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Applied and Environmental Microbiology, September 2001, p. 4077-4083, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4077-4083.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Growth Patterns of Two Marine Isolates: Adaptations
to Substrate Patchiness?
Annelie
Pernthaler,
Jakob
Pernthaler,*
Heike
Eilers, and
Rudolf
Amann
Max-Planck-Institut für marine
Mikrobiologie, D-28359 Bremen, Germany
Received 3 May 2001/Accepted 26 June 2001
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ABSTRACT |
During bottle incubations of heterotrophic marine picoplankton,
some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus
Pseudoalteromonas rapidly multiplied in
substrate-amended North Sea water, whereas the densities of
Oceanospirillum changed little (H. Eilers, J. Pernthaler,
and R. Amann, Appl. Environ. Microbiol. 66:4634-4640, 2000). We
therefore studied the growth patterns of two isolates affiliating with
Pseudoalteromonas and Oceanospirillum in batch
culture. Upon substrate resupply, Oceanospirillum lagged
threefold longer than Pseudoalteromonas but reached more
than fivefold-higher final cell density and biomass. A second, mobile
morphotype was present in the starved Oceanospirillum populations with distinctly greater cell size, DNA and protein content,
and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in
the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the
final cell densities of Oceanospirillum were reduced three times as much as those Pseudoalteromonas, compared to
growth yields in pure cultures. In contrast, the gradual addition of
substrates to stationary-phase cocultures was clearly disadvantageous
for the Pseudoalteromonas population. Different growth
responses to substrate gradients could thus be another facet affecting
the competition between marine bacteria and may help to explain
community shifts observed during enrichments.
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INTRODUCTION |
Prefiltration and confinement of
marine bacterioplankton during enrichments (8, 43),
dilutions (13), and enclosure experiments (36,
37) can result in changes of both taxonomic composition and
phenotypic features of communities. The percentage of cells with higher
per-cell rRNA, DNA, and protein content (8, 13, 15), the
proportion of plate-countable cells (11), and the
proportion of cells exhibiting higher metabolic activity (15, 41) have all been observed to increase. Often the original
community is overgrown by a few genera of frequently cultured marine
gamma-proteobacteria, e.g., Vibrio sp.,
Alteromonas sp., and Pseudoalteromonas sp.
(8, 16, 37), which are, however, most probably not very
abundant members of the bacterioplankton (8).
Are those microbes that are not enriched in bottles or enclosures in
principle unable to grow on the offered substrates? The majority of
pelagic bacteria and archaea are capable of incorporating mixes of
radiolabeled amino acids (21, 29). In previous works, strains related to the genera Roseobacter
(alpha-proteobacteria), Oceanospirillum sp.
(gamma-proteobacteria), and Cytophaga sp. (Bacteroidetes), were isolated from North Sea water
samples on a substrate mix of amino acids and monomers, yet members of
these lineages were not enriched from North Sea plankton during
incubations of filtrates on the same substrates (8, 9).
Bacterioplankton community change upon filtration and/or substrate
addition may thus be a consequence of other features of the enriched
populations, rather than of the ability to utilize a particular
substrate. A considerable proportion of the substrates and bacterial
productivity in coastal pelagic environments are distributed in
microscale patches of variable concentration and size, such as algal
"phycospheres," marine snow, or metazoan fecal pellets (3,
31). The particle-attached and free-living pelagic communities
differ both in phenotypes and in taxonomic composition (1,
7). Individual microbial species or phylogenetic lineages within
the bacterioplankton may consequently differ in their ability to
succeed in habitats with steeper or flatter substrate gradients. We
therefore hypothesized that bacteria which exhibit a more rapid growth
response under batch culture "feast-and-famine" conditions (32) are also favored during enrichments of environmental samples.
Flow cytometry and image-analyzed epifluorescence microscopy are tools
to study growth-related microbial cell features, e.g., size and
macromolecular content, both in whole communities (13, 14, 25,
33, 45) and in individual populations (5, 20). For
example, a high per-cell ribosome content is generally regarded as a
feature of active bacteria in mixed assemblages (2). Pure culture studies show a dependence of total ribosome content on growth
rate in continuous cultures (5, 22, 26, 34). Furthermore, it has been suggested that some bacteria maintain a high rRNA content
(i.e., excess protein synthesis capacity) during nongrowth to be able
to rapidly respond to changes in growth condition (10, 12). If this hypothesis is correct, bacterial strains that
exhibit contrasting patterns of per-cell ribosome concentration during early stationary phase should also differ in their competition for more
or less patchy substrates.
Batch growth experiments with two marine isolates were performed in
pure culture and coculture on low concentrations of organic carbon. The
selected strains are affiliated with gamma-proteobacterial genera that
had exhibited contrasting responses during substrate-amended enrichments of environmental samples in an earlier study
(8) (Table 1). Cell numbers
and sizes and the patterns of rRNA, total nucleic acid, and protein
content per cell were followed during the different growth phases in
pure cultures. The population sizes of the two strains were then
monitored in cocultures to which substrates were either added in one
batch or gradually.
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TABLE 1.
Abundances of Pseudoalteromonas sp. and
Oceanospirillum sp. in enrichments of North Sea filtrates
and FISH detectability of the two studied strains during long-term
starvation (for details, see reference 8)
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MATERIALS AND METHODS |
Batch cultures of single strains.
Growth experiments were
carried out on a synthetic medium previously used for ecophysiological
investigations on a marine Sphingomonas sp.
(39). A mix of monomers and amino acids as described by
Eilers et al. (9) was added to the medium at micromolar concentrations. The two marine strains used in this study,
Oceanospirillum sp. strain KT0923 and
Pseudoalteromonas sp. strain KT0912.10 (45), were both isolated from surface waters in the German Bight of the North
Sea (9). According to 16S rDNA gene sequence analysis, they are phylogenetically most closely affiliated with
Oceanospirillum commune and Pseudoalteromonas
atlantica (95.7 and 99.7% rDNA similarity, respectively). Prior
to the experiments, both strains were maintained on liquid medium for
several growth cycles. Six days after their last reinoculation, 4 liters of freshly prepared medium was inoculated at initial densities
of approximately 105 cells ml
1. Incubations
were performed in two parallels at 15°C and with gentle stirring.
Fifty-ml subsamples were taken at 30-min to 2-h intervals for the first
58 h and at longer intervals thereafter, were fixed for 30 min
with formaldehyde solution (final concentration, 2% [vol/vol]), and
were stored frozen (
80°C) until further processing.
For the competition experiments, strains were inoculated at densities
of approximately 0.5 × 10
5 cells ml
1.
In one set of treatments ("batch cocultures"), substrates were
present in the medium at the time of inoculation. In a second
set
("extended batch cocultures"), portions of the substrate mix
(1%
of total) were added hourly to the medium by a peristaltic
pump. In
addition, two controls without substrates were inoculated
with the two
strains. Subsamples were aseptically taken at several
time points and
were treated as described
above.
Flow cytometry.
Samples were analyzed on a FACStar Plus flow
cytometer (Becton Dickinson, Mountain View, Calif.). Cell counts and
DNA and protein quantifications were carried out as previously
described by simultaneous staining with the fluorescent dyes
HOECHST33342 and SYPRO (Molecular Probes, Leiden, The Netherlands) and
by double excitation with UV and green lasers (265 and 543 nm)
(27, 45). Fluorescence was measured with logarithmic
signal amplification. All measurements were standardized to the
fluorescence of latex beads (FluoroSpheres, yellow green, 2-µm
diameter; Molecular Probes) added to each sample at known
concentrations. Absolute bacterial abundances were determined from the
ratios of beads to bacteria. Objects that showed both DNA and protein
fluorescence above background levels were regarded as bacteria. At
least 2,000 such positive events, excluding beads, were recorded per
sample. To avoid errors due to clustering of cells, samples were
sonicated for 5 s prior to measurements (OmniLab sonicator bath;
Bandelin, Berlin, Germany). Depending on cell concentration, data were
acquired for a few seconds to several minutes. Measurements were
excluded from the evaluation of fluorescence intensities if a
significant drift of signal during the acquisition period was detected.
Analysis of samples from the first experimental vessel revealed
instrument instabilities; therefore, DNA and protein fluorescence
intensities were evaluated from samples of the second experimental
vessel only. The relative number of events in the high- and low-DNA
subpopulations was determined for time points when two separate maxima
of DNA fluorescence were readily distinguishable in histogram plots. Within the DNA-rich cell fraction of Oceanospirillum sp.
populations, the frequency of bacteria with a high or low protein
content was quantified during lag phase.
FISH.
Based on the flow cytometry counts, selected time
points of the growth curves were analyzed by fluorescence in situ
hybridization (FISH). Subsamples were filtered onto white membrane
filters (GTTP, diameter, 47 cm; pore size, 0.2 µm; Millipore,
Bedford, Mass.) and were hybridized with the CY3-labeled probe EUB338
(2) for quantitative FISH. Specific probes for
Pseudoalteromonas sp. and Oceanospirillum sp.
(9) were used to evaluate the competition experiment.
Hybridization and washing buffers were composed as described previously
(9, 17). To minimize differences between quantitative
hybridizations, the handling time between incubation and washing was
standardized. All filter sections from a complete time series were
hybridized simultaneously in one single batch of hybridization buffer.
Samples were air dried and embedded in VectaShield antifading mounting
medium (Vector Laboratories, Burlingame, Calif.).
Image acquisition and analysis.
Gray images of fluorescently
labeled cells were acquired at ×100 magnification on a confocal laser
scanning microscope (LSM 510; Carl Zeiss, Jena, Germany) (calibrated
pixel length, 0.064 µm; 4,096 gray levels). Since the stability of a
laser as excitation light source is superior to that of a mercury arc
bulb, conditions of measurement setups are more readily reproduced.
Probe fluorescence from excitation with a green laser (HeNe, 543 nm)
was recorded at a scanning speed of 30 s. To ensure output stability,
the laser was switched on at least 2 h prior to measurements. To
minimize uncontrolled cell bleaching, microscopic focusing was carried out by rapid prescanning at low laser intensity rather than by illumination with the mercury arc bulb. Background fluorescence was
excluded by appropriate adjustment of the pinhole, which was set to
collect light from a 0.6-µm-thick optical section (corresponding to
the average cell width). This optical sectioning, moreover, provided an
efficient focusing aid during prescanning, as even small deviations
from the optimal focal position resulted in a strong decrease of cell
brightness. We avoided all microscopic fields in which brightness
gradients of stained cells were apparent, because such gradients
probably indicated that the respective filter positions were not
sufficiently horizontal for brightness measurements within a 0.6-µm
slice. A total of 300 to 1,000 individual cells from 10 to 20 images
was analyzed per sample.
Images were processed and measured with the software MetaMorph (version
3.5; Universal Imaging, West Chester, Pa.). Object
edges were
established by Unsharp Masking (
28). The gray image
was
smoothed by a 16- by 16-pixel square, low-pass kernel,
downscaled
to 95% of its original brightness, and subtracted from the
original
image. The resulting image was multiplied by 20, and noise was
reduced by a 5- by 5-pixel neighborhood Median filter. The
edge-enhanced
images from a series were subsequently thresholded
automatically
at a preset intensity (gray value, 200 to 500). The
binary image
served as a mask for size and brightness detection. Edges
were
smoothed by morphological closing, and objects of <25 pixels and
of >1,000 pixels were discarded. Each processed image was examined
and
if required was interactively edited prior to measurement
(exclusion of
irregularly shaped objects and separation of touching
cells). Object
area, perimeter, total, and mean gray values were
recorded. Cell
volumes were calculated from the measured area
and perimeter
(
33). To compensate for potential differences
between
individual hybridization series, a sample from a time
point with a low
standard deviation of mean gray values (
Pseudoalteromonas sp., parallel 2, 100 h) served as the internal standard. In each
series of samples, this internal standard was also hybridized
and
evaluated and brightness values from different hybridization
series
were corrected
accordingly.
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RESULTS |
Pure culture batch growth.
Following transfer to fresh medium,
the lag phase of Pseudoalteromonas sp. (defined as the
period between inoculation and the first doubling of cell numbers) was
significantly shorter (9 [±1] h) than that of
Oceanospirillum sp. (25 [±1] h) (Fig. 1). The highest doubling times of
Pseudoalteromonas sp. and Oceanospirillum sp.
were 2.4 and 3.7 h, respectively. The Pseudoalteromonas
sp. population ceased cell division after 27 (±1) h,
Oceanospirillum sp. after 100 h. At the onset of
stationary phase, Pseudoalteromonas sp. had 18% of the cell
density and 17% of the biomass of Oceanospirillum sp.
Relative 16S rRNA concentration per cell.
Mean per-cell
fluorescence after quantitative FISH with the 16S rRNA-targeted probe
EUB338 was used to estimate changes in rRNA concentrations of
Oceanospirillum sp. and Pseudoalteromonas sp.
during growth (Fig. 2). Both organisms
showed an increase of rRNA content before significant cell
multiplication was detectable, and maximal RNA fluorescence intensity
was approximately double its initial value in both strains. This
maximum occurred during late logarithmic growth in
Oceanospirillum sp. and at the onset of stationary phase in
Pseudoalteromonas sp. The relative per-cell rRNA
concentration of Pseudoalteromonas sp. was significantly elevated during 100 h of nongrowth. In contrast, rRNA fluorescence in Oceanospirillum sp. rapidly decreased to initial values
at the onset of stationary phase.
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FIG. 2.
Mean 16S rRNA concentration of
Oceanospirillum sp. (a) and Pseudoalteromonas sp.
(b) of two separate experiments (means ± 1 standard error). a.u.,
arbitrary units.
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DNA and protein fluorescence and cell sizes.
Both organisms
showed a bell-shaped curve of per-cell protein content during growth
(Fig. 3). The relative protein
fluorescence of Oceanospirillum sp. increased more rapidly
than that of Pseudoalteromonas sp., to about 2.5 times of
its initial value, whereas the maximum protein content of
Pseudoalteromonas sp. was less than double its initial
minimum. During stationary and exponential growth phases, the cellular
protein content of Oceanospirillum sp. ranged from 75 (±15)
fg cell1 to 164 (±6) fg cell1 and that of
Pseudoalteromonas sp. ranged from 53 (±8) to 98 (±2) fg
cell1, respectively. Maximum protein content per cell
during mid-logarithmic growth corresponded with maximal cell volumes
determined from size measurements of hybridized cells (data not shown).
Mean per-cell DNA fluorescence intensity of both organisms
approximately doubled during growth (Fig. 3). Except during
mid-logarithmic growth, two subpopulations with different DNA content
could be readily distinguished in both strains (Fig.
4). The high-DNA fraction represented
about 25% in the Oceanospirillum sp. population even during
stationary phase, whereas in Pseudoalteromonas sp., the high-DNA subpopulation declined to less than 5% in stationary-phase cells.
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FIG. 3.
Mean per-cell DNA and protein content of
Oceanospirillum sp. (a) and Pseudoalteromonas sp.
(b) during batch growth in pure cultures. a.u., arbitrary units.
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FIG. 4.
Relative contribution of the fraction of cells with a
high DNA content (multiple genome copies) in Oceanospirillum
sp. and Pseudoalteromonas sp. The break in the curve
indicates the time period during mid-logarithmic growth where a clear
distinction of two DNA brightness classes was not possible.
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Population heterogeneity of Oceanospirillum sp.
After 1 week of starvation and during lag phase, two distinct cell
types were present in the Oceanospirillum sp. population: a
small, nonmotile rod and a rare, large, fast-moving spirillum. The
latter formed 1 to 2% of all cells at the time of inoculation and was
not apparent during exponential growth or the first 24 h of
stationary phase. FISH with a probe specific for
Oceanospirillum sp. confirmed the purity of the culture
(data not shown). The two subpopulations differed both in their cell
sizes and mean rRNA fluorescence intensity, and the large size classes
exhibited significantly higher rRNA concentrations at the end of lag
phase (Fig. 5a) (analysis of variance,
Scheffé post hoc comparisons, P < 0.05). No such
subpopulations were observed in Pseudoalteromonas sp. (Fig.
5b). Two classes of cells with distinct protein content were
distinguished in the DNA-rich subpopulation of
Oceanospirillum sp. during lag phase (Fig. 6a to
c) but not in
Pseudoalteromonas sp. The Oceanospirillum sp.
subpopulation of DNA-rich cells with distinctively higher protein
fluorescence increased from <2% to 14% ± 2% after substrate
addition and constituted >50% after the first doubling.
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FIG. 5.
Cell size distributions (bars) and distribution of mean
16S rRNA fluorescence (symbols) in different cell size classes
(mean ± 1 standard deviation) at the end of lag phase of
Oceanospirillum sp. (t = 16 h) (a) and of
Pseudoalteromonas sp. (t = 8 h) (b).
Asterisks indicate significant differences between the rRNA brightness
of a size class and the brightness in the size classes of 100 to 200 and/or 200 to 300 pixels (analysis of variance, P < 0.05). a.u., arbitrary units.
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FIG. 6.
(a) Histogram of bimodal distribution of DNA
fluorescence (fl.) in Oceanospirillum sp. at t 16 h. (b) Histogram of bimodal protein fluorescence (fl.) within the
high-DNA fraction of DNA fluorescence in panel a. (c) Relative
abundances of subpopulations with different DNA and protein content in
Oceanospirillum sp. during lag phase (0 to 16 h) and until
the first doubling (25 h). a.u., arbitrary units.
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Competition between Pseudoalteromonas sp. and
Oceanospirillum sp.
Cocultures of the two strains
always reached lower total cell densities ([5.8 ± 1.1] × 106 cells ml1 [mean ± standard
deviation]; n = 6) than the pure cultures of either
strain. During coculture, Oceanospirillum sp. and
Pseudoalteromonas sp. reached 7.5 and 25% of their pure
culture maximum abundances, respectively (Fig.
7). The length of the lag phases and the
duration of exponential growth of both organisms were similar in
cocultures and in pure batch cultures. Thus,
Pseudoalteromonas sp. had already ceased cell division at
the onset of growth of Oceanospirillum sp. (Fig. 7). In both
the batch and extended batch cocultures, where portions of substrates
were added at intervals, Oceanospirillum sp. reached higher
maximal cell densities than Pseudoalteromonas sp. During
extended batch growth, Pseudoalteromonas sp. entered stationary phase when 30% of the total substrate had been added to the
medium. It reached only 40% of the cell numbers attained in the batch
cocultures (Fig. 7b). Total cell counts of Oceanospirillum sp. were similar after 100 h in both treatments. This resulted in
three- to six-times-higher maximal densities of
Oceanospirillum sp. than of Pseudoalteromonas sp.
during extended batch cultivation, whereas the ratio of
Oceanospirillum sp. to Pseudoalteromonas sp. was
1.3 in batch cocultures. No significant growth was observed in
cocultures without substrate addition (data not shown).
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FIG. 7.
Growth of Oceanospirillum sp. and
Pseudoalteromonas sp. in cocultivation experiments. (a)
Batch incubations. (b) Extended batch incubations with gradual
substrate addition during 100 h.
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DISCUSSION |
Facultative eutrophic bacteria.
Marine bacteria are frequently
categorized into oligotrophic and eutrophic species. The latter are
described as readily culturable, rare in bacterioplankton and prone to
increase substantially in cell volume upon addition of substrate
(40). The eutrophic bacterial strategy may represent the
dominant type in some habitats, e.g., brackish waters
(30), but common eutrophic isolates were generally rare in
North Sea bacterioplankton (9). According to the above definition, both Pseudoalteromonas sp. and
Oceanospirillum sp. are eutrophic marine genera.
Although frequently isolated, bacteria affiliating with the genus
Pseudoalteromonas sp. were only occasionally detected on
particles in coastal North Sea waters (
8). High numbers of
Pseudoalteromonas-specific viruses have been observed in
fish
feces (A. Wichels, personal communication), and a number of
species
from this genus are known to be associated with metazoans
(
18).
Growth features that are commonly attributed to the
opportunistic
bacterial strategists were clearly more pronounced in the
studied
Pseudoalteromonas strain, such as the shorter lag
phase upon transfer
to fresh medium, a higher maximal growth rate, and
lower total
cell production (Fig.
1). It should be noticed, however,
that
all these parameters are potentially influenced by the composition
of the cultivation medium. Thus, it would be premature to draw
general
conclusions about the ecological role of the two genera
in North Sea
coastal waters. Nevertheless, our results provide
a model for
understanding the outcome of our previous enrichment
experiments on the
same substrate mix (
8) and illustrate the
potential
effects of substrate gradients on a two-species coculture
system.
The stationary-phase subpopulation with a high DNA content in
Pseudoalteromonas sp. was significantly smaller
(Mann-Whitney
U test,
P < 0.001) than in
Oceanospirillum sp., where it comprised
roughly 25% of all
cells (Fig.
4). Two other marine isolates also
maintained large
DNA-rich subpopulations in pure culture even
during extended periods of
starvation (
20,
24). This contrasts
somewhat with the view
that the fraction of bacteria with a high
DNA content found in pelagic
microbial assemblages is representative
of the growing part of the
community (
14,
25). Presently we
can only speculate if and
how the size of the high-DNA fraction
during nongrowth is related to
cultivation conditions or to the
growth strategy of a
population.
Marine spirilla have been known for several decades both from
cultivation (
44) and in situ observations
(
19). The phylogenetically
closest relative of
Oceanospirillum sp. strain KT0923,
O. commune,
was isolated from tropical surface waters (
4). In coastal
North
Sea plankton, free-living bacteria related to
Oceanospirillum sp. could be visualized in low densities
(5 × 10
3 cells ml
1) by FISH
(
8). The genus apparently includes culturable strains
that
are also present in the bacterioplankton and that are not
oligotrophic
by current definition (
40).
A second phenotype was present in starved
Oceanospirillum
sp. cultures, which was clearly separated from the majority of cells
by
size, higher protein content, motility, and per-cell rRNA concentration
(Fig.
5a and
6c). The rapid increase of such cells in stationary
Oceanospirillum sp. after substrate addition (Fig.
6)
suggests
that cell multiplication mainly originated from within this
subpopulation.
Such heterogeneous growth has been observed before in
marine bacteria.
Upon substrate resupply, only a small fraction of a
nongrowing
Vibrio sp. population regained motility prior to
cell multiplication
(
42). The starvation-induced
motile subpopulation in
Oceanospirillum sp. might thus be
part of a more complex life strategy and, e.g.,
play a role in the
colonization of new substrate patches (
6).
Quantification of FISH staining intensities.
Quantitative
measurements of fluorescence intensities after FISH staining and
image-analyzed microscopy yield two parameters as a potential measure
of the 16S rRNA content per cell, the mean object gray value (optical
brightness [O.B.]) and the total object gray value (integrated
optical brightness [I.O.B.]). I.O.B. is the sum of fluorescence
intensities of every positive pixel of a digitized image of a cell. The
O.B. is the I.O.B. divided by the number of positive pixels, i.e., the
mean pixel intensity.
The total amount of rRNA per cell that can be determined in chemical
assays (
22,
23), slot blot hybridizations
(
28),
or flow cytometry evaluation of FISH-stained cells
(
5) is proportional
to the sum of ribosomes per cell and
is therefore equivalent to
the I.O.B. of a hybridized cell. In batch
culture studies, I.O.B.
might be of limited use, because the bacterial
cell volume substantially
influences the total amount of ribosomes per
cell. Thus, fluctuations
in I.O.B. will to a large extent reflect
changes in cell volume
(
35), even though the mean cell
size and I.O.B. are not expected
to change completely in parallel
during batch
growth.
The O.B., on the other hand, is related to rRNA concentration, i.e.,
the density of ribosomes per unit of cell volume. The
inherent
advantage of the mean cell fluorescence as a measure
of growth or
protein synthesis potential is therefore its independence
of changes in
cell volume. It has been demonstrated that the cellular
ribosome
concentration (or its equivalent, the I.O.B. divided
by the cell
volume) increases with growth rate both in
Desulfovibrio vulgaris and in
Pseudomonas putida during balanced
growth (
26,
34).
The two bacterial strains studied clearly differed in their patterns of
cellular 16S rRNA concentration during the various
phases of their
growth cycle (Fig.
5). During 100 h of stationary
phase, high
ribosome concentrations per cell were observed in
Pseudoalteromonas sp. (Fig.
2b). Such maintenance of excess
rRNA
in a marine
Vibrio sp. during starvation has been
interpreted
as an adaptation to a feast-and-famine existence, to allow
rapid
initiation of protein synthesis upon substrate resupply
(
12).
The more rapid growth response of
Pseudoalteromonas sp. both in
pure culture and in cocultures
(Fig.
7) and its selective enrichment
in substrate-amended plankton
samples (Table
1) provide evidence
for this hypothesis. In contrast,
the ribosome concentration of
Oceanospirillum sp. declined
upon the onset of stationary phase
to the levels of the prestarved
culture. The per-cell rRNA content
of a
Sphingomonas sp.
that is thought to be representative of
the free-living marine bacteria
decreased by 90% upon cessation
of growth (
10). This
development of cellular 16S rRNA concentrations
during batch
cultivation agrees with earlier findings that starvation
periods of
several weeks result in a much more pronounced decline
of FISH
detectability in cultures of
Oceanospirillum sp. than
in
those of
Pseudoalteromonas sp. (
8) (Table
1).
We must, however, caution against overinterpretetion of the observed
differences in ribosome content between the strains.
A higher
measurement frequency might be required to gain a detailed
understanding of the actual development of cellular rRNA content
during
periods of rapid change, e.g., logarithmic growth. More
studies are
required to investigate other aspects which could
potentially affect
the patterns of rRNA concentration during batch
growth. For example, it
is presently unknown if and how the composition
of the cultivation
medium affects the patterns of macromolecular
content. We used an
artificial seawater mix that was specifically
developed for the
isolation of an oligocarbophilic marine
Sphingomonas sp. and
for subsequent ecophysiological investigations (
38,
39),
and this artificial seawater was successfully used for
the isolation of
the two studied strains. Yet this does not prove
that the medium
provided optimal growth conditions for the studied
microbes.
Growth in cocultures.
Numerous bacteria, including several
Pseudoalteromonas species, are known to inhibit other
microorganisms by releasing allelopathic substances (18).
We found no indication for such interactions between the studied
strains. Cell densities of Oceanospirillum sp. decreased
during the first 24 h of nongrowth in the gradual enrichment, but
no such decline was observed during batch cocultures, at higher total
densities of Pseudoalteromonas sp. In contrast, mortality of
stationary-phase Pseudoalteromonas sp. was higher in common
batch culture enrichments. The lower abundances of both populations
added together, compared to the density of either strain in pure
culture (Fig. 1 and 7), rather indicated that cocultivation negatively
affected the growth of both species.
Cocultivation and enrichment mode clearly influenced the growth rates
and total cell production of the two species, but the
duration of both
the lag and of the respective exponential growth
phases was unaffected
by the treatments (Fig.
7). This may allow
predictions about the
performance of particular strains in batch
coculture from parameters
that can be readily determined in pure
culture studies, provided that
cocultivation is performed on the
same
medium.
From the length of the lag phases and the total cell production in pure
cultures, it was predicted that the abundance ratio
of the two strains
in stationary-phase cocultures should be influenced
by the mode of
substrate addition. We hypothesized that
Pseudoalteromonas sp. should dominate in a classic batch enrichment, whereas the
more
slowly but more "efficiently" growing
Oceanospirillum
sp.
(Fig.
1) should be favored in a setup with gradually added
substrates.
This was only partially verified. In batch cocultures the total cell
production of
Oceanospirillum sp. was indeed reduced
to a
much greater extent than that of
Pseudoalteromonas sp.,
compared
to pure cultures (Fig.
1 and
7a). The most obvious advantage
of
Pseudoalteromonas sp. under these conditions was the
shorter growth
delay upon substrate addition, in both pure and mixed
cultures
(Fig.
1 and
7). Therefore,
Pseudoalteromonas sp.
probably consumed
the bulk of available organic matter. On the other
hand,
Oceanospirillum sp. was not only capable of growth on
the fraction of substrate
that was not consumed by
Pseudoalteromonas sp.; it eventually
even reached higher
total densities than the other strain in batch
coculture. This agrees
with the higher total cell production of
Oceanospirillum sp.
in pure culture (Fig.
1).
In contrast, the shorter lag phase of
Pseudoalteromonas sp.
would represent no specific advantage during gradual substrate
addition. The significantly reduced growth of
Pseudoalteromonas sp. in extended batch cocultures
(Mann-Whitney U test,
n = 8,
P < 0.01) (Fig.
7b)
is therefore most likely the consequence of
a lower amount of available
substrate at the onset of cell multiplication.
Less than 20% of the
organic carbon of the batch culture had been
added at that time point.
The gradual addition of substrates to
stationary cocultures of the two
strains did not result in lower
final abundances of
Oceanospirillum sp., and the slopes of cell
increase during
exponential growth of
Oceanospirillum sp. were
unaffected or
even slightly higher in the gradual enrichments
(Fig.
7a and b). In
summary, there is evidence for both strains
that the mode of substrate
addition affected competition between
Pseudoalteromonas sp.
and
Oceanospirillum sp. in batch
coculture.
Conclusions.
Under our specific cultivation conditions,
neither the length of lag phases of the studied strains nor the
duration of logarithmic growth appeared to be affected by
cocultivation. The selective enrichment of Pseudoalteromonas
sp. on a particular substrate mix, as previously observed in pelagic
samples (8), is therefore most likely related to a shorter
growth delay upon addition of these substrates. The
Pseudoalteromonas sp. strain, moreover, maintained high
stationary-phase levels of cellular rRNA, which has been predicted for
marine bacteria with a more opportunistic life strategy. This
hypothesis was supported by the outcome of gradual substrate addition
to cocultures, which resulted in a shift of total cell production
towards Oceanospirillum sp. Gradual enrichment might,
therefore, provide a tool for the directed isolation of bacteria that
are otherwise rapidly overgrown.
| |
ACKNOWLEDGMENTS |
We thank B. Fuchs for fruitful discussions on the topic of
brightness measurements, B. MacGregor for critical reading of the manuscript, and N. Neese for advice on fluorescence staining.
This work was supported by the German Ministry of Education and
Research (BMBF, project BIOLOG) and by the Max Planck Society.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Max-Planck-Institut für Marine Mikrobiologie, Celsiusstrasse 1, D-28359 Bremen, Germany. Phone: 49 421 2028 940. Fax: 49 421 2028 580. E-mail: jperntha{at}mpi-bremen.de.
| |
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Applied and Environmental Microbiology, September 2001, p. 4077-4083, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4077-4083.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
