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Applied and Environmental Microbiology, November 1998, p. 4299-4306, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Seasonal Community and Population Dynamics of
Pelagic Bacteria and Archaea in a High Mountain Lake
Jakob
Pernthaler,1,*
Frank-Oliver
Glöckner,1
Stefanie
Unterholzner,2
Albin
Alfreider,2
Roland
Psenner,2 and
Rudolf
Amann1
Max-Planck-Institut für marine
Mikrobiologie, D-28359 Bremen, Germany,1 and
Institut für Zoologie und Limnologie, University of
Innsbruck, Innsbruck, Austria2
Received 13 May 1998/Accepted 2 September 1998
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ABSTRACT |
The seasonal variations in community structure and cell morphology
of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and
image-analyzed microscopy. Compositional changes and biomass
fluctuations within the assemblage were observed both in summer and
beneath the winter ice cover and are discussed in the context of
physicochemical and biotic parameters. Proteobacteria of the beta
subclass (beta-proteobacteria) formed a dominant fraction of
the bacterioplankton (annual mean, 24% of the total counts), whereas
alpha-proteobacteria were of similar relative importance only during
spring (mean, 11%). Bacteria of the
Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during
most of the year, thus contributing significantly to the total
microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of
ice cover formation, respectively. A set of oligonucleotide probes
targeted to single phylotypes was constructed from 16S rRNA-encoding
gene clone sequences. Three distinct populations of uncultivated
microbes, affiliated with the alpha- and beta-proteobacteria, were
subsequently monitored by FISH. About one-quarter of all of the
beta-proteobacteria (range, 6 to 53%) could be assigned to only two
phylotypes. The bacterial populations studied were annually recurrent,
seasonally variable, and vertically stratified, except during
the periods of lake overturn. Their variability clearly exceeded the
fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.
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INTRODUCTION |
Until recently, microbial ecologist
studying aquatic bacteria faced a basic dilemma: they could either
measure the abundance, biomass, growth rates, activity, etc. of
the "average" bacterium under in situ conditions (e.g.,
see reference 13), ignoring the phylogenetic and
physiological diversity of microbial communities, or they could isolate
and ecophysiologically characterize individual bacterial strains (e.g.,
see reference 36) but were then not able to tell if
these microorganisms were also common in the environment. Consequently,
little knowledge has been gathered about the spatial and temporal
abundance fluctuations of defined phylogenetic groups and of individual
bacterial species in natural habitats. Molecular biological techniques
used to identify microbes in environmental samples have recently
provided new tools to study bacterioplankton biodiversity (e.g., see
references 1, 9, 14, 15, and 19)
and the in situ abundances of bacteria and archaea that could not be
adequately distinguished before (2, 4, 5, 25).
Microbiologists are now in a position to potentially elucidate the
biogeography (24), population dynamics, and successions (28) not only of a few morphologically conspicuous microbes but of a large number of species, most of which might still be uncharacterized.
Fluorescence in situ hybridization (FISH) with rRNA-targeted
oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (3, 5). Based on a
rapidly growing set of 16S (and, to a lesser extend, 23S) rRNA sequence
data, it is probably the phylogenetically most sophisticated (22) approach for whole-cell in situ identification. On the other hand, FISH of plankton samples can be performed with minimal laboratory requirements (16), and evaluation relies on
epifluorescence microscopy, which is a standard technique of aquatic
microbial ecologists, e.g., for counting (30) and sizing
(33) of picoplankton. In contrast to other identification
approaches, FISH largely conserves the gestalt of the targeted
microorganisms, i.e., their morphologies, cell sizes (26,
34), and cellular rRNA content (7, 32). So,
despite the limitations of the method (as discussed in reference 5), its potential for the identification and
cytometric analysis of planktonic microbes is just about to be recognized.
Recent investigations have reported that various freshwater microbial
communities are dominated by bacteria which are phylogenetically affiliated with the alpha and beta subclasses of the class
Proteobacteria (alpha- and beta-proteobacteria,
respectively) and with members of the
Cytophaga-Flavobacterium cluster (2, 6, 16, 19). These observations were based on single or short-term sampling schemes. The instantaneous community composition of the
bacterioplankton, however, may not be representative for different
seasons, and the typical ranges of annual community variability remain
to be established.
The size distribution of planktonic bacteria, and particularly
the appearance of filamentous cells, has come into the focus of aquatic
microbial ecology in the context of studies of predator-prey interactions. It has been shown both in the laboratory (18, 37) and in field experiments (20) that the filamentous
morphotype is a phenotypic adaptation of some microbes to protistan
grazing, but there are probably numerous other causes for bacteria
to elongate far beyond their typical sizes (e.g., see reference
23). Threadlike bacteria have been observed
throughout the year in the plankton of a hypertrophic lake
(41) but were also found in midwinter in an
oligotropic alpine lake (31).
In earlier studies, we demonstrated FISH to be an appropriate tool for
the monitoring of spatial (2) and short-term temporal (26) dynamics of different phylogenetic groups of the
planktonic microbial community in a high mountain lake. Here we report
on the seasonal and vertical abundance distributions of pelagic members of Bacteria and Archaea in Gossenköllesee
and analysis of the community structure at different levels of
taxonomic resolution. We applied published domain- and
group-specific oligonucleotide probes (5) but also
used the sequence information from a 16S rRNA-encoding gene
(rDNA) library obtained from Gossenköllesee bacterioplankton 1 year earlier to construct specific probes targeted at individual
bacterial populations. Particular attention was paid to the changes in
abundance and taxonomic composition of the filamentous bacterial
morphotypes which were recognized as a permanently important fraction
of the planktonic procaryotes in Gossenköllesee. Additionally, we
monitored the seasonal changes in the biomass size distributions of the
nonfilamentous fraction of the pelagic microbial community.
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MATERIALS AND METHODS |
Sampling site and procedure.
The study site,
Gossenköllesee, is a small, oligotrophic high-mountain lake
in the Central Alps (Tyrol, Austria), situated above the timberline at
an altitude of 2,417 m (11). The catchment area of
Gossenköllesee is listed as a United Nations Educational, Scientific, and Cultural Organization Man and Biosphere Reserve. Annual
water temperatures typically range between 0 and 15°C, and thermal
mixing occurs from late October to mid-November and from late June to
early July (44). During the summer months, the lake is
exposed to high levels of solar UV radiation (40). The lake
was ice covered from mid-November 1996 until the end of June 1997, and
during this period, dissolved oxygen decreased to well below saturation
in the lower water layers (44). Table 1 depicts the development of temperature,
dissolved oxygen, conductivity, and the chlorophyll a
concentration during the study period (from reference
44).
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TABLE 1.
Temperature, dissolved oxygen, alkalinity, and
chlorophyll a concentrations in Gossenköllesee during
the study perioda
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Water samples from three depth layers (the surface, 4 m, and
8 m) were collected monthly between 4 July 1996 and 25 June 1997
with a 5-liter Schindler-Patalas sampler. Portions of 100 ml were
filtered with polycarbonate membrane filters (pore size, 0.2 µm;
diameter, 47 mm, Osmonics, Livermore, Calif.) for later in situ
hybridization and fixed as described previously (
16).
Filters
were stored in small petri dishes at
20°C until further
processing.
Additionally, 100-ml subsamples were fixed with formalin
(final
concentration, 2% [vol/vol]) for bacterial abundance and cell
size
determination.
Abundance and biomass size distribution of the microbial
assemblage.
Formalin-fixed subsamples of 5 to 20 ml were filtered
with black membrane filters (pore size, 0.22 µm; diameter, 25 mm;
Osmonics) and stained with 4',6'-diamidino-2-phenylindole (DAPI),
and bacterial abundances were determined by epifluorescence microscopy
(30). At least 200 filaments per sample were counted at a
low magnification (×400). The cell sizes of more than 400 DAPI-stained
bacteria per sample were measured by using the semiautomated image
analysis system described by Posch et al. (31). The analysis
was limited to nonfilamentous bacterial morphotypes (<10-µm cell
length). Cell volumes were estimated from measured area and perimeter
(c.f. reference 31) and converted into cell dry mass
by using the allometric conversion factor of Loferer-Krößbacher
et al. (21). Biomass allocation into 0.4-µm cell length
classes was calculated as described by Pernthaler et al.
(27).
Design and characterization of oligonucleotide probes.
Water samples for DNA extraction were taken at Gossenköllesee on
11 December 1995 from a depth of 3 m. Around 900 ml of
50-µm-prescreened lake water was filtered with hydrophilic filters
(Durapore [pore size, 0.2 µm; diameter, 47 mm]; Millipore Corp.,
Bedford, Mass.) until the filters were completely clogged with plankton
biomass. During filtration, the samples were kept at ambient water
temperatures (2 to 3°C). The filters were cut into smaller sections
and stored at 20°C until further processing. DNA extraction from
the filters was performed by following the protocol of Fuhrman et al.
(14). Almost full-length bacterial 16S rDNA fragments were
amplified by PCR from the extracted DNA by using two general bacterial
16S rDNA primers (38) on a Hybaid OmniGene thermocycler
(MWG-Biotech, Ebersberg, Germany). Amplification conditions were 94°C
for 1 min, 50°C for 2 min, and 72°C for 3 min for 30 cycles after
an initial preheating step of 94°C for 3 min. PCR amplification
products were purified and cloned as described previously
(38). Fifty 16S rDNA clones were partially sequenced (300 to
500 nucleotides) by using a LICOR DNA 4000 automated sequencer
(MWG-Biotech) (38). These partial sequences were added to
the 16S rRNA sequence database of the Technical University of Munich
with the program package ARB (43). Two clones each
affiliated with the alpha (GKS59 and GKS69)- and beta (GKS16 and
GKS98)-proteobacteria were selected, and their 16S rDNA inserts were
fully sequenced. The clone identifications, EMBL accession numbers, and
nearest relatives in the database are presented in Table
2. By using the PROBE_DESIGN tool of
ARB (43), specific oligonucleotide probes were constructed.
In order to minimize the risk of accidentally detecting more than one
population by FISH, we designed two oligonucleotide probes per clone
that were targeted at different positions on the 16S rRNA and each of
which showed at least one mismatch with all other known sequences. All
probes were purchased from Interactiva (Ulm, Germany) and labeled with
the indocarbocyanine dye Cy3. The sequences and target positions of the
clone-specific probes are given in Table 2.
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TABLE 2.
Probes designed from bacterial 16S rDNA sequences that
were retrieved from Gossenköllesee in December 1995
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dequate hybridization conditions were established by slot blot
hybridization of in vitro RNA transcripts from the four 16S
rDNA
inserts with oligonucleotide probes at increasing concentrations
of
formamide (cf. reference
29). Plasmids containing
inserts
GKS16, GKS59, GKS69, and GKS98 were extracted from overnight
cultures
of the respective clones (QIAprep Spin Plasmid kit; Qiagen
Inc.,
Chatsworth, Calif.), linearized with restriction enzyme
NotI (GKS16
and GKS59) or
ApaI (GKS69 and GKS98)
(Boehringer, Mannheim, Germany),
and purified (QIAquick PCR
purification kit; Qiagen Inc.). rRNA
was transcribed with the SP6/T7
rRNA transcription kit (Boehringer),
and products were examined by
polyacrylamide gel electrophoresis
(4%) and ethidium bromide staining.
Transcripts were denatured
with 2% buffered glutaraldehyde and blotted
in triplicate onto
nylon membranes (Magna Charge; Micron Separations,
Westboro, Mass.)
in a Minifold II slot blot apparatus (Schleicher & Schuell, Dassel,
Germany). The membranes were hybridized with
radioactively labeled
(
42) probes GKS16-442, GKS59-1434,
GKS69-1451, and GKS98-1459
at 46°C for 1.5 h and subsequently
washed at 48°C for 30 min.
The hybridization and washing buffers were
identical to those
used for FISH (see, e.g., reference
38). Probe-conferred radioactivity
was quantified
with a PhosphorImager (Molecular Dynamics, Sunnyvale,
Calif.).
FISH.
FISH of filter sections with oligonucleotide probes,
counterstaining with DAPI, and mounting for microscopic evaluation were performed as described previously (16). The group-specific
probes were ARCH915 (domain Archaea), EUB338 (domain
Bacteria), ALF1b (alpha-proteobacteria), BET42a
(beta-proteobacteria), GAM42a (gamma-proteobacteria), and CF319a
(members of the Cytophaga-Flavobacterium cluster). Probe
sequences and respective formamide concentrations in the hybridization
buffers were given by Snaidr et al. (38). Epifluorescence microscopy was carried out as described before (2, 16) on a
Zeiss Axioplan or a Zeiss Axioplan II, each equipped with an HBO 100-W
mercury lamp and a 100× Plan Neofluar objective. At low relative
abundances of hybridized cells (
5% DAPI staining) at least 1,000 DAPI-stained objects were evaluated per sample; otherwise, between 500 and 1,000 cells were inspected. The lower boundaries depicted in Fig.
2, 4, and 5 represent the empirical limits of reliable quantification
by our counting protocol (usually 1% of the DAPI-stained cell counts).
Absolute densities of hybridized bacteria were calculated as the
product of their relative abundances on filter sections (percentage of
DAPI-stained objects) and the DAPI-stained direct cell counts. Both the
absolute numbers and the fractions of hybridized cells were evaluated
separately for filamentous bacteria. Therefore, the lower limits of
these counts differ from those of all of the cells. Two counting
protocols were tested. For bacteria hybridizing with EUB338 and CF319a, the abundances of hybridized cells were quantified as fractions of
DAPI-stained filaments, in analogy with the standard counting technique. For ARCH915, first the fraction of all hybridized cells (filaments and nonfilaments) was determined as a percentage of the
total DAPI-stained cell counts. Then, the relative abundance of
filaments in at least 500 hybridized cells was evaluated. This second
protocol was much less time consuming but turned out to be too
imprecise for the calculation of absolute filament abundances. Accordingly, only the relative fractions of filamentous archaea are
depicted in the results (see Fig. 2).
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RESULTS |
Total assemblage.
Cell densities determined by DAPI staining
ranged from 0.11 × 106 to 0.55 × 106 cells ml
1 (Table
3). Two peaks of total numbers in early
autumn and midwinter were separated by distinct minima in July,
October, and February. Filamentous cells were present in the plankton
throughout the season at densities of 0.076 × 104 to
1.33 × 104/ml1, reaching maximal
abundances in early autumn and continuously decreasing in number during
the period of ice coverage. In early winter, filaments were more
frequent in the lower water layers. Three clearly separated maxima of
nonfilamentous bacterial biomass were found in late summer, midwinter,
and spring (Fig. 1). Between July and
January, a considerable amount (>35% in September and October) of
biomass was present in cells >3.2 µm in length, and during August
and September, the biomass was distributed more evenly over bacterial
size classes. Between March and June, biomass maxima were formed
exclusively by small bacteria (0.4 to 1.2 µm) and were more
pronounced in the deeper water layers.
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FIG. 1.
Seasonal dynamics of the biomass size distribution of
nonfilamentous pelagic procaryotes in Gossenköllesee at different
depths. Values on contour lines indicate micrograms of bacterial dry
mass per liter. The hatched bar outlines the period of ice cover.
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FISH with domain- and group-specific probes.
Between 26 and 82% of all DAPI-stained objects (mean, 55%) could be visualized
with bacterial probe EUB338, and between 68 and 100% of filamentous
cells (mean, 87%) were detected by EUB338 (Table 3). Detection rates
were lowest at a depth of 8 m (mean, 53%) between December and
February for all cells and from October to December for filamentous
forms. Archaea represented from less than 1% to a maximum of 5% of
all DAPI-stained cells (mean, 1.4%) and reached their highest
densities in September and shortly after ice cover formation in late
November (Fig. 2). During September and
October, there was a distinct maximum of the filamentous fraction of
archaea throughout the water column. On an annual average, beta-proteobacteria formed 24% (range, 5 to 41%) of all DAPI-stained cells and 45% (range, 20 to 89%) of the cells detected by the bacterial probe. At all three depths, the beta-proteobacteria reached
their maximal abundances in late summer (August to September) (Fig.
3a). Another maximum during midwinter
(December and January) was followed by a sharp decline in numbers. The
alpha-proteobacteria showed a similar peak in early autumn in the upper
water layers (Fig. 3b). A second peak was observed in May at a
depth of 4 m, succeeding an annual maximum of chlorophyll
a which occurred at the same depth during early spring
(Table 1). Alpha-proteobacteria accounted for 4 to 23% of
all DAPI-stained cells (mean, 11%) and, on average, 21% of the EUB338
counts (range, 7 to 45%). The ratio of alpha- to beta-proteobacteria
ranged from 0.15 in midwinter and early summer to 0.90 during April and
May (annual mean, 0.50), with distinct maxima in autumn (September to
November) and spring (March to May) at the surface and at a depth of
4 m. Gamma-proteobacteria always accounted for less than 2%
of the total cell density (data not shown). Bacteria of the
Cytophaga-Flavobacterium cluster never exceeded 4 × 104 ml or1 10% of all DAPI-stained cells
(mean, 3.5%) (Fig. 3c). Threadlike morphotypes formed up to 80% of
all of the bacteria hybridizing with CF319a in the surface layer during
October but were virtually undetectable during early summer (June and
July; annual mean, 20%) (Fig. 4).
Members of this phylogenetic cluster were a dominant fraction of all
filamentous bacteria for most of the year (mean, 53%), except for two
distinct minima in summer and winter, respectively.
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FIG. 2.
Seasonal abundances of pelagic archaea (lines) and
percentages of filamentous morphotypes (bars). The horizontal bar
outlines the period of ice cover.
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FIG. 3.
Seasonal fluctuations in the abundance of beta (a) and
alpha (b) proteobacteria and (c) bacteria in the
Cytophaga-Flavobacterium cluster in Gossenköllesee.
Note the different scale in panel c. The horizontal bar indicates the
period of ice cover.
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FIG. 4.
Seasonal dynamics of filamentous bacteria of the
Cytophaga-Flavobacterium cluster and their fraction of
bacteria (% EUB). The horizontal bar indicates the period of ice
cover.
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Populations monitored with clone-specific probes.
Each pair of
probes was found to define identical populations, i.e., two probes
targeted at different positions of one rRNA sequence (Table 2) detected
bacterial cells that could not be distinguished in terms of their
numbers in parallel samples and their cell morphologies. As the pairs
of probes appeared to be equivalent in terms of detected population
sizes, evaluation of the seasonal samples was performed by using the
probe that gave the brighter hybridization signal (probes GKS16-442,
GKS69-218, and GKS98-1459). The hybridization of RNA transcripts from
the 16S rDNAs with these probes at various concentrations of formamide resulted in typical sigmoidal melting curves (Fig.
5). Probes targeted to three of the four
clones detected bacterial populations that at least seasonally exceeded
1% of the DAPI-stained cells (Fig. 6a to
c). The sequence of the fourth clone, GKS59, affiliated with
alpha-proteobacteria, was associated with a filamentous morphotype (cell length, 11 to 20 µm) that occurred sporadically throughout the year at low densities (data not shown). The bacteria hybridizing with probes GKS16-61 and GKS16-442 were rod-shaped cells
(lengths, 1.5 to 3.5 µm) and short filaments (8 to 13 µm). The
phylotype GKS16 is affiliated with beta-proteobacteria, and
Rhodoferax fermentans is its closest known relative with a
16S rRNA similarity of 93.9%. These populations were most abundant
between September and January, and the populations were seasonally more
stable at the surface than in the lower water layers (Fig. 6a). The
maximal relative densities were 6% of DAPI-stained cells. At
4 m, we observed a biphasic annual population
development: a distinct spring bloom between March and May was set
apart from the previous autumn to early winter maximum by a decline to
a level below the detection limit during February. A similar drop was
also observed at a depth of 8 m. On both dates in July, when the
lake mixes, population densities were very similar at all three depths.
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FIG. 5.
Hybridization of in vitro RNA transcripts from retrieved
16S rDNA sequences with matching sequence-specific probes at increasing
levels of formamide.
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FIG. 6.
Population dynamics of cells hybridizing with probes
targeted at beta-proteobacteria (GKS16 [a] and GKS98
[b]) and alpha-proteobacteria (GKS69 [c]) The genera with the
highest 16S rRNA sequence similarity with the respective clones are in
parentheses. The horizontal bar indicates the period of ice cover.
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The second beta-proteobacterial population hybridizing with GKS98-826
and GKS98-1459 consisted of rods with cell lengths of
2 to 4.5 µm. They showed a clearly parallel seasonal development
at all depths
(Fig.
6b). After a decline below the detection densities
in
August, the populations reached maximal abundances during the
period of
ice layer formation (4% of DAPI-stained objects) and
then declined
below the limit of detection during the early spring.
On a seasonal
average, cells hybridizing with probes for clones
GKS16 and GKS98
constituted about one-quarter of the beta-proteobacteria,
but this
fraction fluctuated between >50% in the surface layer
during the
formation of the winter ice cover and less than 10%
in the lower water
layers during
February.
Bacteria targeted by probe GKS69-218 or GKS69-1451 were brightly
fluorescent rods with cell lengths of 2.8 to 9.5 µm. The
phylotype
GKS69 clusters with the genus
Sphingomonas within the
alpha-proteobacteria. The respective population appeared sporadically
at densities of 5,000 to 10,000 cells ml
1 between August
and March, mainly in the upper water layers (Fig.
6c).
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DISCUSSION |
Seasonal community changes.
Bacterial in situ identification
and the determination of total biomasses appear to describe
supplementary features of microbial communities. Either analysis
established that the microbial assemblage of Gossenköllesee
continuously transformed, even during the months when the lake was
sealed by the winter ice cover. The icebreak period between June
and July appeared to be a major promoter of community transition (c.f.
reference 26). This is easily conceivable by
considering, e.g., the allochthonous input from the winter ice cover
(35a), the subsequent thermal mixing of the water body (Table 1) and the increased exposition to sunlight. Recently, Sommaruga
and coworkers demonstrated that UV A and B radiation readily penetrates
the water column of Gossenköllesee and that this is sufficient
to directly and indirectly affect bacterial 3[H]thymidine and 14[C]leucine
incorporation (40) and to influence the bacterivory of a
heterotrophic nanoflagellate (39). It was not until
September that alpha- and beta-proteobacteria (Fig. 3), filamentous
bacteria (Table 2) and archaea (Fig. 2), and cells hybridizing with
clone-specific probes (Fig. 6) formed population maxima at the lake
surface. During the autumn thermal mixing and the period of ice cover
formation, the total microbial biomass declined in all layers (Fig. 1),
yet at the same time, small cells of the archaeoplankton (Fig. 2) and
bacteria hybridizing with GKS98-1459 (Fig. 6b) produced distinct annual
maxima. Interestingly, Murray et al. (25) reported strong seasonality of archaeal rRNA concentrations in Antarctic coastal waters, with the highest values occurring during the austral winter. This corresponds to our observation that pelagic archaea were abundant
in the lake's plankton only during autumn and hardly present thereafter.
Successions beneath the winter cover, when the water temperatures
ranged between 0 and 4.2°C (Table
1), were characterized
not only by
two clearly separated blooms of microbial biomass
in the smaller cell
size classes during midwinter and spring (Fig.
1) but also by distinct
changes in community composition (Fig.
2,
3, and
6a and b). Between
January and March, rates of detection
with the bacterial probe dropped
(Table
3), the numbers of beta-proteobacteria
and filamentous
members of the
Cytophaga-Flavobacterium cluster
declined
significantly (Fig.
3a and
4), and bacteria related to
clone GKS16
dropped below the limit of detection in all but the
surface water
layers (Fig.
6a). Simultaneously, the biomass of
very small bacteria
increased (Fig.
1), concomitant with the winter
maxima of chlorophyll
a concentrations (Table
1). The presence
of an active
microbial community beneath the winter ice cover
was also indicated by
the gradual decline of oxygen saturation
in the lowest water layers
(Table 1 in reference
44). The pelagic
bacteria in
Gossenköllesee formed a second biomass maximum in
late spring in
the lower water layers (Fig.
1), in parallel with
a density increase of
both the alpha- and beta-proteobacteria
(Fig.
3a and b) and of the
population hybridizing with GKS16-442
(Fig.
6a). This development
coincided with the decline of the
phytoplankton bloom at a depth of
4 m (Table
1) but also with
the onset of the snowmelt in the
catchment area (
44), as reflected
by decreased lake water
alkalinity at 0 m (Table
1). This water
influx might be the cause
for the reduced bacterial biomass at
the lake surface prior to the ice
break (Fig.
1).
Bacteria of the
Cytophaga-Flavobacterium cluster
bloomed in deeper water layers in summer, at the surface during ice
formation,
and at the depth with the highest chlorophyll
a concentrations
in early spring (Table
1). In summary,
this hints at phylogenetic
diversity within this group masked by the
generality of the probe.
This conclusion is also supported by the
contrasting dynamics
of the filamentous and nonfilamentous morphotypes
(Fig.
3c and
4). A fraction of these threadlike bacteria might
indeed genotypically
resemble small cells with a high potential for
morphological plasticity,
as has been shown, e.g., for marine
psychrophiles (
23) and a
Comamonas
acidovorans strain isolated from a lake (
18). Since
a prominent part of the planktonic filaments in Gossenköllesee
may reach cell lengths of >50 µm (
31), they form a
considerable
fraction of the total microbial biomass. During extended
periods,
most of the threadlike bacteria were affiliated with the
Cytophaga-Flavobacterium cluster (Fig.
4), so that this
phylogenetic group at least seasonally
constituted the largest pool of
bacterial biomass in the
system.
Studying microbial population dynamics by FISH.
It has
been shown that releasing allochthonous microbes into the plankton may
result in their rapid disappearance (see, e.g., reference
7), but the fate and dynamics of autochthonous
bacterioplankton populations is much less well understood. Gordon and
Giovannoni (17) monitored the seasonal density variation of
a bacterial rDNA sequence in the Atlantic, yet they did not attempt to
estimate absolute cell abundances. Recently, Pinhassi et al.
(28) presented a protocol to determine the in situ densities
of individual bacterial species which, however, requires prior
cultivation of the target organisms. Our approach differs from these in
that it analyzes single bacterial cells rather than nucleic acid
extracts. Thus, we could take into account the microscopically obvious
difference between filamentous and nonfilamentous bacteria, which is
certainly of ecological relevance, e.g., considering growth rates
(18) or biomass contribution to the community. Recently,
FISH has been successfully applied in Antarctic coastal water
(25). A similar but indirectly also cultivation-dependent
strategy used to monitor bacterial populations in a lake based on
fluorescently labeled monoclonal antibodies has been described by Faude
and Höfle (10). The two populations investigated in
that study together formed less than 0.1% of the total bacterial
community, which demonstrates the difficulty of specifically isolating
those microbes from the plankton that form larger populations in situ.
Probes designed for single retrieved 16S rRNA sequences of
uncultivated microorganisms cannot be easily tested for specificity.
We
used pairs of probes targeted to distant positions of the 16S
rRNA and
compared abundances and morphologies of hybridized cells
from parallel
samples in order to minimize the risk of detecting
multiple
populations. In addition, melting curve equivalents were
determined by hybridizing the sequence-specific probes against
RNA
transcripts from the retrieved 16S rDNAs (Fig.
5), which have
been
shown to resemble the melting behavior of native rRNA (
29).
On these grounds, it was possible to set hybridization
conditions
that ensured the specificity of the probes for the
respective
rRNA targets. Yet, for definitive confirmation of the
identity
of the targeted 16S rDNA (and not just the target region), it
might be necessary to, e.g., flow sort the respective microbes
and then
amplify and sequence their 16S rDNAs (
45).
Knowing that some bacterial species might have practically identical
16S rDNA sequences (
12), can one actually study the
population dynamics of a single bacterial species by in situ
hybridization?
Considering macroecological principles of competitive
exclusion,
it is probably unlikely to find closely related
microorganisms
co-occurring in a nutrient-poor system dominated by
strong physical
and chemical fluctuations. One must, however, be
aware of the
physiological variability that may be found within
different DNA-DNA
homology groups of one single species
(
35). We thus caution
against premature conclusions about
the ecology of microorganisms
based exclusively on their in situ
occurrence if not backed up
by taxonomic and ecophysiological
studies on
isolates.
However, the knowledge of in situ distributions of microbial
populations at our level of analysis provides a basis for such
research, as it allows us to set the pelagic bacteria into the
traditional framework of plankton ecology. By applying FISH for
single 16S rDNA sequences retrieved from the site, we could
readily
demonstrate that heterotrophic bacterial species (phylotypes)
show distinct vertical abundance patterns (Fig.
6a) and that their
relative importance in the community may be limited to particular
seasons (Fig.
6b). We envisage the microbial assemblage in the
lake to be of a "seed bank" nature in that numerous species might
be permanently present in the system at low abundances but form
larger
populations only during periods of optimal competitiveness.
However,
the causes of the population dynamics observed remain
unknown. We
suggest utilization of the diversity information available
in the
increasing number of planktonic bacterial 16S rDNA clone
libraries
(e.g.,
6,
15,
19) to study the in situ abundances
of
individual microorganisms at different sites and during different
seasons. Such investigations might eventually lead to a better
understanding of the function of individual bacterial populations
in
aquatic environments and of the forces governing their abundances,
biomasses, and cell
morphologies.
| |
ACKNOWLEDGMENTS |
We thank Ramon Rosselló-Mora for fruitful discussions;
Hansjörg Thies, Birgit Sattler, Anton Wille, and Ruben Sommaruga for providing background data; Birgit Rattunde for skillful laboratory assistance; and Kerstin Sahm, Lutgarde Raskin, and Daniel Oerther for
their suggestions on in vitro transcription.
This study was supported by the Austrian Science Foundation (Proj.
P11685-MOB), the European Community (MOLAR, ENV4-CT95-0007), the Deutsche Forschungsgemeinschaft (Am 73/2-4), and the Max
Planck Society.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Max Planck
Institute for Marine Microbiology, Celsiusstr. 1, D-28359 Bremen,
Germany. Phone: 49 421 2028 940. Fax: 49 421 2028.580. E-mail: jperntha{at}mpi-bremen.de.
| |
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Abstract
Introduction
