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Applied and Environmental Microbiology, November 2000, p. 4634-4640, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Succession of Pelagic Marine Bacteria during
Enrichment: a Close Look at Cultivation-Induced Shifts
Heike
Eilers,
Jakob
Pernthaler,* and
Rudolf
Amann
Max-Planck-Institut für Marine
Mikrobiologie, D-28359 Bremen, Germany
Received 28 June 2000/Accepted 29 August 2000
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ABSTRACT |
Enrichment experiments with North Sea bacterioplankton were
performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the
plankton but in a state of dormancy. A metabolic inhibitor of cell
division (nalidixic acid [NA]) was added to substrate-amended (S+)
and unamended (S
) grazer-free seawater samples, and shifts in
community composition and per cell DNA and protein content were
compared with untreated controls. In addition, starvation survival
experiments were performed on selected isolates. Incubations resulted
in rapid community shifts towards typical culturable genera rather than
in the activation of either dormant cells or the original DNA-rich
bacterial fraction. Vibrio spp. and members of the
Alteromonas/Colwellia cluster (A/C) were selectively
enriched in S+ and S, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of
cell populations with distinctively different but generally higher
protein and DNA content in the various treatments. Uncultured dominant
-proteobacteria affiliating with the SAR86 cluster and members of
the culturable genus Oceanospirillum were not enriched or
activated, but there was no indication of substrate-induced cell death,
either. Strains of Vibrio and A/C maintained high ribosome
levels in pure cultures during extended periods of starvation, whereas
Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable -proteobacteria could thus be described as a
"feast and famine" existence involving different activation levels
of substrate concentration.
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INTRODUCTION |
Our knowledge about the phylogenetic
lineages that contribute to the marine bacterioplankton is presently
obtained from three sources: isolation of various bacterial strains
(33, 43), clone libraries of 16S ribosomal DNA (rDNA)
(31, 37, 43), and hybridizations to whole cells or isolated
nucleic acids (22, 33, 38). The results of isolation and of
clone libraries often disagree. During the last decade the discrepancy
between isolation and cloning has commonly been regarded as an
indication of cultivation-induced shifts (4). Yet, since
cloning does not reveal community structure either, this view is
actually based on little experimental evidence. On the contrary,
by using quantitative genome probe hybridizations against
community DNA, some isolates (Sphingomonas and
Caulobacter spp.) have been shown to represent a significant
amount of the total bacterioplankton in brackish Baltic Sea waters
(33). A marine isolate related to Vibrio was
described to exhibit remarkable annual variation in population density,
ranging from undetectably low to 
100 of total community DNA
(38). Is this high relative abundance of typical culturable
bacteria the exception or the rule? In a recent study on North Sea
bacterioplankton (14), we found that the most readily
culturable bacteria on media low in organic carbon, such as
Vibrio, Alteromonas, and
Pseudoalteromonas, did not significantly contribute to
the bacterioplankton community during different seasons, as determined
by fluorescence in situ hybridization (FISH) with specific
oligonucleotide probes. In contrast, a FISH probe targeted to 16S rDNA
clones affiliating with a cosmopolitan -proteobacterial
lineage, SAR86 (1, 14, 18, 31), detected a prominent
fraction (up to 10%) of the microbial community in situ. However, no
corresponding isolates were obtained in spite of extensive cultivation efforts.
It has, however, been claimed that a supposedly typical marine isolate
was undetectable in situ by FISH because of its low per cell ribosome
content (40). This raises the question of whether the
readily culturable bacteria of our previous study were really rare in
situ, or whether they were simply not detectable by fluorescent probes.
If FISH sensitivity limits are interfering with the in situ
quantification of such cells, their "activation" should be
observable during enrichment on substrates successfully used for their
cultivation. If frequently isolated bacteria are, however, found to be
rare in situ, they should then be able to take advantage of
cultivation-associated changes in their environment more rapidly than
their competitors. Monitoring dilutions of North Sea bacterioplankton
with seawater that is free of bacteria by flow cytometry and subsequent
FISH of sorted cells have provided first evidence that members of the
-subclass of the Proteobacteria may indeed be selectively
enriched (17), but it is unknown if those -proteobacteria
were affiliated with typical marine isolates. In this context, the
other side of the observed phylogenetic differences between marine
isolates and rDNA clones needs to be addressed, too: how do so-called
"unculturable" bacteria develop during the early phases of
cultivation attempts or during typical cultivation-associated procedures, such as filtration, confinement, substrate addition, temperature variation, etc.?
We set up enrichments with substrate-amended (S+) and unamended (S)
North Sea water and subsequently analyzed community composition and
changes in bacterial per cell DNA and protein content by FISH and flow
cytometry. The antibiotic nalidixic acid (NA) (27) was added
to half of the treatments. It inhibits prokaryotic DNA replication, yet
allows cells to increase in volume. In our study, NA was not applied
for the quantification of active bacteria. We rather wanted to test if
readily culturable bacteria are frequent but inactive or dormant, and
if consequently their low per cell ribosome content could be the reason
why we found low in situ abundances of such genera by FISH in a
previous study (14). In addition, the FISH detectability of
different -proteobacterial isolates during starvation was monitored.
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MATERIALS AND METHODS |
Sampling site and fixation.
In August 1998, surface water
was collected at a 1-m depth in acid-washed, seawater-prerinsed
50-liter polyethylene containers at station Helgoland Roads (54.09 N,
7.52 E) near the island of Helgoland, which is situated approximately
50 km offshore in the German Bay of the North Sea. Water was stored at
4°C and further processed within approximately 1 h. Samples for
flow cytometry were fixed with formaldehyde (final concentration, 2%
[wt/vol]) and stored frozen. For FISH, portions of 10 to 100 ml of
unfiltered seawater were fixed with formaldehyde (final concentration,
2% [wt/vol]) for several hours, collected on white polycarbonate filters (diameter, 47 mm; pore size, 0.2 µm; type GTTP; Millipore, Eschborn, Germany), and rinsed with distilled water. Filters were stored at 20°C until further processing.
Total cell counts and protein and DNA content per cell.
Determination of total cell numbers and relative DNA and protein
content of bacteria after double staining with Hoechst 33342 and SYPRO
(Molecular Probes, Eugene, Oreg.) was performed by flow cytometry on a
FACStar Plus flow cytometer as described (Becton Dickinson, Mountain
View, Calif.) (48). At least 2,000 Hoechst 33342-positive
cells were counted per sample.
Growth experiments.
For the experimental enrichments,
seawater was gently filtered through cellulose nitrate filters
(diameter, 47 mm; pore size, 1.2 µm; Sartorius AG, Göttingen,
Germany). Half of the prefiltered samples were supplemented with NA (30 mg/liter) (27). Triplicate 150-ml aliquots were incubated at
the in situ temperature (16°C) on a rotation shaker (100 rpm) either
unamended (S) or amended (S+) with a mix of monomers (alanine,
L-aspartate, DL-leucine, L-glutamate, L-ornithine, and
DL-serine [1 µM]; glucose, fructose, galactose,
glycolate, succinate, and mannitol [10 µM]; acetate, lactate,
ethanol, and glycerol [15 µM]). At the beginning of the experiment
and after 20 and 43 h, 10-ml aliquots were fixed for FISH, and
2-ml aliquots were fixed for flow cytometry (see above).
Batch cultures.
For starvation experiments 150-ml triplicate
samples inoculated with either Alteromonas sp. isolate
KT1113 (GenBank accession number AF173965), Oceanospirillum
sp. isolate KT0923 (AF173967), or Vibrio sp. isolate KT0901
(AF172840) (14) were incubated at the in situ temperature
(16°C) on a rotation shaker (100 rpm) in synthetic seawater
(14) to which trace elements, vitamins, and the mix of
monomers used for the field incubation were added. At four time points
within a period of 50 days, 1.5-ml aliquots were fixed for FISH,
immobilized on polycarbonate filters (diameter, 47 mm; pore size, 0.22 µm; type GTTP; Millipore, Eschborn, Germany).
FISH.
Cells on filter sections were hybridized with
group-specific oligonucleotide probes EUB338 (3), ALF968
(20% formamide) (32), GAM42a (30), and CF319a
(29). In addition, probes for subgroups of 
- and
-proteobacteria (Table 1) were used. Counterstaining with 4,6-diamidino-2-phenylindole (DAPI; 1 µg/ml) and
mounting for microscopic evaluation were performed as described previously (3, 21).
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RESULTS |
Changes in community composition.
During a 43-h enrichment,
total cell number increased from 1.6(±0.2) × 106
(mean ± standard deviation, n = 12) cells per ml
by a factor of about 2 in the unamended (S) and about 2.5-fold in the
substrate-amended (S+) samples (Fig. 1).
This difference between S and S+ was not statistically significant
(Student's t test, P > 0.05). After NA
addition, no significant changes in cell numbers occurred during the
first 20 h in both amended and unamended treatments. Total cell
number increased slightly in SNA+ thereafter. In S+NA+, total cell
number almost doubled during the second half of the incubation in spite
of the antibiotic. Detection rates of probe EUB338 ranged around 75% ± 12% (n = 6) in both SNA and SNA+ throughout
the experiment. FISH detection in the substrate-amended treatments
increased from 75% ± 1.5% to 87% ± 1.5% of total cells at the end
of the incubations.
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FIG. 1.
Mean cell numbers of the total bacterial assemblage
(lines) during the enrichment experiments and of cells hybridized with
group-specific fluorescent probes (bars). Solid bars, cells stained
with probe EUB338; dotted bars, -subclass of the
Proteobacteria; open bars, -subclass of the
Proteobacteria; hatched bars, C/F cluster. Error bars
indicate standard deviations (n = 3).
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The amount of cells hybridizing with the group-specific probe for the
-subclass of the
Proteobacteria, GAM42a, increased
from
2.1(±0.4) × 10
5 (
n = 12) cells per
ml 4- to 5-fold and 9- to 10-fold in S
and
S+, respectively. This
effect was enhanced by incubation with
NA. Thirteen percent ± 3%
(
n = 6) of total cells hybridized with
probe GAM42a in
the beginning and 40% (34 to 44%, S
NA+) and 72%
(67 to 76%,
S+NA+) after 43 h of
incubation.
Members of the SAR86 cluster, which are small rods (approximately 0.5 µm in width and 1 µm in length), were detected by FISH
with probe
SAR86-1249. They showed only weak FISH signals and
could not be
enriched during different treatments (Fig.
2). Their
absolute cell numbers remained
constant in both S
NA
and S+NA
treatments (estimated generation
time, 83 h). Incubation with
NA resulted in a continuous decrease
in SAR86 cell numbers within
43 h of incubation. The relative
abundances of this phylogenetic
group dropped from 11.3% (9.0 to
14.1%) to 6.7% (5.2 to 9.1%)
or below the detection level (<1%
DAPI) in the various treatments.
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FIG. 2.
Mean cell numbers of cells hybridized with probes for
various lineages within the - and -proteobacteria in the
different treatments. Error bars without caps indicate ranges of
replicates; error bars with caps indicate standard deviations of
triplicates. Note the different y scale in the bottom
panel.
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Bacteria targeted by the oligonucleotide probe OCE232, specific for the
genus
Oceanospirillum (Table
1), showed only weak
fluorescence and were initially present in small numbers. During
incubation,
Oceanospirillum spp. were not enriched
significantly
in any of the treatments, and their relative abundances
hardly
exceeded the lower limit of FISH detection rates. The
Alteromonas/Colwellia cluster (A/C), as identified by
oligonucleotide probe ALT1413,
showed a different response (Fig.
2).
These bacteria, usually
large cells compared to other marine bacteria,
showed bright FISH
signals. In the beginning of the experiments, they
constituted
approximately 1.5% of total bacteria, but increased
significantly
in the S
, S+, and, during the second half of the
incubation period,
in both NA+ treatments. Concomitant with a rise in
numbers, the
cell volume of these large cells increased even more (Fig.
3).
A/C constituted 6% and 20 ± 2% of total bacteria in S
after 20
and 43 h, respectively. From
abundance changes, we estimated a
generation time (
g) of
9 h. This enrichment was enhanced by the
presence of NA. The
relative abundance of A/C was 33% ± 5% in
the S
NA+ treatment at
the end of the experiment. After a strong
initial increase in absolute
numbers in S+NA
, numbers stagnated
after 20 h of incubation,
resulting in 10% ± 0.2% relative abundance
(
g = 11.8
h). S+NA+ treatments resulted in little increase in
A/C in absolute
numbers.
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FIG. 3.
Relative per cell DNA and protein content (arbitrary
units) of the bacterial assemblages at the beginning and end of the
various treatments.
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Vibrio spp. were enriched more drastically than any other
group during incubation, but only in the S+ treatments (Fig.
2).
The
increase in cell numbers was even stronger in the S+NA+ treatment.
At
the end of the experiments
Vibrio spp. constituted 25% ± 1%
(
g = 6.3 h) and 65% ± 1% of total bacteria in
the S+NA
and S+NA+
treatments,
respectively.
The two other studied groups,
-proteobacteria and
Cytophaga/Flavobacterium (C/F), which constituted 23% (17 to 27%) and 31%
(26 to 34%) of total bacteria in the beginning of
the experiment,
respectively, exhibited much lower growth during the
enrichments.
In absolute numbers, members of the C/F cluster almost
doubled
from 4.9(±0.9) × 10
5 (
n = 8)
to 10.4(±1.5) × 10
5 (
n = 4) cells
per ml in both S
and S+, whereas
-proteobacteria
only grew in the
S
treatments. Both groups decreased little in
their relative
abundances during incubations without NA. In contrast,
-proteobacteria constituted less than half and members of the
C/F
cluster about one third of their original relative abundances
in the
S+NA+
treatment.
The morphologically diverse
Rhodobacter/Roseobacter subgroup
of the
-subclass of the
Proteobacteria constituted 9% ± 3% of
total bacteria and a significant fraction (40%) of
-proteobacteria
(Table
1). Mean cell numbers of
Rhodobacter/Roseobacter increased
from
1.5(±0.5) × 10
5 (
n = 8) by 1.7-fold
and by 2.2-fold in the first 20 h of incubation
in the S
and S+
treatments, respectively, whereas the increase
in NA+ treatments was
smaller. Within the second half of incubation,
numbers of cells
targeted by probe G Rb changed little in all
treatments and dropped
below the original value in the substrate-amended
treatments (Fig.
2).
Their relative abundances in all but the
NA+ treatments decreased
during 43 h of incubation.
Rhodobacter/Roseobacter constituted only about 4% ± 1% in S+ but up to 7% ± 1% of
bacteria
in the S
treatments.
Changes in per cell DNA and protein content.
The flow
cytometric signature of double-stained bacterioplankton cells revealed
treatment-specific changes during the incubations (Fig. 3). At the end
of the experiment, the cytograms from the unamended treatments with and
without NA showed pronounced differences from those of the original
community. In both, a second cell population with higher protein
content was discernible after 43 h. Incubation with substrates
either with or without NA resulted in the appearance of cells with
significantly higher DNA and protein content than in the unamended
sample. In S+NA+ treatments, the fraction of these large cells was much
higher (56.0%) than in S+NA (19.8%).
Starvation experiment.
Representative isolates obtained from
the North Sea (14) hybridizing with probe ALT1413, OCE232,
or G V were starved for more than 50 days (Fig.
4). The percentage of intact bacteria was
determined as the fraction of ribosome-containing cells, i.e., by their
EUB338 signal. A/C and Vibrio spp., which were readily enriched during the field growth experiment, showed no significant loss
of EUB338 detection rate versus DAPI during 50 days. In contrast, the detection rates of Oceanospirillum spp. decreased
rapidly within the first 20 days, and cells from this strain were
almost not detectable in the last half of the starvation experiment. The addition of fresh medium did not result in an increase in FISH-detectable cell numbers of Oceanospirillum within
10 h of incubation.
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FIG. 4.
FISH detection rates of different genera of the gamma
subclass of the Proteobacteria in stationary-phase batch
cultures (probe EUB338, DAPI counterstaining).
Oceanospirillum sp. KT0923,  ; Vibrio sp.
KT0901,  ; Alteromonas sp. KT1113,  .
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DISCUSSION |
Shifts in bacterioplankton community composition during
enrichment.
Enrichment cultures have a long tradition in
microbiology (8). This experimental strategy, arranged
intentionally or not, eventually resulted in the isolation of the
presently known variety of marine bacteria. The classic ZoBell approach
of 1946 (33, 47) has been repeatedly improved or modified,
e.g., by differential filtration (15), dilution
(10), and the use of specific substrates (24).
Knowledge about the spatiotemporal occurrence or physiological features
of particular phylogenetic groups may allow the design of more directed
experiments (12, 23, 36). However, enrichment attempts
always represent substantial interferences with microbial life and
their environment, even in the absence of additional substrates. For
example, prefiltration may influence bacterioplankton composition by
removal of large filamentous and most of the particle-attached cells
(1, 13). Cellulose ester filters of 1.2-µm pore size were
found to retain up to almost 50% of unfiltered bacterial abundances in
coastal waters (19). We could, however, not verify such a
reduction in our samples (means ± 1 standard deviation: unfiltered, 1.52(±0.04) × 106 cells
ml
1, n = 3; prefiltered,
1.57(±0.19) × 106 cells ml1,
n = 9). Even gentle filtration may increase substrate
concentrations (e.g., of dissolved free amino acids) due to damage of
phytoplankton cells (15) and disrupt the link between
dissolved and particulate organic matter (34). The absence
of protistan grazers will relieve bacteria from selective mortality
(41), and confinement will put an end to the dynamic
equilibrium between the formation and decomposition of organic matter
(45).
Already in 1984, Ammerman et al. (
5) and Ferguson et al.
(
15) had shown an increase in population size and average
cell
volume during undirected bacterioplankton growth in unamended
seawater. More recently, FISH in combination with flow cytometry
revealed changes in the taxonomic community composition of North
Sea
bacterioplankton in dilution culture (
17). In our
experiments,
changes in community structure occurred more rapidly than
reported
by Suzuki, who did not detect taxonomic shifts in filtered
seawater
samples for a period of 24 h (
42).
-Proteobacteria had increased
overproportionally already after
20 h of incubation in S
, whereas
-proteobacteria and C/F
members did not (Fig.
1). The addition
of organic substrates in
micromolar concentrations (5.7 mg of
C per liter) did not result in
significantly higher total cell
numbers after 48 h compared to S
(Fig.
1), but in an even more
pronounced change in community structure.
-Proteobacteria increased
from about 15 to 60% and the fraction of
C/F again remained constant,
but
-proteobacteria decreased by half
in relative abundance.
Concomitantly, the development of cell
populations with higher
protein and DNA content was observed in S+ and
S
(Fig.
3), and
these large cells thus mainly belonged to the rapidly
growing
fraction within the
-proteobacteria.
Our study extends previous findings in several respects. We present the
response of several individual groups within the marine
-proteobacteria to different treatments. Evidence is provided
that
the dominant members of this lineage in situ were rapidly
outcompeted
during enrichment culture. In the prefiltered seawater,
about 14% of
total cell numbers, corresponding to approximately
2 × 10
5 cells ml
1, belonged to the
-subclass
of
Proteobacteria. Members of a single
phylogenetic lineage,
the uncultured SAR86 cluster, formed 90%
of all
-proteobacteria in
the beginning of the experiments (Fig.
5). SAR86 belongs to the free-living
fraction of the pelagic bacterioplankton,
as determined by clone
libraries of prefiltered seawater (
1,
14) or visualization
by FISH (
14). Several typical culturable
-proteobacterial
genera were detected in very low numbers, either
attached
(
Vibrio and
Alteromonas) or freeliving
(
Oceanospirillum)
in the original North Sea pelagic
community (
14). In contrast
to
Vibrio and
A/C, SAR86 and
Oceanospirillum were not enriched
in any of
the treatments; the absolute abundances of these groups
remained
constant, and we did not observe a significant increase
in either cell
size or FISH signal intensity. Members of the SAR86
cluster and
Oceanospirillum were therefore neither visibly subjected
to
substrate-accelerated death (
35) nor activated in either
S
or S+. Enhanced mortality of SAR86 was, however, observed as
a
consequence of the antibiotic treatment, and already within
the first
20 h of incubation, the abundance of SAR86 had decreased
significantly in the NA+ incubations (Fig.
2). NA might, therefore,
have acted as a cell toxin for members of this lineage (
2)
or represented a stress factor that caused the lysis of virus-infected
cells (
46).
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FIG. 5.
Percentage of different lineages within the gamma
subclass of the Proteobacteria at the beginning and at the
end of enrichment experiments. Probes: A/C group, ALT1413;
Oceanospirillum, OCE232; Vibrio, G V; SAR86
cluster, SAR86-1249. Factors, increase in -proteobacteria compared
to the original sample.
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On the other hand,
Vibrio and A/C responded rapidly to our
simulated culturing conditions, with lag phases ranging from 5
to
10 h and generation times of between 7 and 12 h, as estimated
from the abundance changes during the 43-h experimental period.
This
corresponds well with the high numbers of
Vibrio-related
sequences found in clone libraries of stationary-phase dilution
cultures from Mediterranean Sea samples (
20), and with the
selective
enrichment of bacteria affiliating with
Alteromonas
macleodii during enclosure incubations in the same system
(
39). Presently
we cannot distinguish between the different
potential causes for
the observed community shifts, such as shorter
response times
to substrate upshifts, but also antibacterial or
autocrine growth
factors released by the rapidly growing groups
(
44).
No activation of dormant culturable bacteria.
The high
relative contribution of microbes affiliated with Vibrio,
Alteromonas, or Colwellia to the colony-forming
bacteria (14, 28) might be attributed to rapid cell
multiplication and short lag times. Alternatively, it may be the
consequence of a high fraction of dormant cells from these genera in
the original community that are activated by culturing effects like
substrate addition or the presence of solid surfaces (26).
The combined incubation with NA and substrates causes an abnormal
increase in cell size, and consequently ribosome content
(11), by delaying cell division until the eventual
appearance of NA-resistant strains (Fig. 3). Therefore, dormant
bacteria that respond to substrate addition should become FISH
detectable in such a treatment. In our experiments, the offered
substrate mix was appropriate to activate, e.g., Vibrio,
Alteromonas, and Colwellia, as it has been
successfully utilized for their isolation previously (14). However, no increase in the relative abundances of these typical culturable bacteria was observed during the initial period of incubation with NA (0 to 20 h) (Fig. 1 and 2). In contrast, there was a clear rise of the two groups during the same incubation period in
the treatments lacking NA. This is evidence that no or few dormant,
FISH-undetectable bacteria affiliated with Vibrio or A/C
were present in the water column. In fact, no initial increase in total
FISH detection rates with the bacterial probe EUB338 was observed
during incubations with NA. This suggests either that in general there
was no activation of dormant cells by our incubation conditions or else
that no cells escaped FISH detectability due to their low ribosome content.
Interestingly, the addition of the cell division-inhibiting agent NA
did not result in the dominance of one particular resistant
bacterial
group irrespective of substrate levels, but rather amplified
the
success of the most competitive lineage within the respective
treatment. In the substrate-unamended enrichments, resistant strains
of
the A/C cluster increased to similar absolute numbers in NA+
as in NA
after 43 h of incubation. However, in the presence of
the
antibiotic, they constituted a much larger fraction, about
one third of
the total community. The addition of substrate always
specifically
favored
Vibrio. This group constituted 65% of total
bacteria in NA+, which was almost three times as much as in the
NA
treatment (Fig.
5). On the other hand, A/C, in spite of being
potentially NA resistant (Fig.
5), was almost completely suppressed
in
substrate-amended NA
treatments, and the antibiotic shifted
the
competition between the two groups towards
Vibrio. It would
be premature to draw general conclusions from an unplanned observation
in a single sample. However, the study of the combined effects
of
growth-promoting and growth-inhibiting factors on microbial
competition
might be a fruitful field for future
investigations.
Enrichable culturable genera: "feast-or-famine"
strategists.
Our data do not support the hypothesis that readily
culturable pelagic bacteria are in general rapidly enriched in filtered or substrate-amended seawater. During extensive cultivation at Hegoland
Roads (14), 33 of 145 different bacterioplankton isolates affiliated with genera which also dominated our enrichments. However, another nine isolates were related to Oceanospirillum, which
did not grow during the incubations. Strains related to
Vibrio and A/C maintained large amounts of cellular
ribosomes during starvation in pure culture, whereas the FISH
detectability of Oceanospirillum declined rapidly (Fig. 4).
A high total per cell rRNA content of nongrowing cells apparently
provides the potential for a more rapid response to changes in growth
conditions (16). We conclude that rapidly enriched
culturable bacteria like Vibrio and A/C are able to maintain
a high potential to react to changes in growth conditions even during
extended periods of nongrowth. This life strategy goes beyond the
simplified dichotomy of r versus K selection (6), and
the growth of the two r strategists A/C and Vibrio was
apparently triggered at different ambient substrate concentrations (Fig. 5). Members of both lineages have been found associated with
marine metazoans (7, 25), which would agree with a concept of a feast-or-famine existence.
This bacterial life strategy will confront microbiologists trying
to culture as yet uncultured bacteria with fundamental
problems.
Some representatives (e.g., A/C) grow on unamended
seawater and
media with a relatively low carbon content
(
14). On the other
hand, they maintain a high potential for
growth during starvation
and show immediate response to the
environmental changes caused
by sampling. Moreover, members of several
readily culturable genera
survived and rapidly resisted the stress
factor NA. In summary,
new strategies are required to enrich and
eventually isolate yet
uncultured bacteria in plankton samples, and for
this purpose
molecular methods that monitor the changes in community
composition
will be
essential.
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ACKNOWLEDGMENTS |
We acknowledge J. Trotter for providing the freeware program
WinMDI. We thank Christian Schütt (BiologischeAnstalt Helgoland, Dept. of Microbiology) for sampling and use of the laboratory facility.
We thank Gunnar Gerdts and Antje Wichels for inspiring discussions.
This work was supported by the Max Planck Society (Germany).
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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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Abstract
Introduction
