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Applied and Environmental Microbiology, April 2001, p. 1636-1645, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1636-1645.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Determination of DNA Content of Aquatic Bacteria by Flow
Cytometry
D. K.
Button1,2,* and
Betsy R.
Robertson1
Institute of Marine
Science1 and Department of Chemistry and
Biochemistry,2 University of Alaska,
Fairbanks, Alaska
Received 6 October 2000/Accepted 24 January 2001
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ABSTRACT |
The distribution of DNA among bacterioplankton and bacterial
isolates was determined by flow cytometry of DAPI
(4',6'-diamidino-2-phenylindole)-stained organisms. Conditions were
optimized to minimize error from nonspecific staining, AT bias, DNA
packing, changes in ionic strength, and differences in cell
permeability. The sensitivity was sufficient to characterize the small
1- to 2-Mb-genome organisms in freshwater and seawater, as well as
low-DNA cells ("dims"). The dims could be formed from laboratory
cultivars; their apparent DNA content was 0.1 Mb and similar to
that of many particles in seawater. Preservation with formaldehyde
stabilized samples until analysis. Further permeabilization with Triton
X-100 facilitated the penetration of stain into stain-resistant
lithotrophs. The amount of DNA per cell determined by flow cytometry
agreed with mean values obtained from spectrophotometric analyses of
cultures. Correction for the DNA AT bias of the stain was made for
bacterial isolates with known G+C contents. The number of chromosome
copies per cell was determined with pure cultures, which allowed growth
rate analyses based on cell cycle theory. The chromosome ratio was
empirically related to the rate of growth, and the rate of growth was
related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb
by this method. In a typical seawater sample the distribution of
bacterial DNA revealed two major populations based on DNA content that
were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell
1
was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell1.
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INTRODUCTION |
Aquatic heterotrophic
bacterioplankton, which are too small for observation by light
microscopy, are commonly visualized with fluorescent DNA stains
(14). The intensity of stain fluorescence as determined by
flow cytometry, together with light scatter data, can help characterize
natural populations (10, 11, 43, 70), determine rates of
growth (16), locate DNA-deficient organisms (49), provide a cell mass basis for comparative and
absolute descriptions of organism affinity for nutrients
(5), and identify low-mass particles (49) as
bacteria in order to quantify a major component of aquatic living
carbon (9).
The mean DNA content of bacterioplankton has been estimated from
analysis of filter-retained material and an organism count together
with the number of organisms observed (17) and from analysis of images of individual cells (36), but mean
values (17, 44) vary more than expected. In early studies,
flow cytometry was used to observe differences among cells in
monocultures of commonly grown large-cell species (60).
Fluorescence from DAPI (4',6-diamidino-2-phenylindole)-bound DNA was
responsible for locating predominant very small oligobacteria
(28). DAPI has been used to estimate the genome sizes of
Synechococcus (3) and oligobacterial
(52) isolates. Stains such as PicoGreen (62), Hoechst 33258, SYBR Green (4, 38), SYTOX Green
(66), Syto 13 (18), YOYO, YO-PRO
(39), and TOTO (24) have also been used, but
the specificity and species dependence of these stains have not been
evaluated. Among these stains the in vitro binding of DAPI by DNA is
best understood (61). DAPI is bright and stable enough and
is minimally affected by DNA conformation (1). To improve
the utility of DAPI as a quantitative probe for DNA in individual
organisms, we studied binding, salt effects, specificity, staining
conditions, and permeation requirements. We show that this stain can be
used to measure DNA content, chromosome size, and chromosome stability,
as well as the distribution of DNA among various types of oligobacteria
or among oligobacteria growing at various rates.
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MATERIALS AND METHODS |
Cultures and seawater samples.
The marine organisms
Cycloclasticus oligotrophus (10),
Marinobacter arcticus (10), and
Sphingomonas sp. strain RB2256 (53) were grown
in synthetic seawater medium containing 1 M Na+
(52) and 1 to 10 µM acetate, mixed amino acids, and
glucose, respectively, as carbon sources. Escherichia coli
DH1 (ATCC 33849) and Brevundimonas diminuta (formerly
Pseudomonas diminuta ATCC 19146) were grown in low-salt (M9)
mineral medium (15) containing 100 µM glucose.
Methanobacterium thermoautotrophicum 
H was grown in
mineral medium supplemented with a stream of methane gas. Cultures were
grown from stock preparations stored in glycerol at 
50°C (20). E. coli and C. oligotrophus
cultures containing subpopulations of cells with up to five genome
copies were produced either by treatment with rifampin or by
constitutive chromosome runout (58) following exhaustion
of the limiting carbon source. Low-DNA-content cells were produced by
20-fold dilution of a C. oligotrophus batch culture in a
medium containing 28 mg of acetate liter1 to obtain
107 cells ml1 and incubation at 20°C.
Seawater was collected at a depth of 15 m with a Niskin bottle
from the R/V Alpha Helix in Thumb Cove off Resurrection Bay in the Gulf of Alaska. Surface water was collected from East Twin Lake
(145 km southwest of Fairbanks, Alaska) by dipping with a baked 3-liter
carafe from the front of an aircraft pontoon while another researcher
was paddling upwind. Other freshwater samples were collected in a
similar manner from a small boat. All samples were preserved with
filtered (pore size, 0.2 µm) formalin (0.5% formaldehyde), placed on
ice, returned to the laboratory, and stored at 5°C in the dark.
Flow cytometry.
Preserved samples were directly stained, and
fresh samples were treated with formaldehyde and refrigerated at least
overnight in the dark before staining. Bacterial populations were
diluted with basal medium to concentrations of about 106
cells ml1, filtered through a 1.0-µm-pore-size filter
(natural samples only), permeabilized with 0.1% Triton X-100, and
stained with freshly diluted DAPI obtained from a frozen stock solution
(0.5 µg ml1) for 60 ± 10 min at 10°C in the
dark (7). An internal standard mixture, consisting of 0.6- and 0.9-µm-diameter beads (Polysciences) was added to each sample.
The smaller particles were used to normalize fluorescence intensity and
correct for instrument drift; the larger particles were added to a
concentration of 1 × 105 particles ml1
(as determined with a Coulter Counter) and used to ratiometrically determine cell population sizes (49).
Measurements were obtained with a modified (
47) Ortho
Cytofluorograf IIs equipped with a 5-W argon laser. Computerized
operation
was accomplished with a Cicero system and Cyclops software
(Cytomation,
Inc.). The laser was tuned to 351.1 and 363.8 nm and was
operated
with a 100-mW output. Blue fluorescence from DAPI was
collected
at 90° to the beam through a 424-nm long-pass dichroic
filter
and a 450- to 490-nm band pass filter. Fluorescence intensity
was determined with a calibrated (
51) 3.5-decade
dynamic-range
logarithmic amplifier with analysis triggered by
fluorescence.
DNA content.
DAPI-DNA fluorescence intensity was converted
from a logarithmic distribution over 256 channels to
103.5 linear channels by Cyclops software. Gains were set
with reference to the 0.6-µm-diameter standard beads, and
formaldehyde-preserved E. coli (5.12 fg of DNA
cell1) (31, 50) was used at the end of each
day to normalize differences in the freshly prepared staining solutions.
To account for differences in the G+C contents of species due to the
AT-binding specificity of DAPI (
32), the apparent DNA
content was adjusted to the
E. coli standard content by
using
the probability (
P) of finding
n adjacent
AT pairs:
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(1)
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where
n is the number needed for binding to
DAPI and
A is the portion of DNA comprised of adenine plus
thymidine. For DAPI,
n is at least 3 (
69), and
the corrected value was considered
the product of the apparent value
and the ratio of
P for
E. coli (0.0657) to
P for the unknown. When the G+C content was known,
P was calculated and related to
P for
E. coli and the DNA content
corrected from the ratio of
P
for
E. coli to
P for the unknown.
For
bacterioplankton a G+C content of 52% was obtained from G+C
measurements for the variable region of rRNA-DNA extracted from
bulk
seawater (L. B. Fandino, personal
communication).
Effect of salt and nucleases.
Salt effects were tested by
diluting 40
salinity filtered seawater, incubating for 1 h, and
then staining. To determine the extent of DAPI binding site loss due to
nucleic acid hydrolysis, cultures were incubated for 75 min at 37°C
with DNase I (2,000 Kunitz units ml1; Sigma Chemical
Co.), RNase A (400 U ml1; Sigma), and proteinase K (10 µg ml1; Sigma) and then permeabilized with Triton X-100
and stained with DAPI as described above.
Spectrophotometric analysis of DNA.
The organisms were
centrifuged at 5,000 × g for 10 min, resuspended in
basal medium at a concentration of 1 × 109 cells
ml1 (as determined with a Coulter Counter ZBI
[Coulter Electronics]), diluted with STE buffer (0.1 M NaCl, 50 mM
Tris HCl, 1 mM Na2EDTA) to concentrations of 0.5 × 107 to 14 × 107 organisms
ml1, lysed with 0.1% sodium dodecyl sulfate for 10 min
at 60°C, and treated with proteinase K (100 µg ml1,
37°C, 30 min) to reduce aggregation. KCl was added to a concentration of 40 mM, the sample was chilled on ice for 30 min, and the resulting potassium dodecyl sulfate precipitate was removed by centrifugation (10,000 × g, 20 min, 5°C) (45). The
supernatant was stained with Hoechst 33258 at a concentration of 0.05 µg ml1, and its fluorescence was determined by using a
spectrofluorimeter (MPF-66; Perkin-Elmer Corp.) with excitation set at
350 nm and emission set at 450 nm (34). The DNA
concentration, uncorrected for G+C content, was obtained from a
standard curve prepared with calf thymus DNA (type I; Sigma).
Biomass.
Biomass values were obtained from a calibrated
Rayleigh-Gans-based forward light scatter standard curve by using
formaldehyde-treated cells with a known formaldehyde-free dry mass and
an aspect ratio of 3 for all organisms (48, 49).
Cell cycle analysis.
C. oligotrophus was grown in
batch cultures at various rates dictated by initial acetate
concentrations of 12 to 200 mg liter1. The rates were
determined by measuring the change in total cell mass by flow
cytometry. DNA contents were determined as described above. The
proportions of cells in the B phase (the period between cell division
and the beginning of replication when cells have one chromosome), the C
phase (when the chromosome is replicating), and the D phase (when there
are two chromosomes [12]) were determined by the peak
reflect method (22) and used along with the growth rate
determined from the rate of biomass increase to calculate (59) the time spent in each phase.
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RESULTS AND DISCUSSION |
Binding.
DAPI attains sufficient binding energy near certain
regions with 3 or more base pairs, such as the duplex d(-AATT-)
of double-stranded DNA and poly[d(A-T)]2, to widen
the minor groove, attach, and fluoresce with an improved quantum yield
when it is excited (1). For M. arcticus, a DAPI
concentration of 0.1 µg ml1 was sufficient to saturate
half the strong binding sites in both one- and two-chromosome cells
(Fig. 1). The binding constant
(Kb) was 4.0 × 105
mol1 for intact cells in 0.6 M Na+ at 10°C
as determined from a Scatchard plot, and this value was close to
1.8 × 105 mol1, the value computed from
the reported value for pure DNA at an equivalent salinity
(69) and corrected for temperature by using an enthalpy
change of 15 kJ mol1, which was obtained from
Arrhenius plots of DAPI binding data versus temperature data
(Fig. 2).
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FIG. 1.
Effect of DAPI concentration on DNA fluorescence
intensity (I). The values are means for the first (1n) and second
(2n) fluorescence peaks from M. arcticus, such as those
shown for C. oligotrophus in Fig. 4A. (Inset) Scatchard
transformation for the 2n cells.
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FIG. 2.
Effect of staining time and temperature on fluorescence
of M. arcticus at different DAPI concentrations.
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DAPI binding requires penetration of the cell envelope, but
gram-positive and lithotrophic organisms can be particularly impermeant
(
29,
67). In the absence of Triton X-100, almost all
M. thermoautotrophicum cells remained in a low-fluorescence
cluster on two-dimensional
histograms or dot plots (Table
1). Prolonged treatment with formaldehyde
resulted in some increase in fluorescence; one-third of the organisms
were repositioned in a moderately bright cluster. Permeabilization
with
Triton X-100 dramatically increased the fluorescence of the
whole
population and resulted in a single bright cluster with
normal
brightness for cultivated bacteria, although individual
chromosomes
were not resolved. About 35% of Resurrection Bay bacterioplankton
were
also made more fluorescent by Triton X-100 treatment (Table
1).
Staining of these resistant organisms was nearly complete
in 10 min and
reasonably stable over time during incubation at
10°C in the presence
of 0.5 µg of DAPI ml
1 and 0.1% Triton X-100 (Table
2). For
E. coli formaldehyde
treatment
alone was sufficient to optimize staining, and for preserved
samples
of
M. arcticus and
C. oligotrophus the
DAPI-DNA fluorescence was
increased by about 5% (data not shown) by
Triton X-100 treatment.
Staining for 3 h or more resulted in a
loss of chromosome resolution.
The organisms belonging to several
easily stainable gram-negative
species then appeared in a single major
cluster that included
the low-DNA cells ("dims") discussed below.
Thus, long staining
times appeared to result in nonspecific staining of
whole organisms
that was detrimental to quantitative measurement, and
cell envelopes
became fluorescent. When a 60-min staining time was used
to integrate
staining with multiple-sample analysis by flow cytometry,
the
results were satisfactory.
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TABLE 1.
Effect of fixative and permeabilization on the
intensities of DAPI-DNA fluorescence of recalcitrant organisms as
determined by flow cytometry
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Preservation.
Formaldehyde-treated, refrigerated
E. coli and C. oligotrophus cells were
indistinguishable after several months from cells that were
freshly prepared in either freshwater or saltwater media on the basis
of either dry mass (49) or apparent chromosome size (Table
2). For M. arcticus there was a loss of resolution in 1n and
2n cells over 11 years, but Coulter Counter populations remained
unchanged (49). For the E. coli used as a DNA
standard, the amount of fluorescence per cell changed ±8.6%
(n = 27) over 18 months (data not shown). This change
in E. coli fluorescence was random, so the small variation
was not due to storage and most likely was due to a change in the stain
working stock solution.
There have been reports of population losses in stored samples
(
63,
64) as determined by microscopic methods. Our
shipboard
DAPI microscopic counts agreed with the flow cytometry counts
only within about ± 30%. Intercalibration with results obtained
in
two other laboratories (unpublished data) by using five independent
preserved seawater samples from two locations gave flow cytometry
count/DAPI count ratios of 0.782 ± 0.12 and 0.92 ± 0.25. The ratios
of flow cytometry cell counts to acridine orange cell counts
were
1.42 ± 0.56 and 0.88 ± 0.66. Values obtained by
microscopic methods
seem to be subjective and to require discrimination
among viruses,
bacteria, and other fluorescent particles.
Examination of heavily
stained cells by flow cytometry (Table
3) revealed only fair
stability in a
population. Samples were normally collected in
triplicate and were
frequently reanalyzed. These samples consistently
gave very stable
population and DNA data. However, an 18-day-old
culture of
C. oligotrophus showed a 38% increase in population
in response to
extended DAPI-Triton X-100 treatment. Two-thirds
of the population was
in an off-scale cluster for which the mass-per-cell
value was large but
the number and level of fluorescence were
low. Additional staining time
resulted in a normal histogram,
and the total cell mass remained
unchanged. Stationary-phase organisms
sometimes produce clumps, and the
data are consistent with formation
of aggregates that were stain
resistant but dissociated with additional
exposure to the Triton
X-100-stain mixture. Thus, Triton X-100
may increase the apparent
number of organisms in old populations,
but the axial-ratio-corrected
cell mass should remain unchanged.
Longer staining times and higher
stain concentrations can increase
the apparent sizes of populations,
perhaps by bringing debris
with nonspecific DAPI binding into the
bacterial regions on histograms.
Because error is exacerbated at the
small end of the size spectrum,
where electronic and particle
contamination are more prevalent,
the variations in total bacterial
biomass are more dependable
than the variations in cell number. Slow
concentration-dependent
changes in stain binding (Fig.
2) demonstrated
the value of carefully
controlled staining conditions for the organisms
analyzed, both
the standard organisms and the unknown organisms.
Standard curves.
DAPI-DNA fluorescence intensity increased
with chromosome number for both E. coli and C. oligotrophus in laboratory media with near perfect linearity and
negligible intercepts (Fig. 3). Changing
the salinity from 13 mM sodium in M9 medium to the salinity in seawater
medium decreased the fluorescence of E. coli 20%, while
diluting the seawater medium used to grow C. oligotrophus so
that the sodium concentration was decreased from 700 to 13 mM increased
the fluorescence only 8%. Severely (40-fold) diluting the media
increased the fluorescence 28 to 42% depending on the number of
chromosomes per organism (Table 4). Based
on the reduction in the 2n/1n fluorescence ratio, the 2n cells
exhibited more of the competitive effects of sodium ions, and the
effects were sufficient to reduce the Kb for
pure DNA by 103-fold (69). The apparent
accessibility of E. coli DNA to salt and the linearity of
the C. oligotrophus fluorescence curve over a range of
chromosome numbers suggest that the DNA contents of unknown
organisms can be estimated by using an E. coli
standard that is equilibrated in the media of the unknown organisms,
provided that extremely high salt concentrations are avoided.
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FIG. 3.
Effect of chromosome number (n) on DAPI-DNA fluorescence
intensity in both fresh and saline media. For E. coli n = 1 to 4, y = 741.1x 48.9 (r2 = 0.995), and y = 569.5x 3.3 (r2 = 0.998). For C. oligotrophus, n = 1 to 3, y = 439x 14.9 (r2 = 0.998), and y = 409.1x 5.4 (r2 = 0.999).
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The background fluorescence values for brownwater lakes such as East
Twin Lake were small, about 8% of the mean value for
the
microflora. The background fluorescence was even less in lakes
without
a golden yellow cast, and the bacterial signals for several
such
lakes were better separated from the background
signals.
Packing.
The concentrations of DNA in bacteria are several
times the concentration found in human diploid nuclei (1%, by volume)
(25) and are sufficient to give measured attenuation of
DAPI-DNA fluorescence in the larger aquatic bacteria (10)
of up to 10%. Distances are too great for resonance energy transfer
(13), but the complex could absorb reemitted photons.
Packing effects were examined by comparing the relative fluorescence
values for cells with one and two chromosomes in numerous E. coli subpopulations, but no packing effects were detected (data
not shown). For C. oligotrophus the change in the amount of
fluorescence per chromosome was also undetectable with cells having one
to five chromosomes (Fig. 4). However,
the dry mass of the organisms also increased, and without an increase
in cell density; the DNA concentration increased only by a factor of
0.5 (Fig. 4C). Fluorescence attenuation due to increased DNA packing
could therefore have escaped detection. The corrections for packing
appeared to be small for the relatively large commonly cultured
bacteria, such as E. coli, due to low DNA concentrations,
and the corrections for aquatic forms were small due to the thin cross
section associated with small size. Therefore, corrections for
fluorescence attenuation due to DNA packing were not incorporated into
measurements of cellular DNA content.
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FIG. 4.
DNA content of C. oligotrophus. (A) DNA
histogram for a stationary-phase culture with chromosome runout. The
coefficients of variation for the one- to five-chromosome peaks ranged
from 4.28 to 8.73%. (B) Linearity between DNA content determined from
the standard curve and modal values for the fluorescence intensity from
panel A. (C) Relationships between DNA content and dry mass and between
DNA content and DNA concentration. Values were calculated by using a
genome size of 3.5 fg cell1 and a dry weight/wet weight
ratio of 0.2; dry mass was determined by using forward light scatter
intensity data (49).
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DNA from extracted populations versus flow cytometry.
The
kinetics of Hoechst staining of pure DNA as determined with a
spectrofluorimeter were similar to those of DAPI staining of whole
cells as determined by flow cytometry. The standard curve was linear
for DNA concentrations between 0.04 and 1.2 µg ml1, and
there was an intercept at 0.08 µg ml1 (data not shown).
The method was applied to three species of bacteria, and the DAPI-DNA
fluorescence from each species was linear with the population of added
cells (Fig. 5). Mean values for the DNA
contents of four species determined by flow cytometry were compared to
average DNA content values based on the amount extracted by wet methods
and the number of cells determined by flow cytometry (Fig.
6). The values were not G+C corrected
since the AT-specific stain DAPI (56) has a binding
mechanism similar to that of Hoechst 33258 (37). Despite
possible errors due to incomplete pelleting and/or resuspension of the
small low-density organisms, there was a good correlation between the
two methods.
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FIG. 5.
Fluorescence intensity as determined with a
spectrophotometer of Hoechst 33258-stained DNA extracted from various
dilutions of cultures of three species of bacteria.
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FIG. 6.
Comparison of apparent DNA contents of DAPI-stained
cells of various species determined by flow cytometry with values
determined by spectrophotometric analysis of Hoechst-stained extracts
of populations measured by flow cytometry (y = 1.04x + 0.12; r2 = 0.899).
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Specificity.
The absence of intercepts in the standard curves
(Fig. 3) is consistent with the absence of strongly binding cellular
components other than DNA. Staining of cell components that bind DAPI
more weakly than DNA binds DAPI should result in a concave-up Scatchard plot. The linearity observed (Fig. 1, inset) suggests that the concentrations were insufficient for nonspecific binding of DAPI to
weak sites, such as G+C-rich regions of DNA (2, 69), where binding is by intercalation, or to other macromolecules, such as
polyphosphate, slime (30), RNA, and cell wall components (33). Nonspecific staining occurs in the presence of
excessive concentrations of DAPI (Fig. 2); however, at the
concentration used, 0.5 µg ml1, no sites with
Kb values sufficient to affect DAPI-DNA
fluorescence were detected. The large Kb value
and the absence of saturation with DAPI (Fig. 2) are consistent with
binding primarily at the AT-n 
3 sites when long staining times
are avoided.
Effective DNase treatment should eliminate DAPI fluorescence if the
stain is specific; the reduction in
C. oligotrophus
fluorescence
was only 88% (data not shown). However, neither
E. coli fluorescence
nor
M. arcticus fluorescence was
significantly affected, confirming
that substantially intact DNA can
persist in Triton X-100-permeabilized
DNase-treated cells
(
26). RNase had no effect on fluorescence,
which is
also consistent with good specificity for DNA, but its
efficacy in
intact cells is unknown. Our data call into question
the practice of
using nuclease treatment to render broad-spectrum
dyes DNA or RNA
specific.
The DNA measurements for intact cells determined by flow cytometry were
similar to those obtained for extracted DNA, as mentioned
above. Our
data corroborated the thermodynamic measurements that
showed that there
was undetectable interference from weaker binding
sites (Fig.
1, inset)
at the concentrations used. In addition,
the agreement between values
for DNA in intact cells and values
for solubilized DNA from these cells
suggests that minimal error
was attributable to changes in medium
chemistry, packing effects,
or binding hindrance by supercoiling
(
61). Thus, under restricted
staining time, concentration,
and temperature conditions used,
DAPI appears to be a specific stain
for the DNA of small nonpigmented
bacterium-like organisms when it is
used with a size parameter
such as forward light
scatter.
Bacterioplankton DNA.
The DNA content of indigenous seawater
bacterioplankton determined by the methods used in this study was less
than previously published values, but the concentrations were
surprisingly high. According to the histogram for a typical
euphotic-zone seawater sample (Fig. 7),
the maximum DNA content can be 10% (dry weight basis) with a range of
4 to 16% for the bulk of the population. The DNA content of an
extinction culture isolate, C. oligotrophus, was also high,
14%, compared to 2.2% for E. coli, but the low dry weight
was mitigated by the dilute cytoplasm of C. oligotrophus (16% solids, compared to 26% solids for E. coli)
(10). None of the small-genome bacterioplankton of
seawater have been isolated. The maximum fraction that we have been
able to cultivate from Alaskan waters by extinction culture techniques
is about 10% (46; unpublished data), and all of the
organisms have moderate genome sizes. Thus, small genome size and
resistance to isolation are covariant. The histogram obtained is
typical for both lakewater and seawater in that there is a single
loosely defined major cluster that accommodates regions rich in 1n and
in 2n cells having fairly small genomes and it is possible that some
larger-genome 1n cells are present in the subpopulation labeled 2n.
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FIG. 7.
Dot plot of euphotic zone marine bacterioplankton
collected from Thumb Cove in Resurrection Bay off the Gulf of Alaska.
The locations of dims, a cluster at the likely position of
Synechococcus, and 1n cells and a possible location of 2n
cells are indicated.
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Dims.
A significant population with low fluorescence
corresponding to a DNA concentration of about 0.5 fg
cell1 appears in most lakewater (9) and
seawater (Fig. 7) samples. This population is usually separated from
the normally fluorescent organisms by a particle-free area in
scatter-fluorescence (cell mass-DNA) histograms. Starvation has been
reported to result in a decrease in the amount of DNA from 30 to 1 fg
per cell for isolate ANT-300 (41). Such low-DNA particles
have been referred to as dims (57) due to the weak
fluorescence when they are stained with DAPI. It has been suggested
that these particles are cytoplasmless ghosts and that such
corpses can constitute most of the bacterium-sized particles
(27). However, cytoplasm is the main contributor of the dipoles that are responsible for light scattering, and the scatter
signal is nearly as large as that for most particles in the main
bacterial cluster. It is noteworthy that DNA staining of mixed species
requires attention to permeabilization; some species are quite
resistant, as mentioned above. Also, stain concentrations must be
adequate so that the stain occupies the intended sites but does not
stain low-affinity sites. The specific affinities for amino acids of
the low-mass half of lakewater bacterioplankton sorted from the total
population were the same as those of the large-cell bacterial fraction
(8), so the small cells can be as active as the large
cells, but we have not specifically determined the uptake rate of the
dims. The particle mass of most viruses is small for detection by flow
cytometry, and our efforts to quantify viruses by using DAPI staining
have been unsuccessful. Based on unpublished observations of numerous
diverse lake and seawater sites and depths, dims usually account for
between 10 and 30% of the total population, values which are less
than some much higher values that have been reported (18,
35).
Low-fluorescence organisms have appeared in starved cultures of
E. coli (
49) and in
M. thermoautotrophicum cultures (Table
1). Repositioning of 37% of
the in situ Resurrection Bay bacteria
into a high-fluorescence cluster
after permeabilization showed
that these organisms are DAPI-impermeant
organisms rather than
low-DNA-content organisms, as would be expected
for a normal unculturable
archaeal population of that size
(
42). The remaining 14% should
represent the true
possibly low-DNA-content forms (dims). Formation
of dims from
acetate-grown
C. oligotrophus cells in the laboratory
(Fig.
8A) after substrate was withheld (Fig.
8B) was demonstrated
by a fourfold decrease in fluorescence and a loss
of the 2n population.
With longer staining times these dims became
indistinguishable
from those with a normal DNA content. Since the
intensity of scattered
light from them, which was largely a measure of
cytoplasmic protein
content, was reduced only slightly, the DNA signal
was preferentially
lost, either through hydrolysis or through leakage.
The rate (3%
day
1) and extent (100%) of transformation
to dims is shown in Fig.
8C. Dims in the seawater sample (Fig.
7)
contained 0.2 to 0.8
fg of DNA cell
1 and weighed 1 to 8 fg (dry weight).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 8.
Formation of low-fluorescence dims from C. oligotrophus cells. (A) Histogram of light scatter (cell mass)
versus DAPI-DNA fluorescence for a normal population. s, 0.6-µm
beads. (B) Population formed after substrate removal. The computed
amount of DNA decreased from 2.1 and 4.2 fg cell1 for the
1n and 2n clusters to 0.2 to 0.6 fg cell1. (C) Rate of
dim formation.
|
|
Data have established that dim cells can be generated from normal
bacteria by starvation. The moderate rate of dim formation
(3%
day
1), coupled with doubling times ranging from 1 day to
1 month,
is consistent with the higher levels of dims that appear in
more
active systems, such as warm surface lakewater and
seawater, than
in deep seawater. One advantage of intact DNA
leakage during dim
formation, if it occurs, is to maintain the
dissolved DNA pool
for cross-species genetic
exchange.
DNA content and growth rate.
Because in situ growth rates of
most aquatic heterotrophic bacteria are difficult to evaluate and the
isolation success rate is low and because large-genome representatives
do not always behave well in continuous cultures for controlled
steady-state growth, the cell cycle of these organisms is not well
established. C. oligotrophus has many oligobacterial
properties, such as a high affinity for nutrients, large surface-to-dry
mass ratio, and low cell mass (10). Mild clumping caused
imperfect behavior in continuous cultures. Acetate supported good
growth, but the associated specific affinity was low (10),
so the rate of growth could be controlled for several days in batch
culture by adjusting the acetate concentration (Fig.
9) before significant substrate depletion
occurred. The number of genomes was apparent and growth rate dependent.
The amount of dry mass increased from 25 to 42 fg cell1
as the number of multiple-chromosome cells increased, and there was an
increase in the amount of cell mass per chromosome (Table 5), which is consistent with a need for
additional cytoplasmic enzyme capacity (8). Cell cycle
parameters showed that there was a rather constant C phase and a nearly
30-fold decrease in the time spent as single-chromosome organisms. Both
the chromosome ratio and the growth rate were linear with acetate
concentration up to the maximum specific growth rate
(µmax) (Fig. 10). Gradual saturation by acetate was absent, as expected for a substrate that
enters by diffusion with truncation at the µmax, as
previously observed in a continuous culture (10), due to
limitation at some downstream metabolic step. The growth rate(µ) is
given directly by the chromosome number:
|
(2)
|
where
XB and
XD are the masses of one- and two-chromosome
organisms, respectively, and
kc is the cell
cycle constant (0.07
h
1 in this case).
kc can be related to the concentration of
substrate
through specific affinity theory (
5):
|
(3)
|
where
aA is the specific affinity
for a substrate,
A is the concentration of that substrate,
and
YXA is the intervening cell
yield. The
advantages of this method are that the problems of
bottle effect and
dissimilar responses to probes common with other
methods are minimized,
mechanism-sensitive specific affinity theory
is used to empirically
link the chromosome ratio to nutrient concentrations
(
5)
and to phenomenological treatments for phototrophs (
40,
65), and requirements for a defined relationship between
µ
max and organism affinity as defined by the
Michaelis-Menten relationship
are eliminated (
6). However,
this method has not been tested
with the abundant small-genome
organisms, problems such as the
causes of chromosome runout
(
58), when suddenly starved organisms
generate multiple
genomes, are not understood, and the distribution
of genome sizes among
bacterioplankton is not yet known with certainty.
Still, the amount of
DNA per cell, like cell size (
55), provides
a potentially
species-independent reflection of the rate of growth
that can be
conveniently measured, and the method seems to reflect
general levels
of activity in near-arctic lakes, where a large
change in specific
affinity occurs over the seasons and the specific
affinity varies with
the amount of DNA per cell (unpublished data).
Without an increase in
the mean genome size during the spring,
which is not anticipated due to
an increase in the supply of major
substrates, such as amino acids, an
increase in the number of
copies per cell with growth rate is likely.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 9.
Growth curves and dry mass-DNA histograms for C. oligotrophus. The histograms show the results obtained after 24 h
of growth. The distributions of light scatter and fluorescence
intensities associated with the cells at three growth rates are given
(lower scales), and these values were converted to dry weights and DNA
contents (upper scales) for the main subpopulations.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 10.
Effect of acetate concentration on the growth rate
( ) and the chromosome ratio of C. oligotrophus determined
from the 2n and 1n chromosome peak heights (Fig. 9) after 17 h
( ) and 24 h ( ) in batch culture.
|
|
Genome sizing.
Since the fluorescence associated with 1n cells
can be resolved by flow cytometry, the genome sizes of isolates can be
estimated easily. Data suggest a small genome size, approximately 1.5 Mb, for the uncultivatable bulk of the bacterioplankton. This is
because slowly growing populations are rich in 1n cells and extra
copies of chromosomes are a particular burden in oligotrophic systems.
If the G+C content of an isolate is different from that of
E. coli, a correction is required (equation
1).
However, a 10%
difference from the standard value results in a 40%
change in
fluorescence, and a value of 3 for
n A · T
combinations may not
be maximal (
1). The method was tested
with some cultures of
organisms with previously reported genome sizes
and with some
isolates (Table
6). One
might question whether flow cytometry
underestimates bacterial DNA
content due to conformations not
accessible to stain, inadequate cell
envelope permeabilization,
high G+C content, absorption by pigments,
exclusion of stain by
sodium ions, self-absorption due to packing, or
incomplete staining
as discussed above. However, our examinations
failed to detect
significant errors, and the last three possible
reasons for underestimates
mentioned above are minimized in small
organisms. The very large
mean value for the dry weight of DNA for
natural populations (12
to 16%) compared to the values for commonly
cultured organisms,
such as
E. coli (1.3%)
(
8), suggests that if the potential
errors, which
generally favor underestimates, are significant,
then the DNA contents
would be even higher than the very high
values measured.
Electrophoretic methods for genome sizing were
problematic for
C. oligotrophus as well (unpublished), and the
values can differ from those obtained by sequence analysis. However,
agreement with results of wet methods in which a different probe
was
used, and failure to find an indication of error aside from
differences
with previously published genome size determinations
are consistent
with reasonable cross-species accuracy, and relative
intraspecies
accuracy should be excellent.
| |
ACKNOWLEDGMENTS |
This work was supported by National Science Foundation Polar and
LExEn programs.
Information was provided by Hugh Ducklow, Tim Hollibaugh, and L. B. Fandino.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Marine Science and Department of Chemistry and Biochemistry, University of Alaska, Fairbanks, AK 99775. Phone: (907) 474-7708. Fax: (907) 474-7204. E-mail: dkbutton{at}ims.uaf.edu.
| |
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Applied and Environmental Microbiology, April 2001, p. 1636-1645, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1636-1645.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
