Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir.

Abstract

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% day(-1)) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-microm filtrate), grazing mortality was approximately 200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.

Full text

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/01/$04.00?0
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
DOI: 10.1128/AEM.67.6.2723–2733.2001
June 2001, p. 2723–2733 Vol. 67, No. 6
Changes in Bacterial Community Composition and Dynamics
and Viral Mortality Rates Associated with Enhanced
Flagellate Grazing in a Mesoeutrophic Reservoir
KAREL SˇIMEK,1,2* JAKOB PERNTHALER,3MARKUS G. WEINBAUER,4KAREL HORNA ´K,2
JOHN R. DOLAN,5JIRI´NEDOMA,1MICHAL MASˇI´N,2AND RUDOLF AMANN3
Hydrobiological Institute of the Academy of Sciences of the Czech Republic1and Faculty of Biological Sciences,
University of South Bohemia,2Na sa ´dka ´ch 7, CZ-37005 Cˇeske ´ Bude ˇjovice, Czech Republic; Max-Planck-Institute for
Marine Microbiology, Bremen, Germany3; Department of Biological Oceanography, Netherlands Institute for Sea
Research (NIOZ), Texel, The Netherlands4; and CNRS ESA 7076, Marine Microbial Ecology Group,
Station Zoologique, F-06230 Villefranche-Sur-Mer, France5
Received 7 November 2000/Accepted 28 March 2001
Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-?m treatment)
or enhance (<5-?m treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time
course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume,
production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial
community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning
and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH)
with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton character-
istics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about
equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the “grazer-
free” (0.8-?m-filtered) treatment, subject only to a relatively low mortality rate (?17% day?1) from viral lysis,
bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE
indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta
proteobacteria. In the grazing-enhanced treatment (5-?m filtrate), grazing mortality was ?200% and viral lysis
resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and
filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of
the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was
detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected
in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of
the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral
concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, sug-
gesting that protistan grazing may stimulate viral activity.
Many experimental studies have shown that protistan pre-
dation yields distinct changes in bacterioplankton communi-
ties. The phenomena typically reported include changes in
average physiological rates, as well as average cell morphology.
For example, protistan bacterivory is associated with increases
in per-bacterium production (e.g., thymidine incorporation
rate) and decreases in average cell size or, perhaps more com-
monly, development of larger, “grazing-resistant” filament or
floc forms (see e.g., references 15, 18, 29, and 49). Recent
studies of grazing effects on natural or mixed communities
(see, e.g., references 19, 26, 38, 40, 45, and 48) have shown that
protistan bacterivory can also affect bacterial community com-
position (BCC). Interestingly, some similar morphological
changes associated with increases in grazing pressure, i.e., de-
velopment of filaments and flocs, can be due to quite variable
shifts in the taxonomic composition of the community (19, 38).
Filament formation is apparently a growth rate-controlled de-
fense mechanism, which is widespread among bacteria (16).
For example, manipulating grazing mortality among natural
communities during different seasons has shown that filaments
can be formed by bacteria of the alpha subclass of Proteobac-
teria in spring and by species of the Cytophaga-Flavobacterium
(CF) group in the summer community (38).
While there is little doubt that protist-induced mortality
yields changes in bacterial communities, protists are not the
sole source of bacterial mortality. Virus-induced mortality is
probably responsible for 5 to 50% of bacterial mortality, de-
pending on the system (see reference 8 for a review). Surpris-
ingly few attempts have been made to make simultaneous
estimates of virus-induced and grazer-induced bacterial mor-
tality (4, 9, 13, 43, 53). Furthermore, the significance of exper-
imentallyinducedchanges
whether virus or protist related, is difficult to assess. While
there are data on the spatial variability of natural bacterio-
plankton communities on a large variety of scales (see e.g.,
references 1, 14, 17, and 51), data on temporal variability are
limited to seasonal changes (see, e.g., references 25 and 47).
Remarkably, over the timescales used in most experimental
studies (hours to days), even data on simple morphological
innaturalbacterioplankton,
* Corresponding author. Mailing address: Hydrobiological Institute
AS CR, Na sa ´dka ´ch 7, CZ-37005 Cˇeske ´ Bude ˇjovice, Czech Republic.
Phone: 420 38 7775873. Fax: 420 38 5300248. E-mail: ksimek@hbu.cas
.cz.
2723

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variability of natural bacterial communities are rare (41). Thus,
protist grazing no doubt affects bacterioplankton, but the sig-
nificance of differential mortality effects remains obscure since
the background variability in BCC over short timescales has
not been examined. Here we present the results of an investi-
gation which addresses these problems.
We conducted a field study employing communities from a
meso-eutrophic freshwater reservoir during the late clear-wa-
ter phase, a period when relatively low protistan grazing pres-
sure is exerted on the in situ bacterioplankton (38). Using a
size fractionation approach to manipulate natural populations,
we monitored changes in bacterioplankton subjected to negli-
gible or to high levels of protistan bacterivory. Communities
were incubated in situ in dialysis bags to minimize experimen-
tal artifacts. Building on our previous study (38), viral abun-
dances and virus-induced mortality were also assessed and the
natural variability of bacterioplankton characteristics was ex-
amined through simultaneous sampling of the reservoir popu-
lation.
Data were gathered on the abundance and size structure of
the bacterial community, on biomass production rates, and on
community composition. Taxonomic shifts were monitored by
fluorescent in situ hybridization (FISH) with oligonucleotide
probes. Changes in bacterial community composition were also
examined by using denaturing gradient gel electrophoresis
(DGGE) and by cloning and sequencing of 16S rDNA genes
retrieved from the different treatments and reservoir water.
Specific oligonucleotide probes were designed based on se-
quence information from cloning. We intended to (i) confirm
previous results which suggested that increasing bacterivory on
populations naturally subjected to low levels of bacterivory
yields large changes; (ii) examine viral dynamics and, based on
the frequency of visibly infected bacterial cells, estimate bac-
terial mortality; and (iii) assess the short-term in situ variability
of BCC. Furthermore, we provide a more detailed analysis of
the “grazing (or mortality)-resistant” community than is found
in similar previous studies on grazing in natural communities
(19, 38), especially with respect to the structure of bacterial
flocs and the identity of dominant filament-forming bacteria.
We also provide preliminary evidence that protistan grazing
may increase viral lysis, suggesting a synergy between grazing-
and virus-induced mortality.
MATERIALS AND METHODS
Study site and experimental design. The experiment was conducted in the
meso-eutrophic R ˇı ´mov Reservoir (South Bohemia) (for details, see reference
36). The sampling site was located above the former river valley (which is 30 m
deep), about 250 m from the reservoir dam. On 28 May 1999, water samples were
collected with a 2-liter Friedinger sampler from a depth of 0.5 m (15 samples)
and a final volume of 30 liters was mixed in a 50-liter plastic container. The
experiment was run during the late clear-water phase (28 May to 1 June 1999;
water temperature, 18 to 20°C; chlorophyll a concentration, 5 to 9 ?g liter?1).
The experimental design and protocol were similar to those described in detail
by Sˇimek et al. (38), partly simplified since two treatments were employed, but
the experiments were done in triplicate.
Treatments represented nominally (i) “bacterivore free,” via filtration through
0.8-?m-pore-size filters, which remove most bacteriovorous protists, and (ii)
“increased bacterivory,” via filtration through 5.0-?m-pore size filters, which
remove the predators (thus allowing rapid growth) of bacterivorous heterotro-
phic nanoflagellates (HNF). Thus, water samples were sequentially size fraction-
ated through 5- and 0.8-?m-pore-size Poretics filters (diameter, 47 mm;
OSMONIC Inc., Livermore, Calif.) into the fractions ?5 ?m (bacteria and HNF
only) and ?0.8 ?m (bacteria only), which were used along with unfiltered
samples taken directly from the surrounding reservoir water (all bacterivores and
HNF predators present); these samples are designated throughout the text as ?5
?m, ?0.8 ?m, and reservoir treatments, respectively. The last ?0.8-?m filtration
step was conducted in sterilized glass Poretics filter holders to minimize HNF
contamination. The water fractions were placed in ?2-liter pretreated (distilled
water rinsed and boiled) dialysis tubes (diameter, 75 mm; molecular mass cutoff,
12,000 to 16,000 Da [Poly Labo]). The dialysis bags were incubated in the
reservoir at a depth of 0.5 m, oriented horizontally in open Plexiglas holders.
Samples (?250 to 300 ml) were taken from each dialysis bag and reservoir water
at 0, 12, 24, 48, 72, and 96 h (t0, t12, t24, t48, t72, and t96).
Bacterial abundance and biomass. Samples were fixed with formaldehyde (2%
[vol/vol] final concentration) stained with 4?-6-diamidino-2-phenylindole (DAPI)
(final concentration, 0.1 ?g ml?1), and enumerated by epifluorescence micros-
copy (Olympus BX 60 microscope). Between 450 and 700 DAPI-stained bacte-
rial cells were recorded at a magnification of ?1,000 with an analog monochrome
charge-coupled device camera (Cohu Inc., San Diego, Calif.) mounted on the
microscope and processed with the semiautomatic image analysis systems
LUCIA D (Lucia 3.52; resolution, 750 by 520 pixels; 256 grey levels [Laboratory
Imaging, Prague, Czech Republic]). Details of the image processing (gray trans-
formation and edge finding) are described by Posch et al. (28). Bacterial biomass
was calculated from the allometric relationship between cell volume and carbon
content as described by Simon and Azam (42) and modified by Norland (24).
Since the cell width showed very little variability, the cell length was the most
important factor in determining cell volume. Cells longer than 4 ?m were
assumed to be ungrazable by most of the bacterivorous HNF (15, 40). Corre-
spondingly, we used this criterion to classify bacteria as biomass in the form of
small cells (?4 ?m) that were susceptible to grazing, as opposed to biomass in
the form of grazing-resistant, bacterial filamentous cells (?4 ?m). Since some of
the filaments in the ?5-?m treatments were even longer than 100 ?m, they were
recorded at a magnification of ?400 and processed separately.
Bacterial production. Bacterial production was measured by a thymidine in-
corporation method modified from that of Riemann and Søndergaard (31).
Duplicate 5-ml subsamples were incubated for 30 min at in situ temperature with
10 nmol of [methyl-3H]thymidine (Amersham) per liter, preserved with neutral
buffered formaldehyde (2% [vol/vol] final concentration) filtered through 0.2-?m
membrane filters (Poretics), and extracted 10 times with 1 ml of ice-cold 5%
trichloroacetic acid (see reference 36 for details). Replicate blanks prefixed by
2% formaldehyde were processed in parallel. An empirical conversion factor
between the thymidine incorporation rate and the bacterial cell production rate
was determined using data from the triplicate ?0.8-?m treatments. The cell
production rate was calculated from the slope of the increase of the natural
logarithm of bacterial abundance over time (0, 24, and 48 h). Our determined
empirical conversion factor (2.3 ? 1018cells mol of thymidine?1) was used for
calculations.
DGGE, clone libraries, phylogenetic tree, and probe design. DGGE of 16S
rRNA gene segments (21) amplified by PCR was performed for samples from
two dialysis bags of each treatment and from reservoir water at t0, t48, and t72.
Oligonucleotide primers 341F and 907R (21) were used to directly amplify an
approximately 550-nucleotide segment of the 16S rDNA genes from cells con-
centrated on polycarbonate filters (7). DGGE was performed by the method of
Muyzer et al. (21), and banding patterns were visualized by ethidium bromide
staining. Two gels were run for each of the 0.8- and 5 ?m-filtered treatments, and
samples from the reservoir served as references on each of the gels.
Three clone libraries were constructed from a reservoir sample at t0, a dialysis
bag containing 0.8-?m-filtered water at t72, and a bag with 5-?m-filtered water at
t72. From cells concentrated on membrane filters, PCR with bacterial 16S rRNA
primers 8F and 1492R (6) was used to amplify almost full-length 16S rDNAs
under the amplification conditions described by Glo ¨ckner et al. (11). The am-
plified rDNA was inserted into the pGEM-T vector (Promega Corp., Madison,
Wis.) and cloned into competent Escherichia coli cells as specified by the man-
ufacturer. Plasmid DNA inserts of 25 clones from each library were digested with
7.5 U of Sau3a-1 (Promega) for 3 h at 37°C. The fragments were analyzed by
agarose gel electrophoresis, and restriction patterns were compared visually.
Plasmid DNA from 38 selected 16S rDNA clones were partially sequenced
(approximately 700 bp) by Taq cycle sequencing with universal 16S rRNA spe-
cific primers using an AB1377 (Applied Biosystems, Inc.) sequencer. Sequence
data were analyzed with the ARB software package (Lehrstuhl fu ¨r Mikrobiolo-
gie, Technische Universita ¨t Mu ¨nchen, Munich, Germany [http://www.micro.bi-
ologie.tu-muenchen.de]). Almost full-length sequences of 15 clones which affil-
iate with the beta subclass of Proteobacteria and the Cytophaga-Flavobacterium-
Bacteroides (CFB) phylum were determined. One sequence was rejected as
chimeric (http://www.cme.msu.edu/RDP/html/analyses.html). A phylogenetic
tree was reconstructed for these sequences by maximum parsimony of all se-
2724SˇIMEK ET AL.APPL. ENVIRON. MICROBIOL.

Page 3

quences of ?1,400 nucleotides in the ARB database, using neighbor joining and
maximum-likelihood analyses on various subsets for the evaluation of topologies
(Fig. 1). Alignment positions at which fewer than 50% of sequences of the entire
data set had the same residues were excluded from the calculations. Using the
PROBE-DESIGN tool of ARB, specific oligonucleotide probes were designed
for defined phylogenetic clusters within the beta Proteobacteria and CFB, which
also contained sequences from the clone libraries (Fig. 1). Two probes were
found to detect significant populations in situ. The sequences targeted by these
probes are depicted in Fig. 1. Hybridization conditions for probe R-BT065
(5?-GTTGCCCCCTCTACCGTT-3?, E. coli positions 65 to 83 [5]), were adjusted
by formamide series applied to different filters from the experiment, and bright-
ness was evaluated by eye. Probe R-FL615 (5?-CACTGCAATCGTTGAGCG
A-3?, E. coli positions 615 to 634) was hybridized to pure cultures of Flectoba-
cillus major at increasing levels of stringency, and fluorescence intensities were
quantified (22). For evaluation of environmental samples, both probes were used
with 35% formamide in the hybridization buffer and incubated for 2 h at 46°C.
FISH with oligonucleotide probes. BCC was analyzed by in situ hybridization
with fluorescence oligonucleotide probes on membrane filters (1, 10). For details
of the hybridization procedure and error estimates, see reference 26. Briefly,
bacterial cells from 10 to 20-ml subsamples were concentrated on white 0.2-?m
filters (47 mm in diameter; Poretics Corp.), fixed on membrane filters by over-
laying the filters with 4% paraformaldehyde in phosphate-buffered saline (pH
7.2), and stored at ?20°C (1). The seven different group-specific oligonucleotide
probes (Interactiva, Ulm, Germany) were targeted to the domain Bacteria (Eu-
bacteria, EUB338), the alpha, beta, and gamma subclasses of the class Proteobac-
teria (ALF968, BET42a, and GAM42a), the CF (CF319a) phylum (2), and two
more narrow phylogenetic clusters (the probes R-BT065 and R-FL615). The
probes were fluorescently labeled with the indocarbocyanine dye Cy3 (BDS,
Pittsburgh, Pa.). After hybridization, filter sections were stained with DAPI, and
the percentage of hybridized bacterial cells were enumerated by epifluorescence
microscopy (Olympus, AX70 PROVIS).
Protozoan grazing and abundance. Protozoan grazing on bacteria was esti-
mated using fluorescently labeled bacterioplankton (FLB) (34) concentrated
from the reservoir water (for details, see reference 38). FLB uptake experiments
were run with water from each of the bags containing 5-?m-filtered water as well
as with unfiltered reservoir water at each time point. Samples (180 ml) were
dispensed into acid-soaked and rinsed 250-ml flasks and preincubated at in situ
temperature for 15 min. HNF and ciliate FLB uptake rates were determined in
short-term FLB direct-uptake experiments with FLB equal to 5 to 15% of the
natural bacterial concentration. Subsamples (25 ml) for protozoan enumeration
and tracer ingestion determinations were taken at 0, 5, 10, 15, 20, and 30 min
after tracer addition and fixed by adding 0.5% of alkaline Lugol solution, im-
mediately followed by 2% (vol/vol) (final concentration) formaldehyde and sev-
eral drops of 3% (wt/vol) sodium thiosulfate to clear the Lugol color (34). We
determined ciliate grazing rates in time series from 5- to 15-min subsamples and
flagellate grazing rates in subsamples from 10 to 30 min. Subsamples (5 ml for
flagellates or 20 ml for ciliates) were stained with DAPI, filtered through 1-?m
black Poretics filters, and inspected via epifluorescence microscopy. Nonpig-
mented HNF and plastidic flagellates were differentiated. At least 40 ciliates and
50 flagellates were inspected for FLB ingestion in each sample. To estimate total
protozoan grazing, we multiplied average uptake rates of ciliates and flagellates
by their in situ abundances as previously described (37, 38).
Viral abundance and viral mortality of bacteria. Viruses in formaldehyde (2%
[vol/vol] final concentration)-preserved samples were stained with SYBR Green
I (10,000? in dimethyl sulfoxide [Molecular Probes chemical S-7567]) and enu-
merated by epifluorescence microscopy (23). Viruses were also enumerated
using transmission electron microscopy as described previously (44). Briefly,
viruses were collected quantitatively onto Formvar-coated, 400-mesh electron
microscope grids by centrifugation in a swinging-bucket rotor (Sorval TH-641;
100,000 ? g for 2.5 h), stained for 30 s with 1% (wt/vol) uranyl acetate, and rinsed
three times with deonized distilled water.
The frequency of visibly infected bacteria (FVIB) and the burst size were
determined by transmission electron microscopy (50). Due to restrictions of
sample volume, subsamples from each replicate were pooled, yielding a single
sample for each treatment for each time point. Replicate grids were prepared
and processed to estimate measurement error. The FVIB was related to virus-
mediated mortality of bacteria using conversion factors relating the frequency of
all infected cells to visibly infected cells and assuming that the latent period is
equivalent to the generation time (30), as was done by Binder (3): mortality ?
(1/g ln 2) {FVIB/[(1 ? e) ? FVIB]}, where g is the ratio of latent period to
generation time (g ? 1) and e is the fraction of the latent period that elapsed
before the appearance of intracellular virus particles (e ? 0.816 [average from
reference 30]). Mortality is a fraction per generation.
Nucleotide sequence accession numbers. The 16S rDNA sequences generated
in this study were deposited in GenBank under accession numbers AF361192 to
AF361205.
RESULTS
Bacterial and protistan community dynamics and activity.
In the grazer-free treatment (?0.8 ?m), bacterial numbers and
total biomass covaried closely (Fig. 2); both increased between
12 and 48 h, with an apparent generation time of about 24 h
(Table 1), and then remained relatively stationary at about
11 ? 106to 12 ? 106cells ml?1. Bacterial production (BP) data
showed a similar trend but with a peak at 72 h followed by a
decrease at the end of the study (Fig. 2). In contrast to bacte-
rial numbers and total biomass, no marked changes in the
mean cell volume of bacteria (MCV) were detected in the
grazer-free treatments.
Microbial interactions in the ?5-?m treatment yielded quite
different trends. After a slight initial increase, bacterial abun-
dance, biomass, and BP dropped between 24 and 96 h (e.g.,
bacterial numbers dropped from ?5 ? 106to 1 ? 106cells
ml?1[Fig. 2]). A shift in bacterial cell morphology was evident
as the bacterial MCV increased with bacterivore abundance,
HNF (from ?3 ? 103to 34 ? 103cells ml?1, -doubling time,
15.4 h [Table 1]), and bacterivory. HNF abundance and grazing
were maximal at 72 h in the ?5-?m treatment and then de-
creased toward the end of the experiment. While aggregate
HNF grazing activity consumed 20 to 40% of BP during the
first half of the study (Fig. 2), it represented about 200% of BP
by the end of the incubation. The high HNF grazing pressure
coincided with a marked, treatment-specific shift in bacterial
size structure and morphology toward the dominance of graz-
ing-resistant bacterial populations, i.e., filament- or floc-form-
ing bacteria (Fig. 3). The clear increase in the MCV of bacteria
reflected an exponentially increasing proportion of bacterial
filaments (cells longer than 4 ?m), accounting for ?7% of the
total bacteria and for ?50% of the total bacterial biomass by
the end of the experiment. Moreover, this process was also
paralleled by a clear shift from the dominance of free-living
single bacterial cells to that of floc-forming bacteria, which
represented ?75% of total bacteria by the end of the incuba-
tion.
Compared to the ?0.8- and ?5-?m treatments, whole-water
samples from the reservoir showed negligible changes in nearly
all parameters throughout the experiment (Fig. 2). The only
exception was a steady increase in ciliate abundance, probably
reflecting decreasing grazer control of ciliates by large zoo-
plankton during the late stage of the clear-water phase in the
reservoir. However, it had a minor effect on the overall rates of
protistan grazing on bacterioplankton in the reservoir.
Changes in bacterial community composition. Of the 16S
rDNA sequences obtained from the R ˇı ´mov Reservoir and
from the different incubations (Fig. 1), 14 affiliated with the
beta proteobacteria and the CF cluster, the majority with re-
cently described cosmopolitan freshwater clades (beta I, beta
II, cf I, cf II, and cf III) (12). Sequences were also affiliated
with gamma proteobacteria, actinobacteria, and other gram-
positive lineages (data not shown). An analysis of BCC based
on FISH with group-specific probes did not show any signifi-
cant differences among the treatments at time zero (analysis of
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FIG. 1. Unrooted phylogenetic trees of 16S rDNA clones from the beta proteobacteria (A) and the CF cluster (B) retrieved from the various
treatments and from the reservoir. Brackets indicate sequences targeted by FISH probes R-BT065 and R-FL615. Superscripts indicate that
sequences with identical restriction patterns were also found in clone libraries from reservoir samples (a) and the 5-?m treatment (b). Scale bars
indicate 10% estimated sequence divergence.
2726SˇIMEK ET AL.APPL. ENVIRON. MICROBIOL.

Page 5

variance and multiple Tukey honest significant distance [HSD]
test, P ? 0.5). Thus, at t0, the experimental manipulations
yielded no marked bias apparent at our level of taxonomic
resolution. During the course of the study, the manipulations
resulted in treatment-specific changes in BCC indicated by
means of both rRNA-targeted oligonucleotide probes (Fig. 4)
and DGGE (data not shown). For the seven different rRNA-
targeted oligonucleotide probes we used in this study, cells
detected by ALF968, GAM42a, and EUB338 showed no clear
trend during the course of the study in any treatment or sample
group. The proportions of bacteria targeted by probe EUB338
ranged between 75 and 85% of total DAPI-stained bacterial
cells (Fig. 4). The proportions of bacteria hybridizing with
ALF968 (data not shown) and GAM42a were consistently low
(?1% and 2 to 3%, respectively).
In the ?5-?m treatment, significant changes in BCC oc-
curred (Tukey HSD test, P ? 0.05) compared with those of the
?0.8-?m treatment and reservoir water. The shift consisted
mainly of an increase in the proportions of members of the
CF319a and BET42a lineages and an increase of a cluster of
beta proteobacteria targeted by probe R-BT065 (Fig. 4), de-
rived from the appearance of a treatment-specific DGGE band
pattern (data not shown). Moreover, most of the very long (?5
?m), thin filaments (Fig. 5), which dominated the biomass of
filamentous bacteria (Fig. 3), were detected by probe R-FL615
targeted to members of the Flectobacillus lineage (Fig. 1),
FIG. 2. Changes in microbial parameters in different size fractionation treatments (?0.8 and ?5 ?m) exposed in dialysis bags compared to
unfiltered reservoir water during the experiment. (Top) Bacterial abundances, biomasses, and mean cell volumes. (Bottom) Abundances of HNF,
ciliates, bacterial production, and total protistan bacterivory subdivided into HNF and ciliate grazing. Values are means for three replicate
treatments, and vertical bars show SDs. Statistical relationships are given in Tables 2 and 3.
TABLE 1. Net doubling times of ungrazed microorganisms in the
predator-free treatment or in the ?5-?m treatment
Organism Doubling time (h)c
Interval (h)
Bacterioplankton
CF319a bacteria
BET42a bacteria
R-BT065 bacteria
R-FL615 bacteria
HNF
24.8 ? 2.2
19.7 ? 2.0
16.6 ? 1.4
8.8 ? 1.6
12.7 ? 1.3
15.1 ? 2.0
0–48a
0–48a
0–48a
0–24a
72–96b
24–72b
aTreatment at ?0.8 ?m (bacterioplankton and different bacterial groups).
bTreatment at ?5 ?m (grazing-resistant R-FL615-positive filaments and
HNF).
cThe values are mean ? SD of three triplicate treatments. Rates were calcu-
lated for intervals with exponential growth.
VOL. 67, 2001 PROTISTS, VIRUSES, AND THE BACTERIAL COMMUNITY2727

Page 6

accounting for 3.4% of total bacteria at t96. No cells targeted by
the probe (more than 100 inspected per sample) were less than
5 ?m long. Finally, the floc-forming bacteria, which by the end
of the experiment numerically dominated the ?5-?m treat-
ment community (Fig. 3), showed a characteristic structure:
the outer floc cells were large and hybridized with BET42a
(Fig. 5), whereas the inner floc cells were detected by probe
CF319a. Growth of these cells was clearly reflected in the
appearance of treatment-specific bands in DGGE after a 48-h
incubation (data not shown). It is noteworthy that neither
R-FL615-positive cells nor flocs were found in the ?0.8-?m
treatment or the reservoir samples.
In the grazer-free treatment (?0.8 ?m), no floc- or filament-
forming bacteria appeared during the course of the study. A
change in BCC occurred, compared to the untreated reservoir
water, in the form of a significant increase in the proportions of
BET42a-targeted bacteria, which was almost entirely due to a
steep increase in the proportions of the cells affiliated to the
R-BT065 cluster (Fig. 4). This treatment also yielded the short-
est doubling time of R-BT065-positive bacteria compared to
other phylogenetic lineages (Table 1). The DGGE banding
pattern clearly indicated a qualitative shift in BCC after 72 h of
incubation as well (data not shown).
In the reservoir population, neither group-specific probes
nor DGGE indicated any changes in BCC during the study
period, which corresponded well to the stability of other mi-
crobial parameters (compare Fig. 2 and 4).
Viral abundance and virus-mediated mortality of bacteria.
Marked differences in viral abundance, FVIC, and virus-in-
duced mortality rates of bacteria were found among the ?0.8-
?m, ?5-?m, and reservoir populations (Fig. 6). Viral param-
eters qualitatively appeared to vary inversely with bacterial
abundance. For instance, in the heavily grazed ?5-?m treat-
ment, by the end of the experiment the largest numbers of viral
particles (?47 ? 106ml?1), FVIC, and calculated virus-in-
duced mortality (30 to 37% day?1) of bacteria were detected,
along with the lowest bacterial abundance (compare Fig. 2 and
6). In contrast, samples from the grazer-free treatment (?0.8
?m), in which bacteria became most abundant, yielded the
lowest estimates of viral abundance (?17 ? 106ml?1), FVIC,
and virus-induced mortality (mean, 17% day?1).
As opposed to the markedly distinct trends between the
grazer-free (?0.8-?m) and grazer-enhanced (?5-?m) treat-
ments, samples from reservoir differed little from the ?5.0-?m
samples. In both reservoir and ?5-?m treatment samples, viral
abundance, FVIC, and estimates of mortality rates increased
steadily from t24to t72and then declined slightly compared to
those for the ?0.8-?m treatment, in which viral parameters
were relatively invariant.
Grazer versus virus-induced mortality. In the grazer-en-
hanced (?5 ?m) treatment, grazing mortality increased
steadily during the experiment from about 25% to peak values
of around 200% of bacterial production rates (Fig. 2) while
virus-induced mortality increased in parallel from about 17 to
37% of bacterial stock per day (Fig. 6). In reservoir water
samples, grazing by protists removed an average of about 50%
and viral lysis removed about 25% of bacterial production. In
comparison, bacterial mortality due to virus-induced lysis in
the treatment without grazers (0.8 ?m) was about 17% (Fig. 6).
Statistical relationships. Pooling data across treatments
(Table 2), bacterial abundance was negatively related to graz-
ing rates as well as to MCV. This corresponded to the time
course data from individual treatments (Fig. 2), showing the
decrease in cell concentrations and increase in average bacte-
rial cell size with increases in grazing. Considering only sam-
ples in which grazers were present (?5-?m and reservoir sam-
ples), MCV was positively related to grazing rates (r ? 0.87,
n ? 35).
In contrast to grazing, the viral concentration was related
neither to bacterial concentration nor to cell-specific incorpo-
ration rates of thymidine. Viral numbers were positively relat-
able to MCV (r ? 0.70, n ? 52) as well as to grazing rates (r ?
0.70, n ? 35). Correlations of FVIC (Table 3) largely followed
those found for viral concentrations. Similar, close relation-
ships between FVIC and MCV, as well as grazing rates (r ?
0.70), were apparent. Infection frequencies were also positively
correlated with ratios of virus to bacterial concentration and
cell-specific thymidine incorporation rates.
DISCUSSION
Overall, our results considerably extend the conclusions of
previous studies concerning mortality-induced shifts in natural
bacterioplankton populations. First, we demonstrated mor-
phological and genotypic stability of an in situ community
when production about equals moderate mortality (50 and
30% day?1due to protists and virus, respectively). In the
reservoir, both bacterial cell concentrations and MCV were
FIG. 3. Results for the ?5-?m treatment. Shown are changes in
proportions of the abundance and biomass of total filamentous bacte-
rial cells and of only filaments targeted by the R-FL615 probe in total
bacterial abundance and biomass (top) and abundance of floc-forming
and of total grazing-resistant bacteria (a sum of floc- and filament-
forming cells) during the experiment (bottom). Values are means for
three replicate treatments, and vertical bars show SDs.
2728SˇIMEK ET AL.APPL. ENVIRON. MICROBIOL.

Page 7

quasi-constant within the study period (Fig. 2). The community
composition showed no significant shifts with time in the pro-
portions of cells detected by the CF319a, BET42a, R-BT065,
and GAM42a probes (Fig. 4), and it is noteworthy that 75 to
85% of all bacterial cells were consistently detected by FISH.
Further evidence of taxonomic stability was provided through
DGGE since there were no obvious differences in the band
patterns of reservoir populations when the band patterns of the
t0, t48, and t72populations were compared (data not shown).
Earlier investigations have shown time course alterations in
manipulated bacterioplankton over periods of a few days, but
the in situ variability was not assessed (19, 20, 38, 45). Our data
show that in situ populations can represent a stable back-
ground against which changes in manipulated populations may
be judged. However, our data also show that a significant
environmental change (e.g., a sudden relief from grazing or
enhancement of grazing or a pulse of organic substrate), which
disturbs the established balance between population-specific
growth and mortality rates of bacteria, results in shifts in BCC
(38).
Our findings with regard to the effects of grazers on bacte-
rioplankton also extend the results of previous studies. Many
changes in natural bacterioplankton communities have been
associated with grazer activity. Major known phenomena are
alterations in the size structure of the bacterial community,
shifts in activity rates, and changes in taxonomic structure (see,
e.g., reference 19, 20, 38, 40, and 45). Perhaps the best studied
is the induction of filament or floc formation. This formation
can be induced in a variety of taxa in natural populations (see,
e.g., references 18, and 38) and may simply be growth rate
associated (16). Interestingly, no filaments were found with
mature phages. This phenomenon has been reported previ-
ously for the oxic layer of the Plußsee Lake (52), indicating that
filaments can be not only resistant to grazing but also—for
unknown reasons—resistant to viral infection. Such a life strat-
egy may allow survival in the presence of competitive domi-
nants in terms of organic matter acquisition.
A probe covering one specific lineage within the CF cluster
(Fig. 1) detected a significant number of filamentous bacteria
in the ?5-?m treatment at t96(Fig. 3). Bacteria targeted by
R-FL615 formed ?25% of total biomass (Fig. 3). This lineage
so far contains one described species, Flectobacillus major. One
FIG. 4. Time course changes in the phylogenetic composition of the bacterioplankton community in size fractionation treatments (?0.8 and
?5 ?m) exposed in dialysis bags compared to unfiltered reservoir water during the experiment. Shown are proportions of beta proteobacteria and
cells detected by probe R-BT065 (top), gamma proteobacteria (middle), CF group (CF319a, top), and total FISH detection rates by the general
bacterial probe EUB338 (bottom). Values are means for three replicate treatments, and vertical bars show SDs.
VOL. 67, 2001 PROTISTS, VIRUSES, AND THE BACTERIAL COMMUNITY2729

Page 8

FIG. 5. Micrographs of bacterial populations in experimental treatments. Total or DAPI-stained bacteria (left) and specifically Cy3-labeled
bacteria (right) are shown. (A and B) Typical bacterioplankton at the beginning of the experiment in all treatments. (C to H) End of the experiment
in the ?5-?m treatment. Shown are the typical floc structure (C and D), with large beta proteobacteria in the outer part of flocs and small bacteria
affiliated to the CF319a cluster usually located in the inner part of flocs (indicated by the position of the arrow at C); typical morphology of bacteria
targeted by the CF319a probe (small cells bound in large flocs and large chain-forming cells) (F); and long filaments in DAPI preparations (G)
targeted by the R-FL615 probe (H).
2730

Page 9

16S rDNA sequence from our clone libraries and several other
sequences from bacteria from various lakes are also affiliated
with this cluster (Fig. 1). A recently described isolate from this
lineage, Flectobacillus sp. strain MWH38, forms filamentous
cells in continuous culture when exposed to a mixotrophic
flagellate (16). This perhaps explains our observation that the
formation of filaments hybridized by R-FL615 was found only
under conditions of increased HNF grazing. The filaments
targeted by the R-FL615 probe also showed a very high growth
rate (? ? 1.31 day?1), although this estimate was based only
on the total number of R-FL615-positive filaments. The fila-
ments were typically composed of a chain of cells, and they
significantly lengthened between t72and t96of the experiment
(from 12.9 ? 7.6 to 25.3 ? 12.2 ?m, mean ? standard devia-
tion [SD]). Therefore, our estimates of the growth rate of this
bacterial group are conservative.
In our experiment, flocs eventually dominated the bacterial
biomass in the grazing-enhanced ?5-?m treatment. We found
that flocs (usually 10 to 50 ?m across) can have a distinct
taxonomic structure, with cells targeted by the BET42a probe
on the surface of the floc and cells targeted by the CF319a
probe consistently in the interior (Fig. 5). While studies em-
ploying FISH have reported on the distinct taxonomic nature
of particle-associated, as opposed to free-living, bacteria (see,
e.g., references 27 and 54), a taxonomic structuring of flocs has
not been revealed previously. However, we can only speculate
about the mechanisms or factors controlling such taxonomic
zonation. Along with the selective HNF grazing, there is the
possibility of specific cometabolism of different phylotypes rep-
resenting different metabolic types on such particles (35).
This study represents one of the very few attempts to directly
estimate both grazer and virus-induced bacterial mortality and
the effects of grazer and virus populations on natural bacterial
communities. In studies focusing on the effects of grazing on
BCC, viral activity is often ignored (19, 38, 45), while investi-
gations of virus-induced bacterial mortality commonly estimate
grazing mortality by simply multiplying grazer abundance by a
conversion factor (see, e.g., references 32, 43, and 51). To our
knowledge, there have been few attempts, using direct esti-
mates of grazer and viral activity, to partition mortality be-
tween grazer- and virus-induced mortality in natural popula-
tions (9, 13). For instance, Fuhrman and Noble (9) using
coastal seawater from southern California, estimated grazing
mortality by using FLB disappearance rates and virus-induced
mortality from (i) declines in radiolabeled DNA, (ii) produc-
tion rates of virus-sized particles, and (iii) frequency of visibly
infected bacterial cells. The three methods of estimating virus-
induced mortality largely concurred and gave estimates similar
to that for grazing, i.e., about 50% day?1(9). Our estimates for
the reservoir bacterioplankton mortality are in a comparable
range; protist grazing was twice as high as virus-induced lysis,
accounting for ?50 and 25% of production, respectively.
We derived virus-induced mortality from infection frequen-
cies. It should be noted that while such estimates appear robust
(9), they are very sensitive to the conversion factors used,
especially the factor relating the portion of the infection period
during which viral particles are detectable (e) (3). When a virus
dilution approach was used for estimating the frequency of
infected cells (FIC) and the virus-induced mortality of bacteria
(M. Weinbaues and M. G. Ho ¨fle, unpublished data) in samples
from the surface water of the Baltic Sea and coastal and off-
shore waters of the Mediterranean Sea, the FIC was only
similar to data obtained by TEM when the highest conversion
factors were used for relating FVIC to FIC (Weinbauer and
Ho ¨fle, unpublished). Since average conversion factors were
used in the present study, the estimates of viral mortality of
bacteria might be an underestimation. While our estimation of
the absolute magnitudes of virus-induced mortality from infec-
tion frequencies perhaps should be treated with caution, the
importance of virus-induced mortality relative to grazing did
differ between treatments.
Cells in the “grazer-free” treatment, subject only to an ap-
parently low mortality rate from virus-induced lysis, (?17% of
production [Fig. 6]) increased markedly in concentration (Fig.
2). While the average cell morphology, as indicated by the
mean cell volume, appeared constant (Fig. 2), there was a shift
in BCC. It consisted of an increase in the proportion of
BET42a-positive bacteria, made up almost entirely of cells
detected by probe R-BT065. This probe is targeted to a small
cluster that contains three 16S rDNA sequences cloned from
the ?0.8-?m and ?5-?m treatments and clone sequences from
FIG. 6. Changes in total viral abundance, FVIC, and bacterial mor-
tality due to virus-induced lysis in size fractionation treatments (?0.8
and ?5 ?m) exposed in dialysis bags compared to unfiltered reservoir
water. Viral abundance values are the means of three replicates; error
bars indicate SD. The FVIC and virus-induced mortality values are the
means of duplicate subsamples from a single pooled sample represent-
ing all three replicates; the vertical bars show the range of the duplicate
estimates. Statistical relationships are given in Tables 2 and 3.
VOL. 67, 2001 PROTISTS, VIRUSES, AND THE BACTERIAL COMMUNITY2731

Page 10

other aquatic habitats (Fig. 1). The cluster represents one
branch of a recently described lineage of cosmopolitan fresh-
water beta proteobacteria (12). The DGGE band patterns of
the t0and t72communities also differed, with the appearance of
distinct bands at t72. Additionally, the significant shift in BCC
was reflected by marked differences in doubling times of dif-
ferent bacterial groups (Table 1), with the highest values being
detected for the R-BT065-positive cells in the “grazer-free”
treatment.
Interestingly, viral concentrations were lowest in the grazer-
free (?0.8-?m) treatment, intermediate in the reservoir pop-
ulation, and highest in the grazer-enhanced (?5-?m) treat-
ment (Fig. 6). The same trend was found when TEM was used
to count viruses in selected samples (data not shown). In ma-
rine mesocosms, a trend of virus concentrations increasing with
flagellate grazer concentrations has been reported (32), while
in another study, roughly parallel trends of bacterivory and
virus-induced mortality were found (13). However, not previ-
ously reported is the close relationship we found between in-
fection frequencies and grazing rates (Table 3).
We cannot reject the possibility that the differences in tax-
onomic composition influence “average” latent periods and
hence the frequencies of visibly infected cells. However, not
only infection frequencies but also viral abundances were high-
est in the ?5-?m treatment. Furthermore, infection rates were
also correlated with ratios of virus to bacterial abundance,
which appears a reasonable relationship. The mechanism be-
hind an apparent synergy between grazing and virus-induced
mortality is unclear. We can only speculate that protist grazing
may reduce bacterial diversity (20) and that there may be a
reciprocal relationship between the diversity of a bacterial
community and virus-induced mortality (46). Alternatively, in-
fection rates may shift with changes in individual cells. Cell-
specific production and activity are stimulated by grazing (29,
33, 39), e.g., due to the release of organic and inorganic nutri-
ents. Higher growth rates might be associated with enhanced
receptor formation on the cell surface, which may result in a
greater chance of phage attachment and thus higher infection
frequencies.
We observed large changes in the bacterioplankton commu-
nities in which grazing was greatly reduced or enhanced rela-
tive to the largely invariant natural reservoir community. The
effects of both flagellate grazing and viruses appear to vary
among different bacterial groups (compare Figs. 2 to 4 and 6
and Table 1). For example, in our experiment, filaments ap-
peared resistant to grazing and viral infection. Given these
differences, the invariant nature of the reservoir community
suggests that among bacteria there are population-specific
growth and removal rates. The changes in the bacterial com-
munities which occurred in the manipulated treatments (?0.8-
and ?5-?m treatments) can thus be attributed to sudden al-
terations in the balance established in situ between population-
specific growth and removal rates of bacteria.
ACKNOWLEDGMENTS
This study was supported by the Grant Agency of the Czech Repub-
lic (research grant 206/99/0028 awarded to K. Sˇimek); by CNRS inter-
national project “Regulation of Heterotrophic Planktonic Pro-
karyotes—PICS 1111”; by instrument grant AS CR “Microanalysis of
microbial communities” program 1036, P 1011802; and by the Max-
Planck Society.
We thank R. Psenner for valuable comments on an earlier version of
the manuscript, F.-O. Glo ¨ckner for assistence with probe design and
ARB, and R. Rossello-Mora for a tutorial on calculating phylogenetic
trees.
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Correlation coefficient (r)n Significance (P)
[Bacteria] vs avg cell vol
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