term incubations with chloramphenicol increased detection rates (42).
A further limitation of the present study originates from the fact that our probes target quite large phylogenetic groups. The alpha subclass of Proteobacteria, for example, encompasses photosynthetic anaerobes like Rhodobacter spp. as well as aerobic heterotrophs like Caulobacter and Hyphomicrobium spp. Furthermore, as a result of the rapid growth of the 16S rRNA sequence database, the coverage of the target groups by the current probe set has been shown to be rather incomplete. This is true for both the domain- and group-specific probes.Conclusions. We have demonstrated the wide applicability of FISH to studies of bacterioplankton composition. FISH can, of course, also be used to observe the development of specific genera or species within the bacterioplankton (45). Additional information on changes in cell size and morphology can be readily obtained. This information is of great ecological importance and allows, e.g., the calculation of biomass distribution to defined groups or the study of the development of grazing-resistant populations (45, 46). FISH studies with high spatial, temporal, and phylogenetic resolution may considerably increase our knowledge of bacterioplankton ecology in the future.
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ACKNOWLEDGMENTS |
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This work has been supported by grant Am73/2-4 of the Deutsche Forschungsgemeinschaft.
We thank Albin Alfreider, Natalia Belkova, Meinhard Simon, Pierre Rossi, and Tamara Zemskaya for making samples available; Alexander Neef for providing access to probe ALF968 before publication; and Günter Jost and Jakob Pernthaler for carefully reading the manuscript and helping with the statistics.
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FOOTNOTES |
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* Corresponding author. Mailing address: Max-Planck-Institut für Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany. Phone: 49 421 2028-930. Fax: 49 421 2028-790. E-mail: ramann{at}mpi-bremen.de.
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