Previous Article | Next Article 
Applied and Environmental Microbiology, November 1999, p. 4863-4872, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Flagellate Predation on a Bacterial Model
Community: Interplay of Size-Selective Grazing, Specific Bacterial
Cell Size, and Bacterial Community Composition
Martin W.
Hahn* and
Manfred G.
Höfle
Microbial Ecology Group, GBF-National
Research Center of Biotechnology, D-38124 Braunschweig, Germany
Received 6 May 1999/Accepted 23 August 1999
 |
ABSTRACT |
The influence of grazing by the bacterivorous nanoflagellate
Ochromonas sp. strain DS on the taxonomic and morphological
structures of a complex bacterial community was studied in one-stage
chemostat experiments. A bacterial community, consisting of at least 30 different strains, was fed with a complex carbon source under conditions of low growth rate (0.5 day
1 when nongrazed)
and low substrate concentration (9 mg liter1). Before and
after the introduction of the predator, the bacterial community
composition was studied by in situ techniques (immunofluorescence microscopy and fluorescent in situ hybridization), as well as by
cultivation on agar media. The cell sizes of nonspecifically stained
and immunofluorescently labeled bacteria were measured by image
analysis. Grazing by the flagellate caused a bidirectional change in
the morphological structure of the community. Medium-size bacterial
cells, which dominated the nongrazed community, were largely replaced
by smaller cells, as well as by cells contained in large multicellular
flocs. Cell morphological changes were combined with community
taxonomic changes. After introduction of the flagellate, the dominating
strains with medium-size cells were largely replaced by single-celled
strains with smaller cells on the one hand and, on the other hand, by
Pseudomonas sp. strain MWH1, which formed the large,
floc-like forms. We assume that size-selective grazing was the major
force controlling both the morphological and the taxonomic structures
of the model community.
| |
INTRODUCTION |
Predation by bacterivorous protists,
particularly by the ubiquitous bacterivorous nanoflagellates, is a
major mortality factor for aquatic bacteria (28, 46, 47).
Several field studies and laboratory investigations have demonstrated
that flagellate grazing potentially influences both the size
distribution of bacterial communities (2, 11, 18, 29, 40)
and their taxonomic composition (11, 20, 30, 31, 40, 41).
Grazing by direct-interception-feeding flagellates takes place in two
steps, encounter and ingestion. The probability of a bacterial cell and
a particular flagellate encountering one another depends on bacterial
cell size and may be influenced by bacterial motility and bacterial
surface characteristics (25). Jürgens and DeMott
demonstrated that the ingestion step is influenced by the physiological
state of the flagellate cells (19). In two flagellate
species, they found significantly lower food selectivity by flagellates
grown under food-limited conditions than by food-saturated flagellates.
The influence of flagellate grazing on the composition of bacterial
communities is based on a complex interplay of several factors.
Probably the most important factors are the above-mentioned selectivity
of flagellate grazing (by size or other qualities) (9, 21, 24,
39), differences in cell size distribution of bacterial species
(11, 12), and different abilities of bacterial species in
grazing defense (11, 30). Furthermore, grazing may influence
the growth conditions of individual bacterial species through the
regeneration of substrates or by reduction of competitors.
We studied the influence of grazing by the flagellate
Ochromonas sp. strain DS on a bacterial model community
under defined experimental conditions to obtain further insights into
this complex interplay. We used chemostats for our experiments in order
to minimize the influences on bacterial composition of changes in substrate supply. In order to involve a high number of bacterial strains, as well as a high number of bacterial defense strategies, in
the studies, we used a complex and nondefined bacterial community obtained from Lake Constance in Germany. The bacteria were fed with a
defined mixture of three different complex media to enable the
permanent coexistence of a high number of strains or species. In the
first step of the experiment, we tried to establish, in two parallel
reactors, nongrazed communities, stabilized in terms of abundance and
morphological and taxonomic structures. In only one of the two reactors
were all three criteria fulfilled; in the second reactor only numerical
and morphological stabilities were observed. In the second step of the
experiments, the bacterivorous flagellate Ochromonas sp.
strain DS was introduced into both reactors and the influence on the
morphological and taxonomic structure of the community, as well as on
the specific cell sizes of selected strains, was investigated. Due to
the lack of taxonomic stability in one reactor before inoculation with
the predator, in respect to the influence on taxonomic composition, the
paper will focus primarily on results from the other reactor. To
exclude the possibility that the changes observed for this single
reactor were caused by chance, we temporarily eliminated the
flagellates, using specific inhibitors, and studied the subsequent
community changes. For further confirmation, we performed separate
experiments on the influence of grazing on the major players isolated
from the experiments presented here (11, 13).
| |
MATERIALS AND METHODS |
Chemostat experiments.
The experiments were carried out with
a one-stage chemostat system (11). This consisted of two
parallel reactors (designated reactors I and II) with working volumes
of 2 liters. The two reactors were fed from one reservoir and mixed by
aeration with sterile air. Sampling and inoculation of the reactors, as
well as all other chemostat work, were done under sterile conditions.
Bacteria were cultured at 15 ± 1°C, in the dark, with NSY
medium containing 9 mg of a complex substrate liter1
(11). The dilution rate was maintained at 0.5 day1 (the bacterial growth rate in the flagellate-free
phase of the experiments was 0.5 day1).
Chemostat reactors were inoculated with a complex bacterial community
obtained from a nonaxenic culture of the bacterivorous flagellate
Ochromonas sp. strain DS. The flagellate and the
accompanying bacteria were isolated from Lake Constance in Germany by
D. Springmann. All subsequent culture work was done under sterile
conditions. The flagellate culture was treated with inhibitors specific
for eukaryotic cells (200 mg each of colchicine and cycloheximide liter1) to separate the bacteria from the flagellates.
The chemostat reactors were initially inoculated with the mixed
bacterial community plus two bacterial strains, Comamonas
acidovorans PX54 (8, 11) and Aeromonas
hydrophila PU7718 (8), both isolated from Lake
Plußsee, Germany.
Chemostat experiments consisted of several phases (reactor I, four
phases, and reactor II, two phases) (Table
1). In the
first phase, the mixed
bacterial community was cultured in the
absence of any predator, and
the second phase started, in both
reactors, with the inoculation of the
flagellate
Ochromonas sp.
strain DS. Only in the case of
reactor I was the flagellate eliminated
by injection of inhibitors (200 mg each of colchicine and cycloheximide
liter
1) specific
for eukaryotic organisms (phase 3).
Microbial abundance.
Determination of total bacterial and
flagellate abundance was done by staining formaldehyde-fixed samples
with 0.1% (wt/vol) DAPI (4',6-diamidino-2-phenylindole), filtration on
0.2-µm-pore-size polycarbonate filters, and enumeration by
epifluorescence microscopy (11).
Characterization of bacterial community composition by
immunofluorescence microscopy.
Two polyclonal rabbit antisera and
two monoclonal antibodies were used to determine the abundance and
specific cell sizes of four different bacterial strains. For the
detection of the Vibrio sp., C. acidovorans PX54,
and A. hydrophila PU7718, an antiserum and antibodies
described previously were used (8, 11). Those antibodies and
the polyclonal antiserum showed no cross-reactivities with other
strains of the investigated mixed bacterial community. For detection of
Pseudomonas sp. strain MWH1, a polyclonal rabbit antiserum
was developed and tested for specificity by immunofluorescence
microscopy. Twenty-six reference strains isolated from the investigated
mixed bacterial community and 14 strains of species of the genus
Pseudomonas were tested. The antiserum showed weak
cross-reactivity with four strains obtained from the investigated
community and with Pseudomonas aeruginosa DSM
50071T, Pseudomonas alcaligenes DSM
50342T, Pseudomonas fluorescens DMS
50090T, Pseudomonas pseudoalcaligenes DSM
50188T, Pseudomonas putida DSM 50222, and
Pseudomonas viridifalva DSM 50338. Determination of the
specific abundance of bacterial strains by immunofluorescence
microscopy was performed as described previously (11).
FISH.
The group-specific composition of the mixed bacterial
community was determined by in situ cell hybridization with fluorescent oligonucleotide probes (fluorescent in situ hybridization [FISH]). The group-specific probes used were as follows: EUB338 (domain Bacteria) (1); BET42a (beta-proteobacteria)
(22); GAM42a (gamma-proteobacteria) (22); and PS
(genus Pseudomonas) (38). Hybridization and
counterstaining with DAPI were carried out according to the method of
Amann et al. (1) and Manz et al. (23). Total bacterial cell numbers (DAPI) and positively probe-hybridized cells
were enumerated by analyzing slides, which were taken of the same
section of a double-stained sample (Zeiss Axiophot).
Characterization of bacterial community composition by
cultivation on agar plates.
Dilution and plating of chemostat
samples on NSY agar (four replicates) were carried out as described
previously (11). Based on several morphological features
(size, color, shape, and surface and border characteristics), 18 different colony types (I to XVIII) were distinguished. Very rare
colony types or irregularly grown colonies (due to contact with other
colonies or the plate border) were pooled in a separate group (type Z).
The relative abundances of the 19 colony types on agar plates were
determined by counting the total numbers of colonies (CFU) and the
type-specific numbers.
Taxonomic analysis of colony types and single strains.
The
morphologically defined colony types were tested for taxonomic
homogeneity by comparison of 5 to 10 isolates by FISH, by using the
phenotypic test system BIOLOG (BIOLOG Inc., Hayward, Calif.) and by
performing low-molecular-weight (LMW) RNA profiles (15, 16).
Isolates of types II, III, and XVI were characterized by clearly
distinct colony morphologies but showed no differences in BIOLOG tests,
LMW RNA profiles, cell morphologies, or reaction with the
anti-Vibrio sp. strain CB5 serum (CB5 belongs to the colony
type III group). Therefore, those three colony types were tested
additionally for taxonomic homogeneity by amplified ribosomal DNA
(rDNA) restriction analysis (ARDRA) (44). Four isolates each
of colony types II, III, and XVI were compared.
Identifications of the strains
Pseudomonas sp. strain MWH1
(
13),
Vibrio sp. strain CB5 (colony type III)
(
11),
Vibrio sp. strain CB2 (colony type II),
C. acidovorans PX54 (
8), and
A. hydrophila PU7718 (
8) were carried out by analysis of
the
16S rDNA sequences (
12).
Determination of nonspecific and specific cell size.
The
cell sizes of DAPI-stained (nonspecific-size) and antibody-labeled
(specific-size) cells were determined by image cytometry as described
previously (11). Specific size data were corrected for
overestimation, as described previously (11).
Assay for size-selective grazing.
The uptake of fluorescent
latex beads of different diameters (Polysciences Inc.) was followed for
estimation of size-selective grazing by Ochromonas sp.
strain DS. Chemostat samples from reactor I (phase 2) received a small
volume of a suspension of beads and were incubated at 15°C. The
suspension contained an equal mixture of beads with diameters of 0.5, 0.75, 1.0, and 2.0 µm in NSY medium. Samples were taken five times at
10-min intervals and were fixed immediately in an equal volume of 4%
ice-cold glutaraldehyde (36). The number of ingested beads
of the different diameters was counted for 100 randomly selected
flagellates. Size-specific grazing rates were calculated, using data on
mean numbers of beads taken up per flagellate from the first four
samplings (no saturation of uptake). The highest grazing rate was set
at 100% for calculation of size-specific relative grazing efficiency.
The experiment was carried out in triplicate.
Statistical analysis.
The data were checked for significance
of differences by t test.
| |
RESULTS |
Influence of flagellate grazing on bacterial abundance, cell size,
and morphology.
In terms of bacterial abundance, cell size, and
bacterial morphology, the nongrazed communities (phase 1), cultured in
the two parallel reactors, showed relatively stable patterns with only
minor differences observed between the two communities. Total bacterial
numbers were (10.3 ± 1.6) × 106
ml1 (reactor I) and (12.5 ± 1.3) × 106 ml1 (reactor II), and the two communities
consisted of only single-celled bacteria (Fig.
1) with mean cell volumes of 0.156 ± 0.003 µm3 (reactor I) (Fig.
2) and 0.125 ± 0.050 µm3 (reactor II). After introduction of the flagellate,
the bacterial abundance and the mean bacterial cell size decreased with
increasing flagellate abundance (Fig. 2). During the grazing-controlled
phase 2, which lasted for 148 (reactor I) and 107 (reactor II) days, the abundance of single-celled bacteria was stable at (1.1 ± 0.4) × 106 ml1 (reactor I) and
(0.9 ± 0.2) × 106 ml1 (reactor
II). Besides the reduction in numbers of single-celled bacteria, the
two communities showed different reactions to flagellate grazing. In
reactor I the initial decrease in mean bacterial cell volume was
followed by a short increase to 0.190 µm3 and thus to a
value higher than those observed for the nongrazed community (Fig. 2).
This maximum in mean cell size was correlated with a maximum in the
relative abundance of mdit.C. acidovorans PX54 (see below) and
the maximal flagellate abundance (Fig. 2). Afterwards, the mean cell
volume of single-celled bacteria decreased again and, simultaneously, a
population of floc-like bacteria was established (Fig. 1 and 2). The
bacterial flocs consisted of as many as 900 rod-shaped cells of similar
size and morphology. For the following 137 days, the
flagellate-controlled bacterial community of reactor I was divided
morphologically into two populations; one consisting of small, single
cells with a mean volume of 0.078 ± 0.016 µm3 and
one consisting of the large bacterial flocs with diameters of 5 to more
than 50 µm. The average cell length of single-celled bacteria
decreased from 0.87 µm (phase 1) to 0.65 µm (the mean value for the
period of phase 2 after the peak).
View larger version (97K):
[in this window]
[in a new window]
|
FIG. 1.
Photomicrographs of bacteria from reactor I stained with
DAPI. (A) Before introduction of the flagellate (phase 1). (B) After
introduction of the flagellate (phase 2). (C) After inhibition of the
flagellate population (phase 3). (D) After reestablishment of the
flagellate population (phase 4). A single cell of the bacterivorous
flagellate Ochromonas sp. strain DS is shown in panel D. The
images represent different sample volumes filtered onto membrane
filters.
|
|
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 2.
Influence of flagellate grazing on the complex bacterial
community cultured in reactor I. The upper panel shows the total
bacterial cell numbers and the percentage of single-celled bacteria.
The lower panel shows the relative abundances (percentages of total
cell numbers) of the three species detected by immunofluorescence
microscopy. The four experimental phases are indicated by vertical
lines. Flag., flagellate; contr., controlled; P. sp. MWH1,
Pseudomonas sp. strain MWH1.
|
|
Also, in reactor II, the mean bacterial cell volume decreased after
introduction of the flagellate (to a mean value of 0.050
± 0.008 µm
3), but no intermediate increase in cell size or
occurrence of
floc-like bacteria was observed in the 81 days after
introduction
of the flagellate. Therefore, the reactor was inoculated
with
a sample (10 ml) from reactor I which contained floc-like
bacteria.
This resulted in the establishment of a population of large,
floc-like
bacteria in reactor II, rather similar to the events observed
in reactor
I.
During flagellate-controlled phases, the numbers of single-celled
bacteria fluctuated in both reactors within a narrow range
of 0.6 × 10
6 to 1.3 × 10
6 ml
1
(Fig.
3) and thus on a level
approximately 10-fold lower than
the level during flagellate-free
phases. In the case of reactor
I, a period covering approximately 10%
of the total flagellate-controlled
phases showed higher abundances of
single cells (1.2 × 10
6 to 2.5 × 10
6 ml
1). The cell numbers of the floc-like
bacteria (
Pseudomonas sp.
strain MWH1) fluctuated (0.2 × 10
6 to 3.3 × 10
6 ml
1)
much more than those of single cells (Fig.
3). During the period
when
the floc-like bacteria were present, the total numbers of
cells
building these flocs made up an average of 55% (reactor
I) and 44%
(reactor II) of the total bacterial cell numbers. The
mean cell size of
bacteria in the flocs (0.206 µm
3) was larger than that of
the single-celled bacteria, and thus,
these bacteria dominated the
total bacterial biomass, with mean
values of 72% (reactor I) and 51%
(reactor II).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Plot of the number of single-celled bacteria against the
number of cells in flocs. The data set represents only samples from the
flagellate-controlled phases of reactor I (circles) as well as from
reactor II (triangles). The open circles represent data from a 20-day
period (about 10% of the total flagellate-controlled phases), and the
solid circles represent data from the other periods.
|
|
Elimination of the flagellate population in reactor I by specific
inhibitors resulted in (i) an increase of bacterial numbers,
(ii) an
increase in the mean bacterial cell size, and (iii) a
decrease in the
floc-like bacteria to levels below the detection
limit (Fig.
1 and
2).
Thus, the bacterial community restored the
morphological structure
typical for phase 1 of both reactors.
The subsequent reestablishment of
the flagellate population resulted,
again, in a decrease of bacterial
numbers and in bidirectional
changes in the morphological structure of
the bacterial community
(Fig.
1 and
2).
Characterization of the taxonomic structure of the bacterial
communities.
The nondefined community was characterized
taxonomically by plating samples on NSY agar, by immunofluorescence
microscopy, and by FISH. The quantitative characterization of the
bacterial communities by culturing was handicapped less by the general
culturability of the bacteria than by the lack of formation of separate
colonies by cells stuck together in the bacterial flocs. During periods with an absence of floc-like bacteria, the mean values of total cell
counts and total CFU showed no significant (P > 0.01)
differences. During other periods, the total cell numbers were higher
than the total CFU, although no significant difference was observed between the numbers of single cells and total CFU (P > 0.01).
On the agar plates, more than 25 morphologically distinguishable colony
types were observed over the total period of the experiment.
Of these
25 types, 18 (designated I to XVIII) were found regularly,
with
percentages of more than 5% of the total colonies. For these
18 colony
types, the respective relative CFU were determined,
and only these
types were tested for taxonomic homogeneity. Only
two (IV and V) of the
18 colony types were found to consist of
morphologically
indistinguishable but taxonomically different
colonies. On the other
hand, colony types II, III, and XVI demonstrated
clear differences in
colony morphology, but no taxonomic difference
was found (by LMW RNA
analysis, ARDRA, or reaction with anti-
Vibrio sp. strain CB5
serum). Sequencing of the 16S rDNA genes revealed
virtually identical
sequences for two tested isolates (CB2 and
CB5) of different colony
morphologies (II and III). Comparisons
of 16S rDNA gene sequences
demonstrated that these isolates were
most closely related to
Vibrio pelagius (sequence similarity,
99.6%
[
26]).
During the absence of bacterial flocs the summarized relative abundance
of the
Vibrio sp. colonies (types II, III, and XVI)
in both
reactors showed no significant differences (
P > 0.1)
from
the relative abundance of the
Vibrio sp. determined by
immunofluorescence
microscopy (Fig.
4,
Table
2). During periods when floc-like
bacteria
were present, the values differed significantly.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
Comparison of the relative abundance of the
Vibrio sp. determined by the plating approach (open circles)
and by immunofluorescence microscopy (solid squares). The plating data
represent the percentage of Vibrio sp. colonies (colony
types II, III, and XVI) among total CFU, and the microscopically
determined data represent the percentage of Vibrio sp. cells
among the total number of single-celled bacteria. The plating data are
based on 100 to 250 counted colonies (total CFU), and the microscopy
data are based on at least 500 counted cells (total single cells).
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Taxa of particular interest and their detection, type of
colony morphology, and range of relative abundance observed during
the experiment
|
|
By immunofluorescence microscopy, 10 to 80% of the bacterial cells in
samples from the two reactors could be identified by
using the four
antisera and antibodies. While, in reactor I, an
average of 49.6% ± 15.9% of the total cell numbers were detectable,
the other reactor had
a lower average detectability of 37.8% ±
17.8%.
The bacterial flocs were formed exclusively by
Pseudomonas
sp. strain MWH1. This was demonstrated by immunofluorescence
microscopy,
by FISH, and by studies with the isolated strain
(
13).
Stability of the nongrazed communities.
During the first 10 days after inoculation of the two reactors, the same succession pattern
was observed in the two communities. Initially, type I colonies
dominated, making up approximately 50% of the total CFU, but they were
replaced extensively by the Vibrio sp. and strains of colony
type V during the following days. For the next 20 days, the community
compositions were very similar as well as stable in the two reactors.
During this period, the Vibrio sp. (approximately 50% of
cells and colonies) and strains of colony type V (25 to 30% of
colonies) dominated in both reactors.
In the further course of the experiment, the communities cultured in
the two reactors showed different types of development.
The community
of reactor I was more or less stable until the inoculation
with the
flagellate (Fig.
2), while the community of reactor II
showed a slow
but steady change in composition (data not shown).
In this reactor, the
percentage of
Vibrio sp. cells and colonies
declined over a
period of 115 days from 50 to 25% and type IV
colonies increased from
less than 10% to fluctuating percentages
of 25 to 60%.
C. acidovorans PX54 established stable populations
in both reactors,
with a mean relative abundance of 0.9% ± 1.7%
(reactor I) and 2.7% ± 1.1% (reactor II). In both reactors
A. hydrophila PU7718
was only found (<1% of total cell numbers) during
the first days
after
inoculation.
Influence of flagellate grazing on the taxonomic composition of the
bacterial communities.
In reactor I the composition of the stable
community changed with the establishment of the flagellate population.
The following succession was observed (Fig. 2): (i) a decrease in the
Vibrio sp. from 51.3% ± 2.5% to 7.1% ± 3.7% of the
total cell numbers; (ii) an increase in C. acidovorans PX54
from 0.9% ± 1.7% to a maximum of 24.5%; (iii) a decrease in
C. acidovorans PX54 to mean values of 0.4% ± 0.5%; (iv) a
simultaneous occurrence and a numerical increase in bacterial flocs
(Pseudomonas sp. strain MWH1 [see below]); (v)
simultaneous with the increase in flocs, an increase in the percentage
of type IV colonies from 3.7% ± 6.5% to a maximum of 80%; (vi) a
steady decrease in the relative abundance of type IV colonies; and
(vii) a simultaneous increase in type V colonies.
The maximum in the absolute abundance of
C. acidovorans
PX54, observed after the introduction of the flagellate (Fig.
2 and
5), was 7-fold higher than the mean
abundance and 3.5-fold higher
than the maximum abundance observed
during the flagellate-free
phase of the experiment.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 5.
Changes in the cell size of the total single-celled
bacteria, of C. acidovorans PX54, and of the
Vibrio sp. after introduction of the flagellate
Ochromonas sp. strain DS into reactor I (center panel). The
upper panel gives the total bacterial and flagellate abundances, and
the lower panel gives the relative abundances of three investigated
bacteria (percentage of total cell numbers).
|
|
The elimination of flagellates by using specific inhibitors resulted in
the recovery of the morphological structure, as well
as the recovery of
a taxonomic structure similar to that of the
nongrazed community (Fig.
1 and
2). The reestablishment of the
flagellate population was followed
by a succession similar to
that observed after the introduction of the
flagellate (Fig.
2).
The main difference was the lack of a maximum of
C. acidovorans PX54.
In reactor II, no significant influence of flagellate grazing on the
taxonomic structure was detectable by the methods used.
Before
inoculation with the flagellate, the relative abundance
of the
Vibrio sp. was already low and those of the taxonomically
heterogenous colony types IV and V were high. Possible changes
within
these two heterogenous groups cannot be excluded. After
inoculation
with a sample from reactor I, the bacterial composition
changed
simultaneously with the establishment of bacterial flocs
consisting of
Pseudomonas sp. strain MWH1
cells.
FISH.
Three samples each from phase 1 and phase 2 of reactor I
were hybridized with four oligonucleotide probes. On average, 80.5% ± 8.2% of the DAPI-labeled cells hybridized positively with the EUB338
probe targeted against all taxa belonging to the domain Bacteria. Samples of the two phases showed no differences in
their reactions to hybridization. Introduction of the flagellate caused no significant change in bacterial composition detectable by the probes
GAM42a and BET42a. The 
-proteobacteria dominated among the
EUB338-hybridizable cells with 77% (phase 1) and 71% (phase 2), and
5% (phase 1) and 10% (phase 2) reacted positively with the BET42a
probe. The PS probe always hybridized positively with cells in
bacterial flocs (Pseudomonas sp. strain MWH1), but signals from single cells were always very weak.
Specific cell size of Vibrio sp. and C. acidovorans PX54 in reactor I.
The mean cell volume of the
Vibrio sp. increased slightly after introduction of the
flagellate from 0.090 ± 0.012 µm3 (phase 1) to
0.124 ± 0.009 µm3 (phase 2) (Fig. 5). A comparison
of the size class distribution of the Vibrio sp. with the
distribution of the total community revealed that the Vibrio
population completely covered the lower-size classes during phase 1 (Fig. 6). The percentages for the
Vibrio sp. found for the three classes from 0 to 0.075 µm3 were slightly higher than those for the total
community, which may indicate an underestimation of the sizes of the
Vibrio sp., but the differences were not significant
(P > 0.1). During the flagellate-controlled phases,
the mean cell size of the total population of single-celled bacteria
was smaller than the mean cell size of the Vibrio sp. (Fig.
5). This resulted in the dominance of other strains in the lower-size
classes (Fig. 6), indicating that the Vibrio population was
strongly replaced by populations of other strains which had smaller
cells under the given growth conditions.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 6.
Influence of size-specific grazing of the flagellate
Ochromonas sp. strain DS (upper graph) on the size
distribution of the bacterial community of reactor I. The bars of the
graph in the middle represent the size class distribution of the
community during phase 1 (mean values of five samples). The hatched
parts of the bars indicate the proportion of Vibrio sp.
cells. The lower graph represents the distribution after introduction
of the predator (five samples from phase 2). The size class >2.0
µm3 is exclusively formed by the Pseudomonas
sp. strain MWH1 flocs (the bars represent mean values for the whole
microcolony population). This size class contained 4% of the bacterial
particles (single-celled bacteria and flocs) but 72% of the bacterial
biomass.
|
|
The mean cell size of
C. acidovorans PX54 increased with
increasing grazing pressure, although afterwards, the cell size
decreased
with the increase of the
Pseudomonas sp. strain
MWH1 population
(Fig.
5). The maximal cell size of
C. acidovorans PX54 occurred
with the maximal cell numbers and
relative abundance of the strain
(Fig.
5).
Size-selective grazing of Ochromonas sp. strain
DS.
The flagellate showed a marked size-selective grazing. Grazing
rates on beads with diameters of 0.5 and 2.0 µm were 10- and 200-fold
lower, respectively, than the rate determined for 1.0-µm-diameter beads (Fig. 6). Based on the uptake of beads, we calculated
size-dependent grazing rates of 19.9 ± 5.5 bacteria
flagellate1 h1 (1.0-µm-diameter beads)
and 1.9 ± 0.05 bacteria flagellate1
h1 (0.5-µm-diameter beads).
| |
DISCUSSION |
The experimental conditions used in our experiments mimicked the
growth rates and abundances of bacteria (12), the growth rates and abundances of bacterivorous flagellates (3, 35, 46), and the mean cell size of bacteria (7, 29, 42)
reported for mesotrophic to eutrophic lakes. On the other hand,
experimental studies like the one presented here are not able to copy a
natural bacterial community. The number of relevant bacterial and
flagellate species is limited, and the presence of species dominating
in natural assemblages is rather unlikely. However, on the level of
morphological community structure, we observed responses to flagellate
grazing (Fig. 6), as predicted for natural bacterial communities by the
empirical model of Güde (10), as well as observed
several times in field studies (10, 29). These general similarities indicate that our system is, to some extent, suitable for
general investigations of the interplay of morphological and taxonomic
changes caused by flagellate grazing.
Bacterial successions after introduction of the flagellate. (i)
Reactor I.
The bacterial community of reactor I was dominated by
the Vibrio sp., C. acidovorans PX54, and
Pseudomonas sp. strain MWH1 over almost the whole experiment
(Fig. 2). These major players responded to the introduction of the
flagellate and to the reestablishment of the inhibited flagellate
population with marked changes in relative abundance and size. These
responses were reproduced in separate chemostat experiments under the
same experimental conditions but involving only one or two of the major
players, as well as the flagellate Ochromonas sp. strain DS
(11, 13). In a chemostat experiment with Vibrio
sp. strain CB5 (one of the Vibrio sp. strains) and C. acidovorans PX54 (11), we also observed, after
introduction of the flagellate, a decrease in the relative abundance of
the Vibrio sp. and a simultaneous increase of C. acidovorans PX54, as well as similar trends in changes in the cell
sizes of the two strains (11) (Fig. 5). In contrast to this
previous study, the increase in cell size of C. acidovorans
PX54 stopped in the present study before the formation of filaments
(>10 µm) occurred. An increase in the cell size and the formation of
filaments by C. acidovorans PX54 is controlled by the growth
rate (12). We assume that in the present study the lack of
further cell elongation was a result of competition, which influenced
the growth rate of the strain. A stronger limitation of C. acidovorans PX54 by competition on substrates in the present study
is indicated by the 80%-lower (in comparison to the nongrazed part of
the reference experiment) biomass of the population. After the
introduction of the flagellate, C. acidovorans PX54 may have
been released from this limitation through a reduction of competitors
as a result of grazing. The later decrease in cell size may have been
influenced by the simultaneous increase of the potential competitor,
Pseudomonas sp. strain MWH1 (Fig. 5).
Formation of flocs by single-celled
Pseudomonas sp. strain
MWH1, after establishment of the flagellate population (Fig.
2),
was
also observed in separate chemostat experiments (
13). These
flocs differed markedly in size from all single-celled bacteria
and
should, in comparison to those, receive protection against
flagellate
grazing. However, differences in fluctuations of the
total abundance of
cells in flocs (
Pseudomonas sp. strain MWH1)
and in the
total abundance of single-celled bacteria (Fig.
2 and
3) demonstrate
that these two groups were controlled by different
mechanisms. We can
largely exclude the possibility that phages
were responsible for the
observed fluctuations of the floc-like
Pseudomonas sp.
strain MWH1 population. An investigation of several
chemostat samples
by transmission electron microscopy revealed
a very low abundance of
<10
4 phages ml
1 (
45). No
differences in phage abundance were found for samples
with low, medium,
and high numbers of bacterial flocs. We assume
that the fluctuations of
the floc-like
Pseudomonas sp. strain
MWH1 were a result of
competition with single-celled bacteria
and/or a consequence of changes
in the proportion of floc-forming
to single-celled
Pseudomonas sp. strain MWH1 (
13).
Based on the comparisons with the reference experiments (
11,
13), we conclude that the total bacterial succession observed
in
reactor I was mainly the result of flagellate
grazing.
(ii) Reactor II.
The bacterial community of reactor II
responded to flagellate grazing through changes in morphology and cell
size, similar to the reaction of the community of reactor I (Table
3), although the methods used to follow
the taxonomic composition were not able to reveal whether a taxonomic
response to the introduction of the flagellate also occurred. However,
several factors may have been responsible for the taxonomic changes
observed during the experimental phase before the introduction of the
flagellate. Firstly, minimal differences in the taxonomic composition
of the inoculi used for the two reactors may be the reason. Secondly, different physiological adaptations of the populations present in the
reactors may have caused the different developments. Such changes in
the physiological features of bacteria cultured in chemostats have been
reported previously (14, 33).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Observed changes in morphological and taxonomic structure
of bacterial communities after introduction of the flagellate
Ochromonas sp. strain DS
|
|
Interplay of morphological and taxonomic structures.
The
bacterial communities of both reactors responded to flagellate grazing
with a marked bidirectional shift in their size distributions (Fig. 2
and 6 and Table 3). These shifts could be easily explained by size
selectivity of the flagellate grazing (2, 9, 29, 39).
However, in the assay used for estimating the size selectivity of the
flagellate grazing, we used fluorescent latex beads. Because these
particles were different in shape, surface qualities, and other traits
from the bacteria cultured in the chemostat, the assay can give only a
rough idea about the size selectivity of the flagellates grazing upon
bacteria. Additionally, a size-independent selectivity of flagellate
grazing was demonstrated (5, 21, 24), as well as a
dependence of selectivity on the physiological state of the flagellate
cells (19). On the other hand, the flagellate population
grew in the chemostat under strongly food-limited conditions. The
flagellate growth rate was 0.5 day1 and thus only
approximately 30% of the maximum growth rate observed under similar
but food-saturated growth conditions. Jürgens and DeMott found
only a weak size-independent selectivity by food-limited flagellates
(19). Therefore, and due to the clear trends in bidirectional shifts of bacterial size distribution, we assume that
size selectivity of flagellate grazing was the major structuring force
controlling the morphological structure of the bacterial community.
However, we cannot rule out the possibility that size-independent selectivity played a minor role in the influence of grazing on the
bacterial community structure.
In terms of changes in the mean size of single-celled bacteria, the
complex bacterial communities of both reactors responded
to flagellate
grazing in a totally different way than five bacterial
species
investigated in separate experiments (
11-13). While the
single-celled part of the complex communities showed a marked
decrease
in mean cell size, each of the five populations responded
to flagellate
grazing by a slight-to-marked increase in cell size.
We previously
demonstrated that the increases in cell size were
not a direct reaction
to flagellate grazing but a reaction to
an increase in bacterial growth
rates due to grazing (
11,
12).
Such a dependence of cell
size on growth rate was also found for
other bacterial species (
4,
6,
37) and may be a result
of an increased need for space for the
ribosomes, which increase
in number as growth rate increases (
4,
32,
34). In the
current experiment, the responses in cell size of
selected taxa
were followed (Fig.
5). These populations showed the
expected
increases in cell size after the reduction of total bacterial
biomass by the predator. The opposite responses of mean cell size
of
single species and mean cell size of the whole single-celled
part of
the total bacterial community indicate that strains with
larger cells
were partly replaced by strains with smaller cells
(Fig.
5 and
6).
However, we cannot rule out the possibility that
some of the minor
players actively decreased their cell sizes
despite the expected
increase in bacterial growth rate. Whether
such a bacterial grazing
defense mechanism exists is
unknown.
We suggest that the observed morphological and taxonomic responses of
the investigated bacterial community to the flagellate
grazing were
mainly a result of the interplay of size-selective
grazing, differences
in cell size distribution of single bacterial
strains, and differences
in the specific responses of bacterial
cell size distribution to
grazing and growth rate (Fig.
7).
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 7.
Schematic model of the interplay of size-selective
grazing, specific bacterial cell size, and morphological as well as
taxonomic structure of the bacterial communities investigated in the
chemostat experiments. The upper part of the model depicts the size
selectivity of protist grazing. The middle part shows the size
distribution of bacterial communities in situations with and without
grazing pressure, as well as their possible strain composition
(indicated by letters). The lower part depicts the size distribution of
single bacterial strains (A to D), as well as grazing-influenced
changes in their size distributions (dotted curves). The arrows
indicate the direction and strength of induced changes in the size
distributions of the strains. Examples for some of the distribution
patterns are shown, and the triggers of changes are given. The key
result is that differences in the distribution patterns of single
bacterial strains produced different grazing mortalities of the
respective populations and thus resulted in changes in the
morphological and taxonomic structures of the bacterial community. The
possible influence of grazing on bacterial competition for substrates
or on changes in substrate supply, as well as size-independent grazing
defense strategies, is not considered. (a), reference
11; (b), reference 12; (c),
reference 13; G, growth rate; ?, unknown.
|
|
In the present study, and in other experiments, we observed the
domination of medium-size single cells in the absence of grazers
(Fig.
6) (
11-13). This corresponds with the model of Güde
(
10)
and with several other observations (
2,
10,
17,
29),
but the reason for the superiority of medium-size strains to
smaller
and larger strains is unknown. One may speculate that larger
cells
have disadvantages in competition for substrates due to a reduced
surface-to-volume ratio. For the smaller cells, this argument
does not
hold true. However, Norland and coworkers (
27), as
well as
Simon and Azam (
43), demonstrated in size-fractionated
bacterial communities that smaller bacterial cells tend to have
a
higher dry-weight-to-volume ratio than larger ones. The water
content
of the cells decreases with cell size, but the percentage
of DNA,
proteins, and cell wall and other cellular components
in the cellular
dry mass increases. This indicates that the evolutionary
process of
development of smaller bacteria requires a compromise
between a general
reduction in cell size and conservation of essential
cellular
functions, which cannot be decreased below a minimum
level (e.g., the
genome size). We assume that this compromise
results in additional
metabolic costs for small bacterial cells
and thus leads to the
superiority of medium-size cells. However,
in situations without
predation, the medium-size cells may have
competitive advantages,
although in the opposite situation these
cells should have higher
grazing losses due to size-selective
predation. This results in
advantages for smaller and larger cells
and, thus, in changes in the
taxonomic and morphological compositions
of bacterial communities (Fig.
7).
Because we found no hints of size-independent grazing defense
strategies of bacteria in our experiments, and due to the general
lack
of knowledge about such mechanisms, we only considered size-dependent
defense mechanisms (Fig.
7). However, in numerous field studies,
marked
morphological shifts of bacterial communities were reported
as
responses to increases in protist grazing pressure (
10,
18,
29,
41). This may indicate that size-independent grazing defense
mechanisms play only a minor role in natural bacterial
communities.
| |
ACKNOWLEDGMENTS |
We thank M. G. Weinbauer for determination of phage
abundances, F. Ziemke for help with the ARDRA, E. R. B. Moore
for 16S rDNA analysis and for linguistic improvements, and K. Seikowsky and K. Dominik for performing LMW RNA profiles, as well as two anonymous reviewers for constructive criticism on the manuscript.
This study was supported by funds from the Bundesministerium für
Bildung, Wissenschaft, Forschung und Technologie (grant BEO-0319433B).
| |
FOOTNOTES |
*
Corresponding author. Present address: Institute of
Limnology, Austrian Academy of Sciences, Gaisberg 116, A-5310 Mondsee, Austria. Phone: 43 6232 3125-29. Fax: 43 6232 3578. E-mail:
martin.hahn{at}oeaw.ac.at.
| |
REFERENCES |
| 1.
|
Amann, R. I.,
B. J. Binder,
R. J. Olson,
S. W. Chisholm,
R. Devereux, and D. A. Stahl.
1990.
Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.
Appl. Environ. Microbiol.
56:1919-1925[Abstract/Free Full Text].
|
| 2.
|
Andersson, A.,
U. Larsson, and Å. Hagström.
1986.
Size-selective grazing by a microflagellate on pelagic bacteria.
Mar. Ecol. Prog. Ser.
33:51-57.
|
| 3.
|
Berninger, U.-G.,
B. J. Finlay, and P. Kuuppo-Leinikki.
1991.
Protozoan control of bacterial abundances in freshwater.
Limnol. Oceanogr.
36:139-147.
|
| 4.
|
Bremer, H., and P. P. Dennis.
1996.
Modulation of chemical composition and other parameters of the cell by growth rate, p. 1533-1569.
In
F. C. Neidhardt, R. Curtiss III, J. P. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Rezikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.
|
| 5.
|
Christoffersen, K.,
O. Nybroe,
K. Jürgens, and M. Hansen.
1997.
Measurement of bacterivory by heterotrophic nanoflagellates using immunofluorescence labelling of ingested cells.
Aquat. Microb. Ecol.
13:127-134.
|
| 6.
|
Chrzanowski, T. H.,
R. D. Crotty, and G. J. Hubbard.
1988.
Seasonal variation in cell volume of epilimnetic bacteria.
Microb. Ecol.
16:155-163.
|
| 7.
|
Cole, J. J.,
M. L. Pace,
N. F. Caraco, and G. S. Steinhart.
1993.
Bacterial biomass and cell size distributions in lakes: more and larger cells in anoxic waters.
Limnol. Oceanogr.
38:1627-1632.
|
| 8.
|
Faude, U. C., and M. G. Höfle.
1997.
Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems.
Appl. Environ. Microbiol.
63:4534-4542[Abstract].
|
| 9.
|
González, J. M.,
E. B. Sherr, and B. B. Sherr.
1990.
Size-selective grazing on bacteria by natural assemblages of estuarine flagellates and ciliates.
Appl. Environ. Microbiol.
56:583-589[Abstract/Free Full Text].
|
| 10.
|
Güde, H.
1989.
The role of grazing on bacteria in plankton succession, p. 337-369.
In
U. Sommer (ed.), Plankton ecology. Succession in plankton communities. Springer, Berlin, Germany
|
| 11.
|
Hahn, M. W., and M. G. Höfle.
1998.
Grazing pressure by a bacterivorous flagellate reverses the relative abundance of Comamonas acidovorans PX54 and Vibrio sp. strain CB5 in chemostat cocultures.
Appl. Environ. Microbiol.
64:1910-1918[Abstract/Free Full Text].
|
| 12.
|
Hahn, M. W.,
E. R. B. Moore, and M. G. Höfle.
1999.
Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla.
Appl. Environ. Microbiol.
65:25-35[Abstract/Free Full Text].
|
| 13.
| Hahn, M. W., E. R. B. Moore, and M. G. Höfle. Role of morphological plasticity in the grazing
defense strategy of the aquatic bacterium Pseudomonas sp.
MWH1. Submitted for publication.
|
| 14.
|
Höfle, M. G.
1983.
Long-term changes in chemostat cultures of Cytophaga johnsonae.
Appl. Environ. Microbiol.
46:1045-1053[Abstract/Free Full Text].
|
| 15.
|
Höfle, M. G.
1988.
Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach.
J. Microbiol. Methods
8:235-248.
|
| 16.
|
Höfle, M. G.
1990.
Transfer RNAs as genotypic fingerprints of eubacteria.
Arch. Microbiol.
153:299-304.
|
| 17.
|
Jürgens, K.
1992.
Is there plenty of food for bacterivorous flagellates in eutrophic waters?
Arch. Hydrobiol. Beih. Ergeb. Limnol.
37:195-205.
|
| 18.
|
Jürgens, K.,
H. Arndt, and K. O. Rothhaupt.
1994.
Zooplankton-mediated changes of bacterial community structure.
Microb. Ecol.
27:27-42.
|
| 19.
|
Jürgens, K., and W. R. DeMott.
1995.
Behavioral flexibility in prey selection by bacterivorous nanoflagellates.
Limnol. Oceanogr.
40:1503-1507.
|
| 20.
|
Jürgens, K.,
J. Pernthaler,
S. Schalla, and R. Amann.
1999.
Morphological and compositional changes in a planktonic bacterial community in response to enhanced protozoan grazing.
Appl. Environ. Microbiol.
65:1241-1250[Abstract/Free Full Text].
|
| 21.
|
Landry, M. R.,
J. M. Lehner-Fournier,
J. A. Sundstrom,
V. L. Fagerness, and K. E. Selph.
1991.
Discrimination between living and heat-killed prey by a marine zooflagellate, Paraphysomonas vestita (Stokes).
J. Exp. Mar. Biol. Ecol.
146:139-151.
|
| 22.
|
Manz, W.,
R. Amann,
W. Ludwig,
M. Wagner, and K.-H. Schleifer.
1992.
Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions.
Syst. Appl. Microbiol.
15:593-600.
|
| 23.
|
Manz, W.,
U. Szewzyk,
P. Ericsson,
R. Amann,
K.-H. Schleifer, and T.-A. Stenström.
1993.
In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes.
Appl. Environ. Microbiol.
59:2293-2298[Abstract/Free Full Text].
|
| 24.
|
Mitchell, G. C.,
J. H. Baker, and M. A. Sleigh.
1988.
Feeding of a freshwater flagellate, Bodo saltans, on diverse bacteria.
J. Protozool.
35:219-222.
|
| 25.
|
Monger, B. C., and M. R. Landry.
1992.
Size-selective grazing by heterotrophic nanoflagellates: an analysis using live-stained bacteria and dual-beam flow cytometry.
Arch. Hydrobiol. Beih. Ergeb. Limnol.
37:173-185.
|
| 26.
| Moore, E. R. B. Unpublished data.
|
| 27.
|
Norland, S.,
M. Heldal, and O. Tumyr.
1987.
On the relation between dry matter and volume of bacteria.
Microb. Ecol.
13:95.
|
| 28.
|
Pace, M. L.
1988.
Bacterial mortality and the fate of bacterial production.
Hydrobiologia
159:41-49.
|
| 29.
|
Pernthaler, J.,
B. Sattler,
K.  imek,
A. Schwarzenbacher, and R. Psenner.
1996.
Top-down effects on the size-biomass distribution of a freshwater bacterioplankton community.
Aquat. Microb. Ecol.
10:255-263.
|
| 30.
|
Pernthaler, J.,
T. Posch,
K. imek,
J. Vrba,
R. Amann, and R. Psenner.
1997.
Contrasting bacterial strategies to coexist with a flagellate predator in an experimental microbial assemblage.
Appl. Environ. Microbiol.
63:596-601[Abstract].
|
| 31.
|
Posch, T.,
K. imek,
J. Vrba,
J. Pernthaler,
J. Nedoma,
B. Sattler,
B. Sonntag, and R. Psenner.
1999.
Predator-induced changes of bacterial size-structure and productivity studied on an experimental microbial community.
Aquat. Microb. Ecol.
18:235-246.
|
| 32.
|
Poulsen, L. K.,
G. Ballard, and D. A. Stahl.
1993.
Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms.
Appl. Environ. Microbiol.
59:1354-1360[Abstract/Free Full Text].
|
| 33.
|
Rosenzweig, R. F.,
R. R. Sharp,
D. S. Treves, and J. Adams.
1994.
Microbial evolution in a simple unstructured environment: genetic differentiation in Escherichia coli.
Genetics
137:903-917[Abstract].
|
| 34.
|
Rosset, R.,
J. Julien, and R. Monier.
1966.
Ribonucleic acid composition of bacteria as a function of growth rate.
J. Mol. Biol.
18:308-320[Medline].
|
| 35.
|
Sanders, R. W.,
D. A. Caron, and U.-G. Berninger.
1992.
Relationships between bacteria and heterotrophic nanoplankton in marine and fresh waters: an inter-ecosystem comparison.
Mar. Ecol. Prog. Ser.
86:1-14.
|
| 36.
|
Sanders, R. W.,
K. G. Porter,
S. J. Bennett, and A. D. DeBiase.
1989.
Seasonal patterns of bacterivory by flagellates, ciliates, rotifers and cladocerans in a freshwater planktonic community.
Limnol. Oceanogr.
34:673-687.
|
| 37.
|
Schaechter, M.,
O. Maaløe, and N. O. Kjeldgaard.
1958.
Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium.
J. Gen. Microbiol.
19:592-606[Abstract/Free Full Text].
|
| 38.
|
Schleifer, K. H.,
R. Amann,
W. Ludwig,
C. Rothemund,
N. Springer, and S. Dorn.
1992.
Nucleic acid probes for the identification and in situ detection of pseudomonads, p. 127-134.
In
E. Galli, S. Silver, and B. Witholt (ed.), Pseudomonas: molecular biology and biotechnology. American Society for Microbiology, Washington, D.C.
|
| 39.
|
imek, K., and T. H. Chrzanowski.
1992.
Direct and indirect evidence of size-selective grazing on pelagic bacteria by freshwater nanoflagellates.
Appl. Environ. Microbiol.
58:3715-3720[Abstract/Free Full Text].
|
| 40.
|
imek, K.,
J. Vrba,
J. Pernthaler,
T. Posch,
P. Hartman,
J. Nedoma, and R. Psenner.
1997.
Morphological and compositional shifts in an experimental bacterial community influenced by protists with contrasting feeding modes.
Appl. Environ. Microbiol.
63:587-595[Abstract].
|
| 41.
| imek, K., P. Kojecka, J. Nedoma, P. Hartman, J. Vrba, and J. R. Dolan. Shifts in the bacterial community
composition associated with different microzooplankton size fractions
in a eutrophic reservoir. Limnol. Oceanogr., in press.
|
| 42.
|
Simon, M.
1987.
Biomass and production of small and large free-living and attached bacteria in Lake Constance.
Limnol. Oceanogr.
32:591-607.
|
| 43.
|
Simon, M., and F. Azam.
1989.
Protein content and protein synthesis rates of planktonic marine bacteria.
Mar. Ecol. Prog. Ser.
51:201-213.
|
| 44.
|
Vaneechoutte, M.,
R. Rossau,
P. De Vos,
M. Gillis,
D. Janssens,
N. Paepe,
A. De Rouck,
T. Fiers,
G. Claeys, and K. Kersters.
1992.
Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA).
FEMS Microbiol. Lett.
93:227-234.
|
| 45.
| Weinbauer, M. G. Unpublished data.
|
| 46.
|
Weisse, T.
1997.
Growth and production of heterotrophic nanoflagellates in a meso-eutrophic lake.
J. Plankton Res.
19:703-722.
[Abstract/Free Full Text] |
| 47.
|
Weisse, T., and H. Müller.
1998.
Planktonic protozoa and the microbial food web in Lake Constance.
Arch. Hydrobiol. Spec. Issues Adv. Limnol.
53:223-254.
|
Applied and Environmental Microbiology, November 1999, p. 4863-4872, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Tarao, M., Jezbera, J., Hahn, M. W.
(2009). Involvement of Cell Surface Structures in Size-Independent Grazing Resistance of Freshwater Actinobacteria. Appl. Environ. Microbiol.
75: 4720-4726
[Abstract]
[Full Text]
-
Young, K. D.
(2006). The Selective Value of Bacterial Shape. Microbiol. Mol. Biol. Rev.
70: 660-703
[Abstract]
[Full Text]
-
Corno, G., Jurgens, K.
(2006). Direct and Indirect Effects of Protist Predation on Population Size Structure of a Bacterial Strain with High Phenotypic Plasticity. Appl. Environ. Microbiol.
72: 78-86
[Abstract]
[Full Text]
-
Pernthaler, J., Amann, R.
(2005). Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations. Microbiol. Mol. Biol. Rev.
69: 440-461
[Abstract]
[Full Text]
-
Brehm-Stecher, B. F., Johnson, E. A.
(2004). Single-Cell Microbiology: Tools, Technologies, and Applications. Microbiol. Mol. Biol. Rev.
68: 538-559
[Abstract]
[Full Text]
-
Fu, Y., O'Kelly, C., Sieracki, M., Distel, D. L.
(2003). Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins. Appl. Environ. Microbiol.
69: 6848-6855
[Abstract]
[Full Text]
-
Hahn, M. W.
(2003). Isolation of Strains Belonging to the Cosmopolitan Polynucleobacter necessarius Cluster from Freshwater Habitats Located in Three Climatic Zones. Appl. Environ. Microbiol.
69: 5248-5254
[Abstract]
[Full Text]
-
Feris, K., Ramsey, P., Frazar, C., Moore, J. N., Gannon, J. E., Holben, W. E.
(2003). Differences in Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient. Appl. Environ. Microbiol.
69: 5563-5573
[Abstract]
[Full Text]
-
Brummer, I. H. M., Felske, A., Wagner-Dobler, I.
(2003). Diversity and Seasonal Variability of {beta}-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning. Appl. Environ. Microbiol.
69: 4463-4473
[Abstract]
[Full Text]
-
Beardsley, C., Pernthaler, J., Wosniok, W., Amann, R.
(2003). Are Readily Culturable Bacteria in Coastal North Sea Waters Suppressed by Selective Grazing Mortality?. Appl. Environ. Microbiol.
69: 2624-2630
[Abstract]
[Full Text]
-
Crump, B. C., Kling, G. W., Bahr, M., Hobbie, J. E.
(2003). Bacterioplankton Community Shifts in an Arctic Lake Correlate with Seasonal Changes in Organic Matter Source. Appl. Environ. Microbiol.
69: 2253-2268
[Abstract]
[Full Text]
-
Hahn, M. W., Lunsdorf, H., Wu, Q., Schauer, M., Hofle, M. G., Boenigk, J., Stadler, P.
(2003). Isolation of Novel Ultramicrobacteria Classified as Actinobacteria from Five Freshwater Habitats in Europe and Asia. Appl. Environ. Microbiol.
69: 1442-1451
[Abstract]
[Full Text]
-
Eguchi, M., Ostrowski, M., Fegatella, F., Bowman, J., Nichols, D., Nishino, T., Cavicchioli, R.
(2001). Sphingomonas alaskensis Strain AFO1, an Abundant Oligotrophic Ultramicrobacterium from the North Pacific. Appl. Environ. Microbiol.
67: 4945-4954
[Abstract]
[Full Text]
-
Pernthaler, J., Posch, T., Simek, K., Vrba, J., Pernthaler, A., Glöckner, F. O., Nübel, U., Psenner, R., Amann, R.
(2001). Predator-Specific Enrichment of Actinobacteria from a Cosmopolitan Freshwater Clade in Mixed Continuous Culture. Appl. Environ. Microbiol.
67: 2145-2155
[Abstract]
[Full Text]
-
Matz, C., Jürgens, K.
(2001). Effects of Hydrophobic and Electrostatic Cell Surface Properties of Bacteria on Feeding Rates of Heterotrophic Nanoflagellates. Appl. Environ. Microbiol.
67: 814-820
[Abstract]
[Full Text]
Top
Abstract
Introduction
