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Applied and Environmental Microbiology, July 1999, p. 3251-3257, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Determination of Total Protein Content of Bacterial
Cells by SYPRO Staining and Flow Cytometry
Mikhail V.
Zubkov,1,2,*
Bernhard M.
Fuchs,2
Heike
Eilers,2
Peter H.
Burkill,1 and
Rudolf
Amann2
Plymouth Marine Laboratory, Plymouth PL1 3DH,
United Kingdom,1 and Max-Planck-Institut
für Marine Mikrobiologie, D-28359 Bremen,
Germany2
Received 22 December 1998/Accepted 20 April 1999
 |
ABSTRACT |
An assay has been developed for measuring protein biomass of marine
planktonic bacteria by flow cytometry. The method was calibrated by
using five species of Bacteria (an Arcobacter
sp., a Cytophaga sp., an Oceanospirillum sp., a
Pseudoalteromonas sp., and a Vibrio sp.)
recently isolated from seawater samples and grown in culture at
different temperatures. The intensity of SYPRO-protein fluorescence of
these bacteria strongly correlated with their total protein content,
measured by the bicinchoninic acid method to be in the range of 60 to
330 fg of protein cell
1
(r2 = 0.93, n = 34). According to the calibration, the mean biomass of planktonic
bacteria from the North Sea in August 1998 was 24 fg of protein
cell1.
| |
TEXT |
The accurate measurement of
bacterial biomass is an outstanding problem of modern aquatic microbial
ecology (examples are shown in references 5 and
19). These measurements are needed for balancing
stocks of organic matter at sea and for relating rates and efficiencies
of heterotrophic bacterial growth to nutrient concentration and grazing pressure.
There are several approaches to determine the biomass of bacteria. The
cell dimensions can be measured by microscopy, and subsequently
biovolume can be computed and transformed into dry weight or cellular
elemental contents of carbon or nitrogen (examples are shown in
references 3, 23, 29, and 30).
Direct elemental composition of bacterial cells can be determined by
using X-ray microanalysis (8, 14). This method provides
accurate measurements, but it is limited by laborious procedures and
difficulties in obtaining absolute calibration. Size fractionation
through a series of nucleopore filters was employed for estimating
average diameters of bacterial cells (4, 33, 35), although
the problem of converting cell biovolume into elemental content
remained unsolved. Flow cytometric enumeration of planktonic bacteria
by using nucleic acid stains has become routine (20-22,
24). Flow cytometry can also provide an estimate of biomass from
the intensity of light scattered by single cells (26).
However, most flow cytometric data for cell size were reported in
arbitrary units (1, 7). Recently the method's capacity to
obtain absolute quantification was improved by using light scatter
theory that accounted for differences in cell shape and relative
refractive index (17, 25). However, the precision of this
method decreases when populations of unknown bacterial species or
complex natural bacterioplankton communities are analyzed.
Another possible technique for measuring individual cell biomass
derives from the quantitative staining of cellular proteins, the most
abundant cell macromolecules, which comprise about one-half of the dry
weight of prokaryotic cells (e.g., 55% in Escherichia coli
[15]). Total protein staining proved to be useful in
monitoring the growth and metabolism of different populations of blood
cells, and in most applications protein stains are not used
alone but are combined with DNA fluorochromes (an example is shown in
reference 28) for better differentiation of cells
from debris in samples. Similarly to light scatter, the measurements of
cellular compounds are generally provided in arbitrary units, and there
have been very few studies where cellular compounds were accurately
calibrated in absolute units (11, 24, 34).
Due to their small size, natural bacteria require a very bright dye to
stain their protein sufficiently strongly to be within the detection
range of modern flow cytometry. Therefore, we chose the recently
developed SYPRO dyes, which were introduced for extrasensitive detection of proteins in gels (12). In the presence of
excess sodium dodecyl sulfate (SDS), nonpolar regions of polypeptides are coated with detergent molecules, forming a micelle-like structure with a nearly constant SDS-to-protein ratio. The SYPRO dye binds to the
SDS coat surrounding proteins and exhibits little protein to protein
variation. A colorimetric assay described by Smith et al.
(31) that uses bicinchoninic acid to chelate Cu+
ions reduced from Cu2+ in the presence of proteins at high
pH was chosen to quantify amounts of protein.
In the present study, we explored the possibility of calibrating the
intensity of fluorescence emitted by whole bacterial cells stained with
SYPRO in femtograms of protein per cell. We used five species of
bacteria of different phylogenetic affiliations: three members of the
gamma-subclass of the class Proteobacteria (a
Pseudoalteromonas sp., an Oceanospirillum sp.,
and a Vibrio sp.), one member of the epsilon-subclass of the
class Proteobacteria (an Arcobacter sp.), and one
member of the class Cytophaga-Flavobacterium-Bacteroides (a
Cytophaga sp.). These bacteria were isolated from seawater samples collected in the North Sea. Using this set of cultured bacteria
for calibration, we assumed that cellular fluorescence of SYPRO-stained
natural bacterioplankton can be extrapolated into cell protein biomass
that can be used directly or, if needed, converted into elemental
biomass of carbon or nitrogen with the knowledge that proteins comprise
about half of bacterial dry weight.
Cultures.
Seawater samples were collected from the North Sea
in the vicinity of the island of Helgoland. The bacteria (Table
1) were isolated on plates with synthetic
seawater medium prepared according to the method of Schut et al.
(27) except for lacking aromatic and polymeric substrates
and containing 1% Bacto agar (Difco, Detroit, Mich.). For
subculturing, peptone (5 g liter1) and yeast extract (1 g
liter1) were added, and cells were incubated at 4, 10, and 20°C in the dark. Cells were harvested at intervals to provide
diverse cell sizes of bacteria. Additionally, dilution culture
experiments were conducted with seawater samples collected at the same
place. Ten milliliters of seawater, screened through a polycarbonate, 0.8-µm-pore-size filter to decrease the number of protozoan
predators, was diluted with 90 ml of seawater filtered through a
0.2-µm-pore-size filter and kept at 20°C in the dark. The
bacterioplankton populations were monitored by regular sampling.
One-milliliter subsamples were fixed with 0.8% (final concentration)
glutaraldehyde or paraformaldehyde every 12 h and kept frozen.
Sample preparation, staining procedure, and total protein
determination.
Cultured bacteria were washed with 1 ml of
aged natural seawater (filtered through a 0.2-µm-pore-size
filter), centrifuged at 4,000 × g, resuspended, and
filtered through 0.8-µm- or 1.2-µm-pore-size polycarbonate
filters (Millipore, Bedford, Mass.) to produce a final
concentration of 2 × 109 to 9 × 109
cells ml1. Subsamples of 10 to 100 µl were transferred
to 2-ml microcentrifuge tubes and kept frozen at 
20°C for
subsequent protein determination in these tubes. Generally, replicated
200-µl subsamples were fixed at 4°C for 1 h with
paraformaldehyde (0.8% final concentration) and then kept frozen at
20°C. Additional parallel subsamples were fixed with glutaraldehyde
or formaldehyde at the same final concentrations, and some
paraformaldehyde-fixed samples were kept at 4°C for comparative study.
The protein biomass of bacteria was directly measured in triplicate
subsamples by using the bicinchoninic acid (BCA) method
(kit number
BCA-1; Sigma, Deisenhofen, Germany), with bovine serum
albumin as a
standard; the coefficient of variance (CV) for replicated
measurements
was below 5%. The average protein content of individual
bacterial
cells was computed from these protein measurements by
dividing the
value obtained for total protein content by the number
of bacterial
cells determined by flow
cytometry.
For flow cytometry, samples of fixed bacteria were thawed at 4°C and
gently vortexed or mixed with a pipette. Cultured bacteria
were diluted
1,000-fold with MQ-water (Millipore, Bedford, Mass.)
filtered through a
0.2-µm-pore-size filter and were simultaneously
stained with Hoechst
33342 (final concentration, 0.4 µg ml
1) (Molecular
Probes, Eugene, Oreg.) and with SYPRO red (in excessive
concentration,
1/10,000 dilution of the commercial stock) (Molecular
Probes) in the
presence of SDS (final concentration, 0.04%) at
20°C for at least 15 min before being analyzed by flow cytometry.
Bacteria grown in dilution
culture were diluted twofold and generally
stained in the presence of
0.01% SDS. To optimize the staining
procedure, the
Cytophaga sp. and
Pseudoalteromonas sp.
were stained
in the presence of various concentrations of SDS
(0.004 to 0.12%)
at salinity from 0 to 75% of natural
seawater.
Standards.
Yellow-green fluorescent microspheres (Molecular
Probes) of 0.5-µm and 2.0-µm diameters were used for the alignment
of the flow cytometer. Although the 0.5-µm beads (CV < 5%)
produced by this company were of superior quality to 0.5-µm
yellow-green fluorescent latex microspheres (CV < 8%)
(Fluoresbrite Microparticles; Polysciences, Warrington, Pa.), the
latter had significantly higher blue fluorescence excited by UV and,
therefore, were more suitable as a universal internal standard of
forward-scatter, blue and red fluorescence.
Flow cytometry.
Stained, replicated samples were analyzed with
a FACStar Plus flow cytometer (Becton Dickinson, Mountain View, Calif.)
equipped with two lasers. The first, argon, laser (Innova 90; Coherent Inc., Palo Alto, Calif.) was tuned to UV multiline-emission (351.1 to
363.8 nm) at 115 mW. The second, Diode-pumped solid-state, laser (DPSS
532; Coherent) had an emission at 532 nm with 200 mW output power.
Light emitted by the first laser and scattered by particles in the
forward direction was focused on a photomultiplier tube through a
360 ± 20-nm band-pass filter. Blue fluorescence from Hoechst
stain bound to nucleic acids was collected through a 460 ± 25-nm
band-pass filter. The fluorescence from SYPRO red, bound to SDS-coated
proteins, excited by the second laser was collected through a 620 ± 60-nm band-pass filter. Data acquisition and analysis were done with
Cell-Quest software (Becton Dickinson). Acquisition was triggered by
blue fluorescence to reduce the noise signal of the particulate and
nonparticulate backgrounds. The ratio of the mean forward light scatter
or fluorescence intensity of a bacterial population to the 0.5-µm
internal standard beads was used to normalize samples and to calculate
bacterial protein content. The bead intensity was taken as 100 units.
The absolute concentration of beads in a standard stock suspension was
determined by epifluorescence microscopy. Four replicates of 100 and
200 µl of the bead solution were filtered on 0.2-µm-pore-size,
polycarbonate filters, 300 to 900 beads were counted in 20 fields of
view under a Zeiss Axioplan microscope by using a 100× Plan Neofluar
objective by two persons, and an average concentration of beads was
calculated (e.g., 26 × 106 beads
ml1, CV 4%). The ratio of bead abundance to number
of bacteria was used to compute the concentration of the latter.
Each of the dual-stained bacterial cultures had distinct flow
cytometric signatures illustrated on bivariate dot plots of
three
recorded parameters (Fig.
1). The fluorescence
intensity
of bacteria stained with SYPRO dye strongly correlated with
measured
cellular protein contents for five cultured species of
bacteria
(
r2 = 0.93,
n = 34) over the
range of 60 to 330 fg of protein cell
1 (intercept
b0 = 53.5 ± 357, slope
b1 = 17.7 ± 0.89) (Fig.
2).
By using linear regression as a
calibration curve, the protein
contents of natural bacteria grown in
dilution culture were calculated.
Bacterial cells of the original
population of bacterioplankton
from the North Sea contained 24 ± 4 fg of protein cell
1 (
n = 12). Their
average protein content increased to above 100
fg of protein
cell
1 during growth in dilution culture and became
comparable to the
protein content of the
Cytophaga sp., the
Oceanospirillum sp.,
and the
Vibrio sp.
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FIG. 1.
Flow cytometric analyses of the bacterial cultures
stained with SYPRO and Hoechst. Bivariate dot plot diagrams of the
intensity of forward scatter versus the intensity of Hoechst-DNA
fluorescence are shown in the left column, dot plots of forward scatter
versus SYPRO-protein fluorescence are shown in the middle column, and
dot plots of SYPRO-protein fluorescence versus Hoechst-DNA fluorescence
are shown in the right column. The symbols used to label bacterial
cultures are the same on all figures.
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FIG. 2.
Comparison of the mean fluorescence intensities of
SYPRO-stained bacteria and corresponding measurements of cell protein
content determined by the BCA method. Open hexagons show protein
contents of bacteria in seawater samples and dilution cultures,
calculated from the intensity of SYPRO-protein fluorescence by using
linear regression. Other open symbols show red autofluorescence of
cultured bacteria in the absence of SYPRO staining.
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|
Methodological questions of quantitative staining of bacteria.
The autofluorescence of bacteria fixed with paraformaldehyde without
SYPRO staining was only 1% that of the fluorescence of stained cells
(Fig. 2). In the absence of SYPRO dye, the fluorescence intensity of
Hoechst staining was somewhat lower (b1 = 0.88 ± 0.033, r2 = 0.8, n = 45)
because of additional low blue fluorescence of SYPRO; however,
forward-scatter intensity remained unchanged (b1 = 0.99 ± 0.014, r2 = 0.99, n = 45)
(Fig. 3). Comparison of dual staining of
bacteria (the Cytophaga sp., the Oceanospirillum
sp., and the Vibrio sp.) fixed with three different
aldehydes revealed no significant difference in SYPRO-protein
fluorescence intensity between glutaraldehyde- and
paraformaldehyde-fixed cells, although formaldehyde-fixed cells had
slightly lower fluorescence (Fig. 4a).
The fluorescence intensity of Hoechst-stained cells fixed with
paraformaldehyde was similar to that of formaldehyde-fixed cells, while
glutaraldehyde-fixed cells had somewhat lower fluorescence (Fig. 4b).
Cells fixed with glutaraldehyde scattered significantly more light than
cells fixed with either paraformaldehyde or formaldehyde (Fig. 4c). The
observed differences probably reflected differences in the fixation
process, since formaldehyde penetrated rapidly into cells but reacted
slowly, while the opposite is true for glutaraldehyde (9,
13). Consequently, paraformaldehyde was chosen as the standard
fixative.
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FIG. 3.
Comparison of the intensities of Hoechst-DNA
fluorescence (a) and forward scatter (b) with and without
SYPRO-staining of bacteria.
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FIG. 4.
Comparison of the intensities of SYPRO-protein
fluorescence (a), Hoechst-DNA fluorescence (b), and forward scatter (c)
of cultured bacteria fixed with paraformaldehyde [PFA] and with
glutaraldehyde (closed symbols, solid line of linear regression) or
with formaldehyde (open symbols, dotted line of linear regression) in
the left column. Comparison of the same parameters (d through f) of
bacteria fixed with paraformaldehyde and kept either frozen [Froz.]
or refrigerated [Refr.].
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|
Storage of fixed bacteria at 4°C for 24 h compared with freezing
at
20° did not affect staining with SYPRO but did result
in
slightly lower Hoechst-DNA fluorescence and higher intensity
of forward
scatter (Fig.
4d through f). Longer storage of refrigerated
samples led
to a decreased SYPRO-protein fluorescence due to an
ongoing fixation
(data not shown). The fluorescence of bacteria
stained in freshwater
gradually increased with the increase of
SDS concentration from 0.004%
to 0.04% and then started to saturate
at 0.08 to 0.12% SDS (Fig.
5) followed by a progressive lysis
of the
cells. In the presence of 25 to 75% seawater, bacteria
started to lyse
at SDS concentrations above 0.02%, whereas the
highest fluorescence
was observed in the presence of 0.008 to
0.012% SDS (Fig.
5). Because
of the relatively low concentration
of bacteria in natural seawater,
the samples of dilution cultures
were diluted only twofold during
staining. The higher ionic strength
of the final solution required only
one-quarter of the concentration
of SDS in the staining mixture (0.01%
compared to 0.04% used for
staining in MQ-water) to obtain similar
protein staining (Fig.
5). The fluorescence intensity of cultured
bacteria stained with
SYPRO in half-diluted seawater in the presence of
0.01% SDS was
slightly higher than in freshwater with 0.04% SDS
(
b1 = 1.14 ±
0.035,
r2 = 0.98,
n = 8).
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FIG. 5.
Contour plots showing the complex dependency of the mean
intensities of SYPRO-protein fluorescence of cultured
Cytophaga sp. (a) and Pseudoalteromonas sp. (b)
on the concentration of SDS and salinity of the staining solution. The
dots indicate actual measurements at particular combinations of the two
variables.
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|
Other cell compounds (e.g., DNA) and properties (e.g., light scatter)
were used to estimate the biomass of microorganisms
(
6,
11,
25,
34). We compared our results of whole-cell
protein staining with
corresponding DNA staining and forward-scatter
intensity of five
species of cultured bacteria and bacteria grown
in dilution cultures
(Fig.
6). Cell DNA content was found to
allow
for an accurate estimation of total cell carbon biomass in
unicellular
phytoplankton (
34). This relationship seems to
be more problematic
for bacteria, since bacterial cells with different
protein contents
have similar DNA contents (Fig.
6a). Furthermore, cell
biomass
of bacterial isolates can vary widely depending on growth media
and temperature, while the cells retain the same DNA content (e.g.,
Vibrio sp.) (Fig.
6a). At the same time, cells with the same
protein
content can have one or two copies of DNA (Fig.
1)
(
Vibrio sp.,
Cytophaga sp., and
Oceanospirillum sp.), and some species can
have even more
copies (
18).
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FIG. 6.
Comparison of the mean intensities of SYPRO-protein
fluorescence of cultured bacteria (n = 86) (closed
symbols) and bacteria grown in dilution cultures (n = 24) (open hexagons) with corresponding intensities of Hoechst-DNA
fluorescence (a) and forward scatter (b).
|
|
Forward light scattering of bacteria helps to resolve bacterial
populations by flow cytometry and has been used to determine
the
biomass of these populations (
16,
25,
26,
32). Forward
scatter and SYPRO-protein fluorescence intensity correlated on
a
cell-to-cell basis for individual species of bacteria that appear
almost spherical when examined by microscopy (Fig.
1,
Vibrio
sp.
and
Arcobacter sp.), and this was also observed for
mixed populations
in dilution cultures. However, our data did not show
pronounced
correlation of these two parameters (Fig.
6b). The intensity
of
light scattered by bacteria of rod or spirillum shapes (e.g.,
the
Cytophaga sp. and the
Oceanospirillum sp.) was
highly variable,
while their protein content was a much more
conservative characteristic.
Therefore, it seems that cellular protein
staining is a more direct
way of determining bacterial biomass than
species- and shape-dependent
light scattering
properties.
In quantifying the total protein content of whole cells, one has to
keep in mind an unavoidable controversy. It is impossible
to stain all
protein molecules, simply because only some of these
molecules and/or
parts of the molecule surfaces are accessible
to a dye in fixed cells.
It is possible to determine total cellular
proteins, but in so doing,
cells would be disrupted and could
not then be counted by flow
cytometry. Therefore, one should be
satisfied if the fluorescence
intensity of stained proteins in
fixed cells correlates with the
independent measurement of total
protein of lysed
cells.
Agreement between the values computed from the fluorescence intensity
of SYPRO-stained bacteria, the values computed from
total protein
measurements by the BCA method, and the numbers
of cultured bacteria
counted by flow cytometry (Fig.
2) did not
depend on the species or
physiological state of the bacteria.
Cultured bacteria used for
calibration were grown at different
temperatures to vary their protein
contents so that, for example,
the mean biomass of
Vibrio
sp. ranged from 100 to 250 fg of protein
cell
1. The
smallest
Cytophaga sp. cells, containing 60 fg of protein
cell
1, had about 4 to 6 times more protein (assuming
equal cell protein
and carbon contents) than oceanic bacterioplankton
(
8,
10)
and only 2 to 3 times more protein than the
planktonic bacteria,
containing 24 fg of protein cell
1,
collected in the coastal zone near Helgoland. Our measurement
of
protein content of coastal bacterioplankton agreed with a direct
measurement of 30 fg of carbon cell
1 (
10),
under the same assumption of equal cell carbon and protein
contents
(
15). Consequently, we conclude that, by using a calibration
based on cultured bacteria, the protein biomass of individual
bacterial
cells of different sizes and physiological states observed
in natural
marine environments of various productivities can be
estimated
accurately. This method is an improvement over other
flow cytometric
methods for determining microorganism biomass
(
2,
6,
11,
24,
25), because it is a direct method
for determination of protein
content of individual cells without
biases due to species and/or shape.
It does not require preliminary
knowledge of individual populations of
bacterioplankton and therefore
can be directly applied to the analysis
of natural
samples.
| |
ACKNOWLEDGMENTS |
We thank M. A. Sleigh and J. Pernthaler for critical reading
and helpful comments on an earlier version of the manuscript.
We are grateful to the Max-Planck-Society for the support of this work.
The research of M.V.Z. was supported by the postdoctoral research
fellowship (GT5/98/16/MSTB) from the Natural Environment Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Plymouth Marine
Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, United Kingdom. Phone: 44 1752 633422. Fax: 44 1752 633101. E-mail:
mvz{at}wpo.nerc.ac.uk.
| |
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Applied and Environmental Microbiology, July 1999, p. 3251-3257, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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