Download CHOI, JOON W., EVELYN B. SHERR, AND BARRY F. SHERR

Limnol. Oceanogr., 41(6), 1996, 1161-l 168
0 1996, by the American Society of Limnology and Oceanography, Inc.
Relation between presence-absenceof a visible nucleoid and
metabolic activity in bacterioplankton cells
Joon W. Choi, Evelyn B. Sherr, and Barry F. Sherr
College of Oceanic and Atmospheric
Corvallis 9733 l-5503
Sciences, 104 Ocean Admin.
Bldg., Oregon State University,
Abstract
We investigated the report of Zweifel and Hagstriim that only a portion of marine bacteria contain
nucleoids -- the DNA-containing
regions of procaryotic cells-and that such bacteria correspond to the active
or viable fraction of bacterioplankton.
In Oregon coastal waters, 21-64% of bacteria had visible nucleoids;
numbers of nucleoid-visible
(NV) bacteria were greater than numbers of metabolically active bacteria, based
on cells with active electron transport systems (ETS) and intact cell membranes. During log growth of a
marine isolate, proportions of NV and ETS-active cells approached 100%. In stationary growth phase, the
fraction of ETS-active cells decreased rapidly, while that of NV cells remained high for 7 d. When starved
cells of the isolate were resupplied with nutrient (50 mg liter-l peptone), total cell number did not increase
during the initial 6 h, but the proportion of NV cells increased from 27 to lOO%, and that of ETS-active
cells from 6 to 75%. In an analogous experiment with a bacterioplankton
assemblage, a similar trend was
observed: the number of NV cells doubled during the initial 6 h prior to an increase in total cell counts.
These results show that some bacteria without visible nucleoids are capable of becoming NV cells, and thus
have DNA in a nucleoid region not detectable with the method used here.
Enumeration of bacterioplankton
by staining with the
fluorochromes
acridine orange or 4’,6-diamidino-2phenylidole
(DAPI) and then counting via epifluorescence microscopy is one of the most common methods
used in aquatic microbiology.
Zweifel and Hagstriim
(1995) presented evidence that only a fraction of the cells
counted with these methods appear to be living bacteria.
They based this observation on the presence or absence
of a nucleoid-a
region packed with condensed DNA in
the bacterial cell. The standard direct count method based
on DAPI staining of aldehyde-preserved marine bacteria
(Porter and Feig 1980) was shown to be nonspecific for
DNA; a modification
of the method resulted in efficient
binding of DAPI to DNA and removal of most nonspecific DAPI staining of other cell components (Zweifel and
Hagstrijm 1995). In seawater taken from the Baltic Sea,
the North Sea, and the Mediterranean
Sea, Zweifel and
Hagstriim reported that from 2 to 32% of bacterioplankton appeared to contain nucleoids. They inferred from
their results, which included laboratory culture experiments, that cells with nucleoids corresponded to the portion of the bacterial assemblage that is active or viable
and that cells without nucleoids were “ghost” cells that
did not contain DNA and were therefore incapable of
growth.
Acknowledgments
This work was supported by DOE grant DE-FG06-92ER6 1423
to B.F.S. and E.B.S.
We thank Kerstin Amthor, Keith Moore, Mike Rappe, and
Barbara Sullivan for their assistance with the LIVE/DEAD
BacLight staining method, Mike Rappe for identification of the
bacterial strain used in the experiments, and Steve Giovannoni
for discussions during preparation of the manuscript. We also
thank Steve Newell and two anonymous reviewers for their
comments during revision.
The observation of Zweifel and Hagstrijm is important
and of immediate concern to microbial ecologists. Have
previous determinations
of bacterioplankton
abundance
included a large background of nonliving “bacteriogenic
particles” that masked actual variation in the abundance
of living bacteria? In this report, we confirmed for Oregon
coastal seawater that only a portion of bacterioplankton
cells contained visible nucleoids. By using two fluorescence-based indicators of cell activity, we also found that
the number of metabolically active cells was less than the
number of nucleoid-visible
(NV) cells except when bacteria were in log-phase growth. In addition, we obtained
evidence from a cultured marine isolate and a natural
bacterial assemblage that when conditions become favorable for growth, cells without visible nucleoids are able
to develop nucleoids. This indicates that non-NV cells
are probably not devoid of DNA.
Materials and methods
Bacterial samples and cultures-We used either bacterioplankton assemblages in seawater collected from Yaquina Bay, Oregon, or a bacterial isolate (identified as
Pseudomonas putida by 16s rDNA partial sequence analysis) cultured from a sample of Oregon coastal seawater.
This isolate was maintained on plates made with 1.5 g of
agar in 1 liter of 0.2~pm filtered, autoclaved seawater
enriched with 100 mg liter- l peptone, and grown in liquid
medium on 100 mg liter-l peptone in 0.2-pm filtered
seawater.
Total cell counts- Samples were fixed with 5% final
volume of 37% borate-buffered Formalin, stained with 5
pg ml-l final concentration of DAPI for 5 min (Suzuki
et al. 1993), and passed onto 25-mm, 0.2~r.Lrnblack-stained
membrane filters. The filters were mounted onto slides
1161
Choi et al.
1162
and examined at 1,250 x with a Zeiss Universal microscope outfitted for epifluorescence microscopy and Zeiss
filter set 4877-02 (excitation 365 nm/barrier 420 nm).
Nucleoid-visible cells-The
original
method of Zweifel
and Hagstrom (1995) included lowering the seawater salinity to < 127~0,incubation of bacteria with detergent to
promote effective binding of DAPI
to DNA,
and a
2-propanol rinse to visualize nucleoids by removing most
of the nonselectively bound DAPI. We modified this protocol somewhat. We found it necessary to fix marine bacterial cells with Formalin to avoid significant cell loss in
the staining process. Without fixation, cells tended to swell
when salinity was lowered and were more susceptible to
breakage during filtration. Samples were fixed in glass
vials with 5% final volume of 37% borate-buffered Formalin freshly dispensed from a 3.8liter container. Formalin held in smaller containers for a few days seemed
to lose effectiveness as a fixative in this application.
After fixation, 0.1-2 ml of each sample was passed onto
0.2-pm black-stained membrane filters. About 5 ml of
deionized water was added to the filter tower before adding the seawater sample, and 5 ml of deionized water was
added after sample addition in order to lower the salt
content and to remove residual Formalin. Once the sample was settled onto the filter, the filter was rinsed with
another 5 ml of deionized water. The vacuum was relieved, then the surface of the filter was flooded with a
solution of 10 pg ml-l DAPI made up with deionized
water and held in the dark for at least 5 min. The DAPI
solution was drawn through the filter, and the filter was
rinsed three times with 1 ml of warm (60-70°C)
2-propanol. The 2-propanol was introduced carefully onto
the side of the filter tower, about halfway down, to avoid
floating the cells from the surface of the filter and thus
creating uneven distribution.
After the final alcohol rinse
was filtered to dryness, the filter was removed from the
tower and placed on a dry paper towel until completely
air-dried (5 min). The filter was then mounted onto a
slide with immersion oil and cover slip; once mounted,
the preparations can be stored at -20°C for weeks.
With this procedure, nucleoids were visualized as bright
yellow spots within dimly blue-fluorescing cells. Many of
the cells without visible nucleoids were difficult to discern,
so the nucleoid preparation could not be used for total
cell counts.
Cells with active electron transport systems (E Ts) - Unpreserved subsamples were incubated with 10 mM final
concentration
of the reagent 5-cyano-2, 3-ditoyl tetrazolium chloride (CTC, Polysciences) for up to 2 h in the
dark at the same temperature as the bacterial culture. CTC
is nonfluorescent and membrane-permeable
in its oxidized state but becomes highly fluorescent and precipitates when it is reduced as the terminal electron acceptor
in the electron transport system (Rodriguez et al. 1992).
We previously determined that a 2-h incubation with a
10 mM concentration of CTC yielded optimum results.
Samples were then fixed with 5% final concentration Formalin, counterstained with DAPI (5 pg ml-l), passed onto
0.2~pm black-stained membrane filters, and mounted onto
slides. Preserved liquid samples can be stored at 4°C for
2 d, but once the samples were mounted onto slides with
immersion oil, the number of cells scored as CTC-positive decreased by half within 1 d. In our experience, preserved liquid samples can also be quick-frozen in liquid
nitrogen and stored at -20°C (or less) for weeks. Cells
were visualized at 1,000 x by DAPI fluorescence; a green
excitation filter set (Zeiss 4877-l 5, exitation 546 nm/
barrier 590 nm) was then used to check each cell for red
fluorescence of CTC.
Intact membrane vs. compromised membrane bacteria -Unpreserved
subsamples were incubated with 3 ~1
ml-l sample of 1 : 1 premixed stains of LIVE/DEAD
BacLight re,agent (Molecular Probes) for 15 min in the
dark; the sample was then settled onto a 0.2~pm blackstained membrane filter and mounted onto a slide. We
visualized green-fluorescing cells with a blue excitation
filter set (Zeiss 4877-09; excitation 450-490 nm, barrier
520 nm) and red-fluorescing cells with the green excitation filter set.
The BacLight reagent consists of two separate nucleic
acid stains that fluoresce either green or red when excited
by blue or green light, respectively. The green-fluorescing
stain can cross cell walls and membranes of both healthy
and damaged bacterial cells. The red-fluorescing stain in
theory only enters bacterial cells with compromised or
damaged membranes and outcompetes the green-fluorescing stain for binding sites. Thus, healthy cells with
undamaged membranes fluoresce green, and cells with
compromised or damaged membranes fluoresce red. In
preliminary tests with this reagent, up to 90% of cultured
bacteria in log growth showed green fluorescence, but after
heat-killing, all cells fluoresced red.
According to the manufacturer (Molecular Probes), the
staining characteristics of bacterial cells are unaffected by
preservation with aldehyde fixative. Our preliminary results indicate that this is the case. For up to a week after
fixation, cells from a bacterial culture fixed with 4% final
concentration of fresh EM-grade glutaraldehyde showed
the same proportion of green and red-fluorescing cells as
did cells in a sample of unpreserved culture. However,
once stained with the BacLight reagent and settled onto
a filter, bacterial cells had to be inspected within 2 h for
consistent results. We also found that a pre- and postfiltration rinse with 0.5 N sodium carbonate buffer, pH 9
(Sherr et al. 1993), and use of Aqua-Poly/Mount
(Polysciences, Inc.) to mount the filters onto slides reduced the
problem of rapid fading of stained cells.
Seawater samples- Surface seawater was collected from
a dock near the mouth of Yaquina Bay, Oregon, in late
summer. In the laboratory, l-liter samples were passed
through a 0.45-pm membrane filter and held at 10°C in
the dark on a shaker table. Subsamples of the filtered
seawater were processed for the total number of bacteria,
nucleoid-visible
(NV) cells, ETS-active bacteria, and bacteria with intact or compromised membranes at various
times up to 5 d after sampling.
1163
Visible nucleoids in bacteria
Laboratory culture experiments-The marine isolate
was grown in liquid medium on 100 mg liter- l peptone
to stationary phase. In a preliminary growth experiment,
several subsamples of the isolate were taken over a growth
curve for determination
of total count, NV cells, ETSactive cells, and cells with intact or damaged membranes
(BacLight reagent).
In a second experiment, cells of the marine isolate
scraped from colonies growing on agar were suspended
in unamended 0.2-pm-filtered
seawater and kept at 10°C
for 2 d. A l-ml aliquot was then inoculated into 100 ml
of seawater enriched with 100 mg liter-l peptone. Initial
bacterial abundance was - 6 x 1O5cells ml-l, and the proportion of NV cells was 35% and that of ETS-active cells
was 8%. Triplicate subsamples of the culture were taken
for total counts, NV cells, and ETS-active cells, at 12 h,
once a day for 7 d, and then at several-day intervals to
24 d.
A third experiment with the marine isolate was carried
out to examine the change in cell number in each of the
three categories over the first 12 h of incubation of starved
bacteria, with low initial proportions of NV and ETSactive cells, resupplied with nutrient. Bacteria were harvested from an agar plate on which colonies of the marine
isolate had been growing for 1 month at 10°C. The bacteria were starved in nonnutrient seawater for 4 d at 10°C.
At the end of this time, the population contained 27%
NV cells and 6% ETS-active cells. Equal volumes (75 ml)
of the starved culture and of 100 mg liter-l peptoneenriched, 0.2~pm-filtered seawater were mixed, yielding
a final peptone concentration of 50 mg liter- l. Triplicate
subsamples were taken for total cell number, NV cells,
and ETS-active cells every 2 h for 12 h.
Experiment with seawater bacterioplankton -An experiment analogous to the short-term experiment with
the laboratory culture was carried out with a bacterioplankton assemblage in coastal seawater. In November,
a sample of seawater was taken from the holding tank at
the Hatfield Marine Science Center (Newport, Oregon).
The tank is filled daily from a subsurface intake at high
tide to minimize the freshwater influence of the Yaquina
River. The seawater was aged in the dark for 2 weeks at
16°C and then passed twice though 0.8~pm filters. A portion of the seawater was mixed with an equal volume of
100 mg liter-l peptone-enriched
seawater (final concn,
50 mg liter-’ peptone). The seawater culture was sampled, as above, every 2 h for 12 h. At the beginning of
the experiment, the fraction of NV cells was 16% and
that of ETS-active cells 8%.
Results
Nucleoid-containing cells - Nucleoids were easily visible in cells of the marine isolate, which were 0.5-2 pm
long. In log-growth cultures, two or more nucleoids were
often visible in individual cells. In seawater samples, larger cells also often had two or more nucleoids. On cursory
inspection, we observed nucleoids in some of the smallest
sized bacterioplankton
cells, although the presence of vis-
Table 1. Proportional abundance (as percent of total DAPI
counts) of nucleoid-visible
(NV), intact membrane (int-mem),
compromised membrane (camp-mem), and ETS-active cells in
natural bacterioplankton
assemblages from Oregon coastal seawater. Seawater samples were passed through 0.4%pm pore-size
membrane filters and held in the dark at 10°C for up to 5 d.
Total bacterial abundances were estimated via direct counts of
DAPI-stained samples by means of epifluorescence microscopy.
Seawater
sample
SW- 1, fresh
Id
3d
SW-2, 2 d
SW-3, 5 d
SW-4, fresh
Id
Bacteria
(lo5 ml-l)
0.9
2.3
7.4
13.0
10.9
14.1
23.2
NV
Intmem
Compmem
ETSactive
29
55
34
38
64
21
27
14
34
19
21
9
-
91
65
93
78
85
-
3;
20
20
10
7
10
ible nucleoids seemed to be most common
cells of the bacterioplankton
assemblage.
in the larger
Components of bacteria in seawater and lab culture (Table I, Fig. I) - Total bacterial abundance in seawater samples ranged from 0.1 to 1.3 x lo6 cells ml-l. Proportion
of cells with visible nucleoids ranged from 29 to 64% of
total DAPI counts. The proportions of ETS-active cells
(8-39%) and of cells with intact membranes (green-fluorescing cells with BacLight reagent, 9-34%) were less
than the proportion
of NV cells (Table 1). Cells with
apparently damaged membranes (red-fluorescing cells with
BacLight reagent) comprised 65-93% of total counts. The
sum of intact membrane and compromised membrane
cells was about the same as total DAPI counts in these
samples (Table 1).
We used data from both of the seawater samples and
from the preliminary
growth experiment of the marine
isolate and plotted the number of ETS-active cells against
the number of intact membrane cells (Fig. 1A) and against
the number of cells with visible nucleoids (Fig. 1B). Across
3 orders of magnitude of bacterial abundance, the number
of ETS-active cells was similar to the number of intact
membrane cells in individual
samples (Fig. 1A). However, the number of ETS-active bacteria was generally
less- often substantially less- than the number of NV
cells (Fig. 1B).
Marine isolate growth curve (Fig. 2) -Total abundance
increased from < 1O6 cells ml-l at time zero to lo8 cells
ml-l on day 4, after which total cell number remained
constant to the end of the experiment (Fig. 2A). Virtually
all cells had visible nucleoids and were ETS-active during
log growth. The number of ETS-active cells and of NV
cells did not decline until the stationary growth phase.
The number of NV cells started to decrease after day 8,
and the number of ETS-active cells after day 6 (Fig. 2A).
The increase and decline in ETS-active and in NV cells
with time is shown most clearly when expressed as a
fraction of total DAPI counts (Fig. 2B). The percentage
of both categories increased to 100% during log growth,
Choi et al.
1164
7.5 -
A
3 6.5 s
S
z 5.5 0”
J
4.5 3.5
-ODAPI
-a-NV
+
Active
1
I
I
I
I
4
5
6
7
8
12’ 1 B
7.5
-I
B
4.5
3.5
I
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I
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I
4
5
6
7
8
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after which the percentage of ETS-active cells dropped
quickly to ~50% by day 4; the percentage of NV cells
remained at 100% until day 7. During the subsequent
decline of both categories to 46% NV and 9% ETS-active
cells by day 24, the number of NV cells remained substantially greater than the number of ETS-active cells (Fig.
2B).
Short- term experiment with the marine isolate (Fig. 3) We repeated the first 12 h of the marine isolate growth
experiment to determine whether the number of NV and
ETS-active cells increased prior to increase in total cell
number during lag phase. This experiment was designed
to test the idea that inactive cells and cells without visible
nucleoids could become active and develop nucleoids
visible with the DAPI stain-propanol
wash procedure.
14
Time (day)
Log1 0 Active
Fig. 1. A. Log-log plot of relation of abundance (cells ml-l)
of bacteria scored as having intact membranes via the BacLight
reagent (intact) to abundance of cells scored as having active
electron transport systems (ETS) via the CTC reagent (active).
Solid line denotes a 1: 1 correspondence between the two parameters. B. Log-log plot of relation of abundance (cells ml-l)
of bacteria scored as having visible nucleoids (NV) to abundance
of ETS-active cells (active). Solid line denotes a 1 : 1 correspondence between the two parameters. In both plots, the lower
cluster of data was obtained from samples of bacterioplankton
in Oregon coastal waters and the upper cluster from a timeseries culture experiment with a marine bacterial isolate.
7
Fig. 2. Results of a long-term (24 d) culture experiment with
the marine :isolate. A. Change in abundance with time (cells
ml-l) of total bacterial counts (DAPI), nucleoid-visible
cells
(NV), and ETS-active cells (active). B. Changes in NV and active
cell abundance with time expressed as a percentage of total
DAPI counts. Error bars represent 1 SE of three replicate samples.
During the first 6 h of the experiment, there was no significant increase in total bacterial abundance, but the
number of NV and ETS-active cells increased dramatically: to 100 and 75% of total cell counts (Figs. 3A, B).
From 6 to 1.2 h, all three categories were similar in abundance (Fig. 3A). When these data are plotted as cumulative increase in cell number (i.e. difference from timezero abundance for each category), the sharp increase in
NV cells and in ETS-active cells during the first 6 h (when
total number showed no change) is clear (Fig. 3C). Furthermore, Fig. 3C shows a close coupling between cumulative increase in NV cells and in ETS-active cells. It
was apparent that during the first 6 h, increase in NV and
active cells resulted from development of ETS activity
and visible nucleoids by previously inactive cells that had
no observable nucleoids at the start of the experiment.
After 6 h, cell increase resulted from division of the active
NV cells.
Short-term experiment with seawater bacteria (Fig. 4)We repeated the short-term
experiment
described above
,
Visible nucleoids in bacteria
4-
1165
_ A
3-
+
DAPI
-w-NV
--.k- Active
B
-a- %NV
-A--
C
6
.s
with a mixed species assemblage of bacteria from a carboy
of coastal seawater that had been held at 16°C in the
laboratory for 2 weeks. The initial fraction of NV cells
was 16% and that of ETS-active cells was 8%. As in the
lab culture experiment, the total abundance of bacteria
did not increase during the first 6 h (Fig. 4A). The increase
in NV and ETS-active cells was smaller for the bacterioplankton compared to the lab culture; however during
the first 6 h, the proportion of NV cells doubled and the
proportion of active cells increased by 2.5 times (Fig. 4B).
DAPI
NV
Active
1
6
12
Time (h )
Time (h )
Fig. 3. Results of a short-term (12 h) culture experiment
with starved cells of the marine isolate. A. Change in abundance
with time (cells ml-l) of total bacterial counts (DAPI), nucleoidvisible cells (NV), and ETS-active cells (active). B. Changes in
NV and active cell abundance with time expressed as a percentage of total DAPI counts. C. Cumulative change in abundance with time (cells ml-l) of total bacterial counts (DAPI),
nucleoid-visible
cells (NV), and ETS-active cells (active) determined by subtracting initial abundances in each category from
abundances determined for triplicate subsamples at each time
point. Error bars represent 1 SE of three replicate samples.
-O-m-A-
% Active
Fig. 4. As Fig. 3, but of a short-term (12 h) culture experiment with starved cells of a marine bacterioplankton
assemblage.
When the results were plotted as cumulative increase in
abundance in each of the three categories (Fig. 4C), the
increases in NV cells and ETS-active cells during the
experiment were tightly coupled, as also observed for the
lab culture experiment (Fig. 3C). The increase in NV cells
during the initial 6-h period was accompanied by a decrease in cells without visible nucleoids. We estimated
that by 6 h, about 20% of cells initially without visible
nucleoids had developed nucleoids.
Discussion
ModiJication of the staining protocol for visualizing nucleoids in bacterial cells-In our hands, the method outlined by Zweifel and Hagstrijm (1995) did not yield
sistent results. The main problem was inadequately
bacterial cells. Fixation gives cells physical rigidity
allows fluorochromes to penetrate cell membranes
readily. A variable fraction of unfixed cells seemed
confixed
and
more
to be
1166
Choi et al.
lost during the dilution with freshwater, incubation with
detergent, and 2-propanol rinse. Our modified method
used Formalin fixation to overcome these problems. Residual aldehyde on the bacterial cell surface can bind
nonspecifically with DAPI and mask fluorescence of the
nucleoids. Rinsing Formalin-fixed
cells with deionized
water and then washing them with warm 2-propanol resulted in effective DAPI binding to DNA and elimination
of most nonspecific fluorescence in bacterial cells. Our
modification of the method seemed to be robust for both
laboratory isolates and mixed species assemblages ofmarine bacteria.
Bacterial cells without visible nucleoids are not necessarily devoid of DNA -As did Zweifel and Hagstrijm
(1995), we observed that the combined DAPI-staining
and propanol wash procedure results in nucleoids fluorescing a bright yellow. We suggest that the yellow color
is precipitated DAPI because we have noticed that DAPI
crystals caught on filters also fluoresce bright yellow. The
concentration of DNA-bound
DAPI in the nucleoid region may be so great that the DAPI is effectively precipitated. Precipitation of DNA due to the alcohol wash may
also contribute to precipitation
of DAPI.
Ofgreater interest is the suggestion ofZweife1 and Hagstrijm that cells without observable nucleoids are devoid
of DNA and are thus empty “ghost” cells incapable of
dividing. According to our results, this is not necessarily
the case. In a starved laboratory culture resupplied with
nutrient, 100% of cells without visible nucleoids developed yellow-fluorescing
nucleoids before cell abundance
began to increase (Fig. 3). In an analogous experiment
with a natural bacterioplankton
assemblage, -20% of
cells without visible nucleoids developed visible nucleoids before significant increase in total cell counts (Fig.
4). It is possible that the peptone enrichment (50 mg
liter-l) used in this experiment provided adequate growth
conditions for only a fraction of the non-NV cells in the
bacterial assemblage.
These results suggest that cells in which there are no
visible yellow-fluorescing
nucleoid regions may still have
DNA. In cells without visible nucleoids, the DNA may
be in a compact nucleoid region but may not be of sufficiently high concentration to result in precipitation
of
DAPI and thus yellow fluorescence. Alternatively,
nonNV cells may have a relaxed nucleoid region in which
the DNA is sufficiently dispersed to prevent precipitation
of DAPI bound to it. The volume of the nucleoid is known
to increase under certain conditions, for example loss of
the intracellular IS+ pool due to damage to the cytoplasmic membrane (Dawes and Sutherland 1992).
If our concept is correct, then nucleoid-containing
cells,
as identified by the Zweifel and Hagstriim method, would
have condensed nucleoid regions with either multiple
copies of the genome or a larger genome size, while nonnucleoid cells would have less DNA, perhaps in a less
compact nucleoid region, and perhaps a genome of smaller size. The latter case might be true if genome size dif-
fered among species of marine bacterioplankton,
which
is untested at present. There are two supporting pieces of
evidence for the idea that marine bacteria contain varying
amounts of DNA per cell, either due to varying numbers
of copies of a genome or to species-specific differences in
genome size. First, Simon and Azam ( 1989) estimated
that the smallest marine bacterioplankton
cells, 0.0260.05 pm3, had a DNA content (2.5 fg cell-l) half as great
as that of the largest bacterioplankton
cells (0.40 hm3, 5
fg DNA cell-l) they considered.
Second, Li et al. (1995) used flow cytometric analysis
of bacterioplankton
stained with a blue-excitable nucleic
acid-binding fluorochrome and found two distinct groups
of marine bacterioplankton
based on cell-specific DNA
content. One group of bacterial cells had 2-5 times as
much DNA per cell as the other group. The DNA-enriched group of cells formed a greater fraction of total
bacteria in coastal waters and in chlorophyll-rich
waters
than in oligotrophic open-ocean water (Li et al. 1995).
Furthermore, the number of bacteria detected via flow
cytometry as having DNA equaled total bacterioplankton
abundance determined by direct counts of DAPI-stained
cells, which is evidence that virtually all suspended bacteria counted via epifluorescence microscopy do contain
DNA. We infer that the DNA-enriched
bacteria of Li et
al. (1995) correspond to the cells with visible nucleoid
regions and. that bacteria with less DNA per cell correspond to cells without visible nucleoids.
These results are not necessarily at odds with those of
Zweifel and Hagstriim (1995). In their growth experiment
with a marine bacterial isolate, all cells had visible nucleoids at the beginning of the experiment. These bacteria
had been in log growth and were subjected to reduced
substrate for only 2 d. Consequently, they were not likely
to be in a starved state, as was our marine isolate at the
beginning of the growth experiments described here (Figs.
2 and 3). In a second growth experiment with a bacterioplankton assemblage, Zweifel and Hagstrijm reported
a constant number of cells without visible nucleoids during the log and early stationary phase of the growth curve.
However, they did not sample this culture during the
initial 12 h of the experiment, and they did not add any
nutrient to the culture. An increase in NV cells caused
by development of visible nucleoids by previously nonNV cells during the first day of incubation, such as we
observed (Fyig. 4), may have been missed. Conversely, the
culture conditions in their experiment may not have stimulated non-NV, presumably dormant cells to develop visible nucleoids and begin growth.
Not all nucleoid-containing cells are active-The relative fractions of active vs. dormant cells in bacterioplankton assemblages has been a long-standing puzzle for microbial eco:logists. Initial studies based on autoradiography (e.g. Reimann et al. 1984; Douglas et al. 1987; Pedros-Alio
and Newell 1989) or the formazan redox
compound INT (Tabor and Neihof 1982; Newell et al.
1988) as indices of active cells showed that the percent
.
Visible nucleoids in bacteria
active component of the bacterioplankton assemblage was
variable and often <50%.
More recent studies with newly available fluorescent
reagents have corroborated these results. Porter et al.
(1995) used fluorogenic ester compounds, which are colorless until enzymatically
cleaved within bacterial cells
to yield a fluorescent product, and reported that the proportion of metabolically active cells was only 7% in oligotrophic lake water and up to 75% in polluted river
water. de1 Giorgio and Scarborough (1995) used the fluorogenic redox compound CTC and reported that across
a spectrum of lakes of differing trophic state, the proportion of cells with active ETS ranged from 15 to 33%
and was positively related to concentrations of inorganic
nutrients and chlorophyll in lake water. de1 Giorgio and
Scarborough also evaluated available data on percent viable cells in marine systems and concluded that variation
in abundance of active cells, which ranged from <5 to
> 50%, was one order of magnitude greater than variation
in total cell counts. Gasol et al. (1995) used CTC to demonstrate that metabolically
active bacteria were on average twice as large (0.12 ,um3) as inactive bacteria in
coastal seawater. In addition, after 3 d of incubation of
0.8-pm-filtered
seawater, the percent active cells in the
bacterial assemblage increased from 6 to 43% (Gas01 et
al. 1995).
Zweifel and Hagstrijm (1995) proposed that cells with
visible nucleoids represented a maximum estimate for
the number of metabolically active cells because the presence of a nucleoid would not ensure that a bacterial cell
was growing. Ours is the first study to directly compare
the abundance of cells with visible nucleoids and abundance of active cells. Number of active cells was determined in two ways: as cells with an active ETS via the
CTC reagent and as cells scored as having an intact as
opposed to damaged cell membrane via the BacLight
reagent. We found that the two methods yielded similar
abundances of active cells in the seawater and bacterial
culture samples tested (Table 1, Fig. 1A). However, the
number of active cells was typically less (and often substantially less) than the number of NV cells (Table 1, Fig.
1B). The abundance of NV cells was greater than that of
ETS-active cells during the stationary phase of a marine
isolate growth curve as well (Fig 2).
However, the numbers of NV and of ETS-active cells
were similar during log-phase growth of marine bacteria.
During exponential growth of a bacterial isolate, 100%
of cells contained nucleoids and > 80% of cells scored as
having an active ETS (Fig. 3). In two short-term growth
experiments, one with the bacteria isolate and one with
seawater bacterioplankton,
there was an equivalence in
the cumulative increase of nucleoid-containing
cells and
of ETS-active cells (Figs. 3C and 4C).
Conclusions
Zweifel and Hagstrijm (1995) demonstrated that marine bacterioplankton
visualized via the standard DAPI
direct count method can be segregated into two distinct
1167
groups of cells: those with and those without visible nucleoids. This is an important finding that should greatly
stimulate the field of microbial ecology. We propose that
cells without visible nucleoids are not devoid of DNA,
but rather have less total DNA or a less condensed nucleoid region than do cells with visible nucleoids. The
distinction between the two groups of cells is, in our opinion, whether there is a sufficiently high concentration of
DAPI bound to DNA in the nucleoid region to form a
visible yellow-fluorescing
DAPI precipitate.
Our results also suggest that determining the proportion
of cells with visible nucleoids in marine bacterioplankton
assemblages can provide useful information
about the
relative activity of such assemblages. Although nucleoidvisible cells did not always seem to be metabolically active, in our study growing cells were ETS-active and had
visible nucleoids (Figs. 3C and 4C). The proportion of
nucleoid-visible
cells was lowest in starved marine bacteria and highest in bacterial populations or assemblages
in log-phase growth (Figs. 3 and 4). We infer that the
presence of a nucleoid detectable by the Zweifel and Hagstriim method is indicative of a bacterial cell that is either
actively growing or has been active in the recent past (on
the order of days to weeks). The correspondence between
abundances of NV cells and of colony-forming
units reported by Zweifel and Hagstrijm supports this view. Cells
without visible nucleoids may be in a dormant (or extremely slow growing) or starvation-survival
state (e.g.
Morita 1985; Kjelleberg et al. 1993).
It is possible that some cells without visible nucleoids
could be devoid of DNA. However, the observation of
Li et al. (1995) that the number of bacterioplankton
cells
with DNA was similar to the number of cells determined
by DAPI direct counts supports the idea that cells without
visible nucleoids do have DNA. The results of both our
study and that of Li et al. argue against the idea that
empty ghost cells form a significant fraction of bacterial
particles.
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Submitted: 3 January , 1996
Accepted: 18 March 1996
Amended: 1 May 1996