Growth patterns of two marine isolates: adaptations to substrate patchiness?
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.
- SourceAvailable from: Josefa Antón[show abstract] [hide abstract]
ABSTRACT: In a previous study (S. G. Acinas, F. Rodríguez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27-40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 micrometer. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria, Alteromonas macleodii, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated gamma Proteobacteria predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the alpha Proteobacteria of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the A. macleodii cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.Applied and Environmental Microbiology 03/1999; 65(2):514-22. · 3.68 Impact Factor
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ABSTRACT: In his commentary Azam summarizes new work indicating that microbes in the ocean lead much different lives than we had supposed. They ingest both dissolved and particulate carbon and colonize particles of marine snow and other detritus. They can have enormous influence on the overall carbon flux in the ocean's ecosystem.Science 04/1998; 280(5364):694-696. · 31.20 Impact Factor
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ABSTRACT: Two hundred and eighteen strains of nonfermentative marine bacteria were submitted to an extensive morphological, physiological, and nutritional characterization. All the strains were gram-negative, straight or curved rods which were motile by means of polar or peritrichous flagella. A wide variety of organic substrates served as sole sources of carbon and energy. The strains differed extensively in their nutritional versatility, being able to utilize from 11 to 85 carbon compounds. Some strains had an extracellular amylase, gelatinase, lipase, or chitinase and were able to utilize n-hexadecane and to denitrify. None of the strains had a yellow, cell-associated pigment or a constitutive arginine dihydrolase system, nor were they able to hydrolyze cellulose or agar. The results of the physiological and nutritional characterization were submitted to a numerical analysis which clustered the strains into 22 groups on the basis of phenotypic similarities. The majority of these groups were separable by a large number of unrelated phenotypic traits. Analysis of the moles per cent guanine plus cytosine (GC) content in the deoxyribonucleic acid of representative strains indicated that the peritrichously flagellated groups had a GC content of 53.7 to 67.8 moles%; polarly flagellated strains had a GC content of 30.5 to 64.7 moles%. The peritrichously flagellated groups were assigned to the genus Alcaligenes. The polarly flagellated groups, which had a GC content of 43.2 to 48.0 moles%, were placed into a newly created genus, Alteromonas; groups which had a GC content of 57.8 to 64.7 moles% were placed into the genus Pseudomonas; and the remaining groups were left unassigned. Twelve groups were given the following designations: Alteromonas communis, A. vaga, A. macleodii, A. marinopraesens, Pseudomonas doudoroffi, P. marina, P. nautica, Alcaligenes pacificus, A. cupidus, A. venustus, and A. aestus. The problems of assigning species of aerobic marine bacteria to genera are discussed.Journal of Bacteriology 05/1972; 110(1):402-29. · 3.19 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Sept. 2001, p. 4077–4083 Vol. 67, No. 9
Growth Patterns of Two Marine Isolates: Adaptations
to Substrate Patchiness?
ANNELIE PERNTHALER, JAKOB PERNTHALER,* HEIKE EILERS, AND RUDOLF AMANN
Max-Planck-Institut fu ¨r marine Mikrobiologie, D-28359 Bremen, Germany
Received 3 May 2001/Accepted 26 June 2001
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 Pseudoaltero-
monas 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.
Prefiltration and confinement of marine bacterioplankton
during enrichments (8, 43), dilutions (13), and enclosure ex-
periments (36, 37) can result in changes of both taxonomic
composition and phenotypic features of communities. The per-
centage 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 bacterio-
Are those microbes that are not enriched in bottles or en-
closures 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 (al-
pha-proteobacteria), Oceanospirillum sp. (gamma-proteobac-
teria), 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 sub-
strates and bacterial productivity in coastal pelagic environ-
ments are distributed in microscale patches of variable con-
centration 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 spe-
cies or phylogenetic lineages within the bacterioplankton may
consequently differ in their ability to succeed in habitats with
steeper or flatter substrate gradients. We therefore hypothe-
sized 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 micros-
copy are tools to study growth-related microbial cell features,
e.g., size and macromolecular content, both in whole commu-
nities (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 main-
tain a high rRNA content (i.e., excess protein synthesis capac-
ity) during nongrowth to be able to rapidly respond to changes
in growth condition (10, 12). If this hypothesis is correct, bac-
terial strains that exhibit contrasting patterns of per-cell ribo-
some 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 gam-
ma-proteobacterial genera that had exhibited contrasting re-
sponses during substrate-amended enrichments of environ-
mental 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
* Corresponding author. Mailing address: Max-Planck-Institut fu ¨r
Marine Mikrobiologie, Celsiusstrasse 1, D-28359 Bremen, Germany.
Phone: 49 421 2028 940. Fax: 49 421 2028 580. E-mail: jperntha@mpi
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.
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 Oceano-
spirillum 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 approx-
imately 105cells 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 fro-
zen (?80°C) until further processing.
For the competition experiments, strains were inoculated at densities of ap-
proximately 0.5 ? 105cells 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 asep-
tically 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-?m 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). Depend-
ing on cell concentration, data were acquired for a few seconds to several
minutes. Measurements were excluded from the evaluation of fluorescence in-
tensities if a significant drift of signal during the acquisition period was detected.
Analysis of samples from the first experimental vessel revealed instrument in-
stabilities; 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. pop-
ulations, 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
?m; 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 an-
tifading 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 ?m; 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-?m-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 micro-
scopic 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-?m 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 sub-
tracted 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 measure-
ment (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.
Pure culture batch growth. Following transfer to fresh me-
dium, the lag phase of Pseudoalteromonas sp. (defined as the
period between inoculation and the first doubling of cell num-
bers) was significantly shorter (9 [?1] h) than that of Oceano-
spirillum 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
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)
Abundances in enrichments (105cells ml?1) (mean ?SD)
during incubation period of:
FISH detectability during starvation (% of total cells)
(mean ? range) during incubation period of:
0 h24 h48 h0 days35 days53 days
4 (?2.6) 0.06 (?0.2) 0.33 (?0.72) 4 (?0.6)
ab.l., below limit for FISH counting (?1% total counts).
bOceanospirillum sp. data from reference 8.
4078PERNTHALER ET AL.APPL. ENVIRON. MICROBIOL.
100 h. At the onset of stationary phase, Pseudoalteromonas sp.
had 18% of the cell density and 17% of the biomass of Oceano-
Relative 16S rRNA concentration per cell. Mean per-cell
fluorescence after quantitative FISH with the 16S rRNA-tar-
geted 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 de-
tectable, and maximal RNA fluorescence intensity was approx-
imately 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 Pseudoalteromo-
nas 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.
DNA and protein fluorescence and cell sizes. Both organ-
isms 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 cell?1to 164 (?6)
fg cell?1and that of Pseudoalteromonas sp. ranged from 53
(?8) to 98 (?2) fg cell?1, respectively. Maximum protein con-
tent 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 fluo-
rescence 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.
populationeven during stationary
Pseudoalteromonas sp., the high-DNA subpopulation declined
to less than 5% in stationary-phase cells.
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 Oceanospiril-
lum sp. confirmed the purity of the culture (data not shown).
FIG. 1. Batch growth of Oceanospirillum sp. and Pseudoalteromo-
nas sp. in two separate experiments.
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.
FIG. 3. Mean per-cell DNA and protein content of Oceanospiril-
lum sp. (a) and Pseudoalteromonas sp. (b) during batch growth in pure
cultures. a.u., arbitrary units.
VOL. 67, 2001 GROWTH PATTERNS OF TWO MARINE ISOLATES4079
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, Scheffe ´ post hoc
comparisons, P ? 0.05). No such subpopulations were ob-
served 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 Oceano-
spirillum 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
Competition between Pseudoalteromonas sp. and Oceanospi-
rillum sp. Cocultures of the two strains always reached lower
total cell densities ([5.8 ? 1.1] ? 106cells ml?1[mean ?
standard deviation]; n ? 6) than the pure cultures of either
strain. During coculture, Oceanospirillum sp. and Pseudoaltero-
monas 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 sub-
strates were added at intervals, Oceanospirillum sp. reached
higher maximal cell densities than Pseudoalteromonas sp. Dur-
ing extended batch growth, Pseudoalteromonas sp. entered sta-
tionary phase when 30% of the total substrate had been added
to the medium. It reached only 40% of the cell numbers at-
tained 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 ex-
tended 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).
Facultative eutrophic bacteria. Marine bacteria are fre-
quently categorized into oligotrophic and eutrophic species.
The latter are described as readily culturable, rare in bacterio-
plankton and prone to increase substantially in cell volume
upon addition of substrate (40). The eutrophic bacterial strat-
egy may represent the dominant type in some habitats, e.g.,
brackish waters (30), but common eutrophic isolates were gen-
erally rare in North Sea bacterioplankton (9). According to the
above definition, both Pseudoalteromonas sp. and Oceanospi-
rillum 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 meta-
zoans (18). Growth features that are commonly attributed to
the opportunistic bacterial strategists were clearly more pro-
nounced 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 conclu-
sions about the ecological role of the two genera in North Sea
coastal waters. Nevertheless, our results provide a model for
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.
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.
4080PERNTHALER ET AL.APPL. ENVIRON. MICROBIOL.
understanding the outcome of our previous enrichment exper-
iments on the same substrate mix (8) and illustrate the poten-
tial effects of substrate gradients on a two-species coculture
The stationary-phase subpopulation with a high DNA con-
tent 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 ma-
rine 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 com-
munity (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 phylo-
genetically closest relative of Oceanospirillum sp. strain
KT0923, O. commune, was isolated from tropical surface wa-
ters (4). In coastal North Sea plankton, free-living bacteria
related to Oceanospirillum sp. could be visualized in low den-
sities (5 ? 103cells ml?1) by FISH (8). The genus apparently
includes culturable strains that are also present in the bacte-
rioplankton and that are not oligotrophic by current definition
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 ad-
dition (Fig. 6) suggests that cell multiplication mainly origi-
nated 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 Oceano-
spirillum sp. might thus be part of a more complex life strategy
and, e.g., play a role in the colonization of new substrate
Quantification of FISH staining intensities. Quantitative
measurements of fluorescence intensities after FISH staining
and image-analyzed microscopy yield two parameters as a po-
tential measure of the 16S rRNA content per cell, the mean
object gray value (optical brightness [O.B.]) and the total ob-
ject 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
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.
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.
VOL. 67, 2001 GROWTH PATTERNS OF TWO MARINE ISOLATES 4081
I.O.B. are not expected to change completely in parallel during
The O.B., on the other hand, is related to rRNA concentra-
tion, 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 inde-
pendence 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 dur-
ing balanced growth (26, 34).
The two bacterial strains studied clearly differed in their
patterns of cellular 16S rRNA concentration during the vari-
ous 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 mainte-
nance 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 hypoth-
esis. In contrast, the ribosome concentration of Oceanospiril-
lum 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 concen-
trations 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)
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 concen-
tration during batch growth. For example, it is presently un-
known 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 me-
dium provided optimal growth conditions for the studied mi-
Growth in cocultures. Numerous bacteria, including several
Pseudoalteromonas species, are known to inhibit other micro-
organisms 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 pop-
ulations 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 produc-
tion 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., com-
pared 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 andmixedcultures (Fig.
Pseudoalteromonas sp. probably consumed the bulk of avail-
able 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 Pseudoalteromo-
nas sp. in extended batch cocultures (Mann-Whitney U test,
n ? 8, P ? 0.01) (Fig. 7b) is therefore most likely the conse-
quence 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 Oceanospi-
rillum 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, nei-
ther 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
Pseudoalteromonas sp. strain, moreover, maintained high sta-
tionary-phase levels of cellular rRNA, which has been pre-
dicted for marine bacteria with a more opportunistic life strat-
4082PERNTHALER ET AL. APPL. ENVIRON. MICROBIOL.
egy. 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 en-
richment might, therefore, provide a tool for the directed iso-
lation of bacteria that are otherwise rapidly overgrown.
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.
1. Acinas, S. G., J. Anto ´n, and F. Rodrı ´guez-Valera. 1999. Diversity of free-
living and attached bacteria in offshore Western Mediterranean waters as
depicted by analysis of genes encoding 16S rRNA. Appl. Environ. Microbiol.
2. Amann, R. I., W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identifi-
cation and in situ detection of individual microbial cells without cultivation.
Microbiol. Rev. 59:143–169.
3. Azam, F. 1998. Microbial control of oceanic carbon flux: the plot thickens.
4. Baumann, L., P. Baumann, M. Mandel, and R. D. Allen. 1972. Taxonomy of
aerobic marine eubacteria. J. Bacteriol. 110:402–429.
5. Binder, B. J., and Y. C. Liu. 1998. Growth rate regulation of rRNA content
of a marine Synechococcus (cyanobacterium) strain. Appl. Environ. Micro-
6. Blackburn, N., T. Fenchel, and J. Mitchell. 1998. Microscale nutrient patches
in planktonic habitats shown by chemotactic bacteria. Science 282:2254–
7. DeLong, E. F., D. G. Franks, and A. L. Alldredge. 1993. Phylogenetic diver-
sity of aggregate-attached vs. free-living marine bacterial assemblages. Lim-
nol. Oceanogr. 38:924–934.
8. Eilers, H., J. Pernthaler, and R. Amann. 2000. Succession of pelagic marine
bacteria during enrichment: a close look on cultivation-induced shifts. Appl.
Environ. Microbiol. 66:4634–4640.
9. Eilers, H., J. Pernthaler, F. O. Glo ¨ckner, and R. Amann. 2000. Culturability
and in situ abundance of pelagic bacteria from the North Sea. Appl. Environ.
10. Fegatella, F., J. Lim, S. Kjelleberg, and R. Cavicchioli. 1998. Implications of
rRNA operon copy number and ribosome content in the marine oligotrophic
ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Environ. Mi-
11. Ferguson, R. L., E. N. Buckley, and A. V. Palumbo. 1984. Response of
marine bacterioplankton to differential filtration and confinement. Appl.
Environ. Microbiol. 47:49–55.
12. Fla ¨rdh, K., P. S. Cohen, and S. Kjelleberg. 1992. Ribosomes exist in large
excess over the apparent demand for protein synthesis during carbon star-
vation in marine Vibrio sp. strain CCUG 15956. J. Bacteriol. 174:6780–6788.
13. Fuchs, B. M., M. V. Zubkov, K. Sahm, P. H. Burkill, and R. Amann. 2000.
Changes in community composition during dilution cultures of marine bac-
terioplankton as assessed by flow cytometric and molecular biological tech-
niques. Environ. Microbiol. 2:191–201.
14. Gasol, J. M., and X. A. G. Mora ´n. 1999. Effects of filtration on bacterial
activity and picoplankton community structure as assessed by flow cytometry.
Aquat. Microb. Ecol. 16:251–264.
15. Gasol, J. M., U. L. Zweifel, F. Peters, J. A. Fuhrman, and A˚. Hagstro ¨m. 1999.
Significance of size and nucleic acid content heterogeneity as measured by
flow cytometry in natural planktonic bacteria. Appl. Environ. Microbiol.
16. Giuliano, L., E. De Domenico, M. G. Ho ¨fle, and M. M. Yakimov. 1999.
Identification of culturable oligotrophic bacteria within naturally occurring
bacterioplankton communities of the Ligurian Sea by 16S rRNA sequencing
and probing. Microb. Ecol. 37:77–85.
17. Glo ¨ckner, F. O., R. Amann, A. Alfreider, J. Pernthaler, R. Psenner, K.
Trebesius, and K.-H. Schleifer. 1996. An in situ hybridization protocol for
detection and identification of planktonic bacteria. Syst. Appl. Microbiol.
18. Holmstro ¨m, C., and S. Kjelleberg. 1999. Marine Pseudoalteromonas species
are associated with higher organisms and produce biologically active extra-
cellular agents. FEMS Microbiol. Ecol. 30:285–293.
19. Jannasch, H. 1967. Enrichments of aquatic bacteria in continuous culture.
Arch. Mikrobiol. 59:165–173.
20. Joux, F., P. Lebaron, and M. Troussellier. 1997. Changes in cellular states of
the marine bacterium Deleya aquamarina under starvation conditions. Appl.
Environ. Microbiol. 63:2686–2694.
21. Karner, M., and J. A. Fuhrman. 1997. Determination of active marine
bacterioplankton: a comparison of universal 16S rRNA probes, autoradiog-
raphy, and nucleoid staining. Appl. Environ. Microbiol. 63:1208–1213.
22. Kemp, P. F., S. Lee, and J. LaRoche. 1993. Estimating the growth rate of
slowly growing marine bacteria from RNA content. Appl. Environ. Micro-
23. Kerkhof, L., and P. Kemp. 1999. Small ribosomal RNA content in marine
Proteobacteria during non-steady-state growth. FEMS Microbiol. Ecol. 30:
24. Lebaron, P., and F. Joux. 1994. Flow cytometric analysis of the cellular DNA
content of Salmonella typhimurium and Alteromonas haloplanktis during star-
vation and recovery in seawater. Appl. Environ. Microbiol. 60:4345–4350.
25. Li, W. K., J. F. Jellet, and P. M. Dickie. 1995. DNA distributions in plank-
tonic bacteria stained with TOTO or TO-PRO. Limnol. Oceanogr. 40:1485–
26. Moeller, S., C. S. Kristensen, L. K. Poulsen, J. M. Carstensen, and S. Molin.
1995. Bacterial growth on surfaces: automated image analysis for quantifi-
cation of growth-related parameters. Appl. Environ. Microbiol. 61:741–748.
27. Monger, B. C., and M. R. Landry. 1993. Flow cytometric analysis of marine
bacteria with Hoechst 33342. Appl. Environ. Microbiol. 59:905–911.
28. Oerther, D. B., J. Pernthaler, A. Schramm, R. Amann, and L. Raskin. 2000.
Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge
wastewater treatment systems. Appl. Environ. Microbiol. 66:2154–2165.
29. Ouverney, C. C., and J. A. Fuhrman. 2000. Marine planktonic Archaea take
up amino acids. Appl. Environ. Microbiol. 66:4829–4833.
30. Pinhassi, J., and A. Hagstro ¨m. 2000. Seasonal succession in marine bacte-
rioplankton. Aquat. Microb. Ecol. 21:245–256.
31. Ploug, H., H. P. Grossart, F. Azam, and B. B. Jorgensen. 1999. Photosyn-
thesis, respiration, and carbon turnover in sinking marine snow from surface
waters of Southern California Bight: implications for the carbon cycle in the
ocean. Mar. Ecol. Prog. Ser. 179:1–11.
32. Poindexter, J. S. 1981. Oligotrophy: feast and famine existence. Adv. Microb.
33. Posch, T., J. Pernthaler, A. Alfreider, and R. Psenner. 1997. Cell-specific
respiratory activity of aquatic bacteria studied with the tetrazolium reduction
method, cyto-clear slides, and image analysis. Appl. Environ. Microbiol.
34. 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.
35. Ruimy, R., V. Breittmayer, V. Boivin, and R. Christen. 1994. Assessment of
the state of activity of individual bacterial cells by hybridization with a
ribosomal RNA targeted fluorescent probe. FEMS Microbiol. Ecol. 15:207–
36. Scha ¨fer, H., L. Bernard, C. Courties, P. Lebaron, P. Servais, R. Pukall, E.
Stackebrandt, M. Troussellier, T. Guindulain, J. Vives-Rego, and G.
Muyzer. 2001. Microbial community dynamics in Mediterranean nutrient-
enriched seawater. FEMS Microbiol. Ecol. 34:243–253.
37. Scha ¨fer, H., P. Servais, and G. Muyzer. 2000. Successional changes in the
genetic diversity of a marine assemblage during confinement. Arch. Micro-
38. Schut, F. 1994. Ph. D. thesis. University of Groningen, Groningen, The
39. Schut, F., E. J. De Vries, J. C. Gottschal, B. R. Robertson, W. Harder, R. A.
Prins, and D. K. Button. 1993. Isolation of typical marine bacteria by dilution
culture growth maintenance and characteristics of isolates under laboratory
conditions. Appl. Environ. Microbiol. 59:2150–2160.
40. Schut, F., R. A. Prins, and J. C. Gottschal. 1997. Oligotrophy and pelagic
marine bacteria: facts and fiction. Aquat. Microb. Ecol. 12:177–202.
41. Sherr, E. B., B. F. Sherr, and C. T. Sigmon. 1999. Activity of marine bacteria
under incubated and in situ conditions. Aquat. Microb. Ecol. 20:213–223.
42. Stretton, S., S. J. Danon, S. Kjelleberg, and A. E. Goodman. 1997. Changes
in cell morphology and motility in the marine Vibrio sp. strain S14 during
conditions of starvation and recovery. FEMS Microbiol. Lett. 146:23–29.
43. Suzuki, M. T. 1999. Effect of protistan bacterivory on costal bacterioplank-
ton diversity. Aquat. Microb. Ecol. 20:261–272.
44. ZoBell, C. E. 1946. Marine microbiology. A monograph on hydrobacteriol-
ogy. Chronica Botanica Company, Waltham, Mass.
45. Zubkov, M. V., B. M. Fuchs, H. Eilers, P. H. Burkill, and R. Amann. 1999.
Determination of total protein content of bacterial cells by SYPRO staining
and flow cytometry. Appl. Environ. Microbiol. 65:3251–3257.
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