FEMS Microbiology Ecology 35 (2001) 11^17
www.fems-microbiology.org
Feasibility of using GFP-expressing Escherichia coli, coupled with
£uorimetry, to determine protozoan ingestion rates
Jacqueline D. Parry
b
a;
*, Karen Heaton a , Janice Drinkall a , Harriet L.J. Jones
b;1
a
Division of Biological Sciences, I.E.N.S., Lancaster University, Lancaster LA1 4YQ, UK
NERC Centre for Population Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PU, UK
Received 23 March 2000 ; received in revised form 5 October 2000; accepted 5 October 2000
The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP),
coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once
cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli
and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium
against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A
decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy,
with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within
the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the
ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate
abundance. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Protozoan ingestion ; GFP-expressing Escherichia coli; Fluorescently labelled microsphere; Fluorescently labelled bacteria ; Fluorimetry
1. Introduction
In recent years, the interest in determining global carbon budgets has been increasing [1]. At the very base of
aquatic food-webs, photosynthetic by-products from algae
[2] and humic substances [3] provide a carbon source for
bacteria and, for this carbon to enter the metazoan food
chain, the bacteria must be consumed. Protozoa, particularly heterotrophic £agellates and ciliates, are the major
consumers of bacteria and are considered to form an important trophic link between the bacteria and metazoa
[4,5]. It is therefore important to determine the rates at
which bacteria are processed, to aid estimates of overall
carbon dynamics in aquatic systems.
To date, the majority of studies on protozoan prey-processing have been directed at the ¢rst stage, i.e. ingestion/
grazing rates, and a number of methods have been derived
* Corresponding author. Tel. : +44 (1524) 593489;
Fax: +44 (1524) 843854; E-mail : j.parry@lancaster.ac.uk
1
Present address: Division of Environmental Sciences, University of
East Anglia, Norwich, Norfolk NR4 7TJ, UK.
(e.g. [6^10]). The use of £uorescently labelled bacteria
(FLB) [8], coupled with epi£uorescence microscopy, has
undoubtedly been the most widely used to date, however,
criticisms of this method include the fact that the prey cells
are not live and that microscopy counts are time consuming. Thus, the feasibility of a new method for determining
protozoan ingestion/grazing rates was tested here, and employed an Escherichia coli population which had been genetically engineered to express the green £uorescent protein (GFP), i.e. it was a live-£uorescing prey particle.
GFP originates from the jelly¢sh Aequorea victoria and
in vivo it emits green light (509 nm) upon excitation with
the blue light (395 nm). GFP was ¢rst characterised by
Shimomura [11] and in 1992, Prasher et al. [12] cloned
the gfp gene, which can be expressed by both prokaryotic
and eukaryotic cells and is non-toxic [13]. GFP remains
the only protein in which the £uorochrome is directly encoded in the amino acid sequence and hence, no cofactors
or substrates (except for oxygen) are required for £uorescence [14], unlike those required for the analysis of luminescence in luxAB-expressing bacteria [15]. The potential
use of GFP-expressing bacterial cells was based on evidence that once cells are subjected to acidi¢cation, the
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 1 0 6 - 9
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
Abstract
12
J.D. Parry et al. / FEMS Microbiology Ecology 35 (2001) 11^17
£uorescence decreases [16]. Acidi¢cation occurs within the
food vacuoles of protozoa [17,18], thus each prey cell ingested should lose its £uorescence within the ¢rst passage
through the protist, whether or not it is digested. Hence,
the rate at which prey £uorescence is lost will equate to the
rate at which the prey are processed/ingested. The performance of this prey against the two most popular surrogate prey states used to date, £uorescently labelled microspheres (FLMs) and FLB, was tested using three methods
of analysis, i.e. direct and indirect counts using epi£uorescence microscopy and £uorimetry.
2. Materials and methods
ature before use. All prey suspensions were sonicated for
at least 10 min (Camlab, Frequency, 50/60 Hz), visually
checked for the presence of aggregates and then enumerated.
2.3. Maintenance of protozoa
Twenty-nine protists were employed in this study (Table
1) and each was subcultured every 2 weeks by transferring
ca. 5 ml of the stationary-phase culture into fresh medium
(Table 1). Each culture was then supplemented with a
mixed bacterial community, which had been previously
isolated from that particular protozoan culture.
2.4. Enumeration of organisms
Subcloning E¤ciency1 E. coli HB101 (FP mcrB mrr
hsdS20 recA13 supE44 ara14 galK2 lacY1 proA2 rpsL20
xyl5 leu mtl1) (Life Technologies) was transformed with
the vector pBAD-GFPuv (Clontech) following the procedure provided by Life Technologies. GFPuv is a mutant of
the original pGFP with the construct placed under the
pBAD promoter of the arabinose operon and its regulatory gene, araC [19]. The pBAD promoter is induced by
arabinose and repressed by glucose. An ampicillin resistance gene is also present. Excitation and emission wavelengths are the same as the wild-type (395 and 509 nm,
respectively). The GFP produced is expressed in a soluble
form and therefore much brighter (U18) than the wildtype GFP [19].
2.2. Preparation and maintenance of prey particles
Suspensions of yellow/green (YG) FLMs (0.49-Wm diameter) (Polysciences, via Park Scienti¢c Ltd., Northampton, UK) were prepared in sterile distilled water and
stored at 4³C. Live-£uorescing E. coli cells were cultured
at 35³C on Luria^Bertani (LB) agar plates [20] supplemented with ampicillin (0.006% w/v) and arabinose
(0.2% w/v). Spread plates (ca. s 500 colonies plate31 )
were incubated for 48 h before a suspension was prepared
in the desired experimental medium which was then stored
at 20³C for 16^24 h. Preliminary experiments showed that
this method yielded 100% £uorescent cells with the highest
level of £uorescence per cell (10 times brighter than a 0.49Wm YG FLM), while the storage period reduced variation
in bacterial cell size (0.75U2.6 Wm). Heat-killed £uorescently stained E. coli cells were prepared following the
method of Sherr et al. [8]. A cell suspension, prepared as
described above, was stained with 5-([4,6-dichlorotiazin2-YL]amino). Fluorescein (DTAF) in Chalkley's medium
[21]; the recommended bu¡ered saline solution was found
to be detrimental to this particular strain. Stained cells
were stored at 320³C and were thawed at room temper-
All prey types were ¢xed before enumeration with icecold glutaraldehyde at a ¢nal concentration of 0.05% (v/v)
which was the lowest concentration found to be lethal
while allowing minimal reduction (17%) in the E. coli £uorescence. A known volume of ¢xed suspension was ¢ltered
onto a 0.2-Wm black polycarbonate track etch screen membrane (Poretics) and viewed under blue light (BP350-460/
LP515 ¢lter block) at a ¢nal magni¢cation of U1250, using a Leitz Laborlux epi£uorescence microscope illuminated by a 100-W mercury bulb. More than 400 prey
particles were counted on each of three membranes, before
conversion to particles ml31 . Samples were also stained
with 4,6-diamidinophenylindole (DAPI) at 0.0001% (w/v)
concentration (following Porter and Feig [22]) to determine the total concentration of E. coli cells and the number of protozoa. These membranes were viewed under UV
light (BP340-380/LP430 ¢lter block) with more than 400
E. coli cells membrane31 being counted, or 20 ¢elds of
view membrane31 for protozoa, before conversion to cells
ml31 . The proportion of live E. coli cells £uorescing was
then determined and only those E. coli suspensions which
contained 100% £uorescing cells were used in experiments.
2.5. Quantifying £uorescence
Fixed samples (4 ml) were analysed with a Perkin Elmer
203 £uorimeter, illuminated with a xenon lamp and set at
excitor and emission wavelengths 395 and 509 nm, respectively. YG FLMs (0.49-Wm diameter) were employed as
standard £uorescent particles to which the £uorescence
of test samples could be compared. The zero and 100 £uorescence units of the machine (ca. y-axis) were set against a
distilled water blank and a known concentration of microspheres (M100 ), respectively (ca. x-axis). The £uorescence
value of the test sample (FLt ) should then fall between 0
and 100 in order to equate the £uorescence emitted to a
concentration of microspheres (EM) using:
EM ˆ …FLt UM 100 †=100
taking into account the £uorescence of the controls, e.g.
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
2.1. Transformation of E. coli HB101 with the
pBAD-GFPuv plasmid
J.D. Parry et al. / FEMS Microbiology Ecology 35 (2001) 11^17
medium alone. An arbitrary £uorescence value of 2U1037
was assigned to a single microsphere (reasons not discussed) and multiplication of EM by 2U1037 yielded a
relative £uorescence (RF) ml31 value for each sample.
Even though E. coli preparations consistently gave RF
cell31 values of 1^2U1036 , this parameter was still
checked before use in experiments.
2.6. The e¡ect of pH on E. coli £uorescence
2.7. Preliminary screening of protozoa for the ingestion of
live-£uorescing E. coli
Each protozoan culture, in late exponential phase, was
incubated with live-£uorescing E. coli cells (ca. 107 cells
ml31 ) at either 15 or 20³C (Table 1) for up to 2 h. Every
3 min, samples (ca. 20^50 Wl) were viewed as slide preparations with visible light (to locate a protozoan cell) and
blue light (to detect E. coli cells inside the protozoan cell)
at a ¢nal magni¢cation of U500. More than 20 protozoan
cells were examined per sample. Those species which did
not contain £uorescing prey in their food vacuoles during
the 2-h period were incubated with non-transformed E. coli
cells (ca. 108 cells ml31 ) and evidence of any increase in
protozoan abundance was noted.
2.8. Grazing of Tetrahymena pyriformis on the three prey
particles
Three types of prey, live-£uorescing E. coli, DTAFstained E. coli and FLMs, were employed in a more de-
Table 1
Source of protozoa, and culture media, used in a preliminary experiment to determine the number of species capable of ingesting E. coli HB101 transformed with the vector pBAD-GFPuv
Protozoan species
Flagellates:
Anthophysa vegetans
Bodo designis
Bodo saltans
Cercomonas sp.
Diaphanoeca grandis
Dinobryon cylindricum
Dinobryon divergens
Dinobryon setularia
Entosyphon sulcatum
Jakoba libera
Ochromonas danica
Ochromonas tuberculata
Paraphysomonas imperforata
Paraphysomonas vestita
Petalomonas cantuscygni
Poteriochromonas malhamensis
Rhabdospira spiralis
Spumella elongata
Stephanoeca diplocostata
Ciliates :
Colpoda in£ata
Cyclidium glaucoma
Euplotes daidaleos
Paramecium aurelia
Stentor coeruleus
Tetrahymena pyriformis
Vorticella similis
Amoebae:
Sacchamoeba limax
Valkamp¢a avara
Vexillifera bacillipedes
Source
Culture medium
Ingestion of E. coli
CCAP 905/1
DJ Patterson
CCAP 1907/2
HLJ Jones
BSC Leadbeater
HLJ Jones
CCAP 917/1
CCAP 917/2
CCAP 1220/1B
DJ Patterson
CCAP 933/2B
CCAP 933/27
BSC Leadbeater
CCAP 935/14
CCAP 1259/1
CCAP 933/1C
CCAP 1271/5
CCAP 955/1
BSC Leadbeater
SPL
ASW
CH
CH
ASW (15³C)
DM (15³C)
DM (15³C)
DM (15³C)
CH
ASW
DM (15³C)
DM (15³C)
ASW
CH
ASW
DM (15³C)
S/W (15³C)
SPL (15³C)
ASW
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
yes
no
yes
yes
CCAP
CCAP
CCAP
CCAP
CCAP
CCAP
CCAP
1615/2
1616/1
1624/15
1660/3A
1682/1
1630/1W
1690/2
CH
CH
S/W
S/W
S/W
CH
CH
yes
yes
no
yes
yes
yes
yes
CCAP 1527/3
CCAP 1588/1A
CCAP 1590/1
NN
NN
NN
yes
yes
yes
ASW: arti¢cial seawater medium; CH: Chalkley's medium; DM: diatom medium; SPL: Sigma Cereal Leaf-Prescott liquid; S/W: soil/water biphasic
medium ; NN: non-nutrient (amoeba saline) medium; CCAP: Culture Collection of Algae and Protozoa, CEH Windermere, Far Sawrey, Ambleside,
Cumbria, UK (see www.ceh.ac.uk/ccap/ for further information and media recipes). Unless otherwise stated, the incubation temperature was 20³C.
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
Distilled water was adjusted with 1 M NaOH or 1 M
HCl to yield a range of pH values from 2 to 11. Volumes
(10 ml) at each pH were inoculated with live-£uorescing
E. coli and the change in RF ml31 over a 60-s period was
recorded. This was repeated ¢ve times. Those cells within
water at pH values 6 7 were left for 15 min before the
medium was adjusted to pH 7 using a predetermined volume of 1 M NaOH. Any change in the RF ml31 was
recorded, to determine whether regeneration of £uorescence occurred. DTAF-stained E. coli cells were subjected
to pH values 6 7 and monitored for 2 h.
13
14
J.D. Parry et al. / FEMS Microbiology Ecology 35 (2001) 11^17
3. Results and discussion
More than 85% of the protozoan species tested ingested
the live-£uorescing E. coli (Table 1). Those that did not,
showed no increase in abundance when fed with nontransformed E. coli cells, suggesting that it was the prey
species itself, rather than the fact that it had been transformed, which resulted in selection. Non-consumption of
E. coli in this study could have been due to the experimental conditions. For example, Euplotes daidaleos contained
a number of photosynthetic endosymbionts and may not
have been feeding heterotrophically under the light regime
used (80 WE m32 s31 ). Alternatively, E. coli cells may have
been too large for ingestion. For example, Diaphanoeca
grandis only consumes very small bacteria (personal observation). Prey-size selection, particularly by heterotrophic
£agellates [25,26], has been thought to limit the use of a
single marker species for determination of protozoan ingestion rates in situ as ideally, the prey particle used
should be consumed by all protozoa. However, Monger
and Landry [27] concluded that prey size dependency by
direct interception feeding £agellates is modest and the use
of a prey particle which is equivalent to the average size of
the picoplankton should be reasonably representative of
the dynamics of the community. This has been substantiated with the use of E. coli as a single species prey, yielding protozoan generation times equivalent to those recorded with natural bacteria [7,9]. In addition, Pelegari
et al. [28] considered E. coli HB10B to be a useful prey
for studies involving protozoa because it does not grow in
seawater and the use of osmotically shocked E. coli, as
opposed to heat-killed bacteria, avoids the denaturation
of organic compounds that may a¡ect POC and/or DOC
egestion rates, or digestibility. Thus, GFP-expressing
E. coli could be a useful marker prey for both freshwater
and marine studies, particularly since it is a live prey
which retains any motility, as motility may overshadow
the in£uence of size on the selective feeding of planktonic
protists, under some circumstances [27,29].
During preliminary screening (see Section 2.7) there was
some di¤culty in deciding whether or not the protozoa
were ingesting the GFP-expressing E. coli. In some cases
the prey cells were clearly visible inside the protozoan cell
for the full 2-h period whereas in others, they were only
visible within the ¢rst few minutes of the experiment. This
di¤culty could have stemmed from inter-species variation
with regards to the timing of food vacuole acidi¢cation
within the di¡erent protozoa, on which, there is little information. Acidic conditions a¡ected the stability of the
GFP, causing a rapid decrease in E. coli £uorescence. For
example, 99% loss of £uorescence occurred after 15 s at
pH 2 (Fig. 1), agreeing with Casey and Nguyen [16], also
working with E. coli HB101. Signi¢cant loss of £uorescence may have occurred earlier, but sampling could not
be obtained before 15 s. There was also concern that £uorescence might be regenerated when the pH of the food
vacuole returned to pH 7 (usually by 11 and 120 min in
Paramecium and Tetrahymena, respectively [17,18]), as
studies on the isolated protein had shown that, under
some conditions, 90% of the original £uorescence could
be regenerated following acidi¢cation [30]. Those cells
which had been subjected to pH values 6 7 for 15 min
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
tailed grazing experiment with the ciliate T. pyriformis.
Each prey type was inoculated into six £asks containing
60 ml Chalkley's medium, to give an initial concentration
of 5U107 particles ml31 , in excess of indigenous bacteria
(1.25U106 cells ml31 ). All £asks were incubated at 20³C
and the RF ml31 and prey concentration in each £ask
were determined over a 2-h period to con¢rm that the
£uorescence of the prey types was stable in the absence
of a predator. Three of the six £asks were then inoculated
with 5 ml of a late exponential culture of T. pyriformis (ca.
47U16 Wm in size) to give an initial concentration of 560
cells ml31 (test £asks). The three remaining £asks were
inoculated with 5 ml Chalkley's medium to allow for
any reduction in RF ml31 and prey particle concentration
due to dilution (control £asks). These £asks were sampled
hourly for 8 h, then at 16, 20 and 24 h to determine the
fate of the prey via the two indirect methods. The ¢rst
involved monitoring the decrease in `free' prey over time
via epi£uorescence microscopy (see Section 2.4). Samples
were also stained with DAPI to determine any change in
ciliate concentration over time and whether the prey had
aggregated. The ingestion rate was determined by dividing
the decrease in prey concentration over the 24-h period by
the concentration of ciliate present. When live-£uorescing
E. coli was employed as the prey, its digestion ultimately
led to an increase in ciliate abundance. Ingestion rate was
thus calculated by using the equations of Frost [23] as
modi¢ed by Heinbokel [24]. The second indirect method
monitored RF ml31 values over time by £uorimetry and
equated these values to an E. coli concentration, in order
to determine the grazing rate in a similar manner to that
used for the previous indirect method. However, for the
¢rst 5 h of the experiment, no discernible decrease in RF
ml31 was recorded in the test £asks and data from the last
19 h of the experiment were used to determine ingestion
rate. Both indirect methods took account of changes recorded in the control £asks, which were minimal, i.e. no
loss of cells or £uorescence in the absence of the ciliate.
To determine the grazing rate by the direct method,
each £ask system was miniaturised to 0.2 ml within wells
of a 96-well microtitre plate. This was required since the
experimental period was extremely short (120 s) and the
use of multi-tip pipettors allowed the three replicates to be
¢xed at exactly the same time. Fixed samples were viewed
as slide preparations and the number of prey particles
within at least 30 ciliate cells was recorded. The ingestion
rate was then determined from the increase in the average
number of prey ciliate31 over time.
J.D. Parry et al. / FEMS Microbiology Ecology 35 (2001) 11^17
Fig. 1. The change in £uorescence of a population of E. coli HB101,
transformed with the vector pBAD-GFPuv, after being subjected to a
range of pH values in distilled water. Values for pH 8 and 9 are not
shown, for reasons of clarity.
ing this direct method, even though a short incubation
time of 2 min was employed. Some skill was also required
to distinguish ingested particles from those resting on the
surface of the protozoan, e.g. trapped in cilia. In addition,
automation of this method has so far proved unsuccessful
[33].
Neither of the two indirect methods could be used to
determine ingestion rates with FLM. This was not unexpected, as FLM are inert particles which are not digested,
or a¡ected by acidi¢cation, and are thus continuously recycled by the protozoan. Because of this property, they
have been successfully used to determine the inherent vacuole passage times of protozoa [29,34^37].
Both indirect methods showed a linear decrease in £uorescing E. coli concentration over time (R2 0.96) due to the
use of such a high initial prey concentration (5U107 E. coli
ml31 ). Estimated ingestion rates were equivalent but signi¢cantly lower than that calculated by the direct method
(Table 2) (P 6 0.05). After the addition of a high concentration of £uorescent prey particles to a grazing system,
ingestion rates will be increased until limited by the vacuole passage time. It is thus probable that the indirect methods yielded an average ingestion rate over the 24-h period
whereas the direct method yielded the elevated, instantaneous ingestion rate over the ¢rst 2 min of the experiment.
At present, it is not known whether it was the low pH of
the food vacuole and/or digestion which caused prey £uorescence loss within T. pyriformis. However, as for this
particular ciliate, (i) the vacuolar pH decreases to 6.0^
5.5 and the action of acid phosphatases begins 10 min
after food vacuole formation [18], (ii) only 10% of ingested
E. coli are digested within the ¢rst passage through the
ciliate [38] which takes 1.5^2 h [18] and (iii) the vacuolar
pH reduces to pH 4.0^3.5 1 h after formation [18], it seems
reasonable to assume that a combination of both occurred. This may not be the case for all protozoa. For
example, Paramecium food vacuoles acidify to pH 2 within
Table 2
Ingestion rates ( þ S.E.M.) of T. pyriformis feeding on three prey particles, determined by (i) the increase in prey abundance within the ciliate
over 120 s (direct method), (ii) the decrease in prey concentration within
the medium over 24 h (`free' cells) and (iii) the decrease in £uorescence
over a 19-h period (£uorimetry)
Prey particle and method used
Direct method :
FLM
DTAF-E. coli
Live GFP-E. coli
`Free' cells:
FLM
DTAF-E. coli
Live GFP-E. coli
Fluorimetry :
FLM
DTAF-E. coli
Live GFP-E. coli
Ingestion rate (prey ciliate31 h31 )
207 þ 32
1048 þ 85
964 þ 126
ND
1440 þ 120
537 þ 138
ND
ND
693 þ 39
ND: Could not be determined using this method.
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
showed no regeneration of £uorescence after the pH was
returned to 7. Therefore, upon ingestion into the food
vacuole and exposure to subsequent pH changes, loss of
£uorescence should be irrecoverable. The £uorescence of
DTAF-stained E. coli cells was stable over the pH range
tested, yielding no discernible change in RF ml31 even
after 2 h. The only noticeable feature was that the cells
tended to form small aggregates at pH values less than 4.
Acidi¢cation within food vacuoles of T. pyriformis is
known to occur 1 h after food vacuole formation [18]
thus direct counts within 2 min allowed comparison of
the extent to which the three £uorescent particles were
consumed by this ciliate. YG FLMs were ingested at a
signi¢cantly lower rate (207 þ 32 FLM ciliate31 h31 )
than live and DTAF-stained E. coli (964 þ 126 and
1048 þ 85 prey ciliate31 h31 , respectively) (Table 2). The
reason for the lower ingestion rate might have been due to
the FLMs being smaller than E. coli cells. No signi¢cant
di¡erence in the calculated ingestion rates was recorded
for T. pyriformis feeding on either heat-killed stained or
live unstained E. coli (two-tailed t-test, P s 0.5) which
agrees with other workers using other FLB [29]. However,
heat-killing bacterial cells, used in the production of FLB,
has often raised concerns, particularly since it can prevent
digestion of some species [31]. It is not possible to produce
live DTAF-stained cells, and this investigation did not
succeed in producing heat-killed GFP-expressing E. coli,
as the proportion of £uorescing cells after various heattreatments was unacceptably low. This is despite the fact
that GFP has previously been shown to retain its £uorescence up to temperatures of 60³C [30] and no di¡erence in
the £uorescence intensity of E. coli V850 (transformed
with a variety of vectors) was recorded after heating at
70³C for 30 min [32]. In this study, heat-killing of the
E. coli cells did not appear to a¡ect calculated values of
instantaneous ingestion rate and all prey particles were
easily visualised within the ciliate food vacuoles. However,
as the experiment progressed, it became increasingly di¤cult to count accurately prey in packed food vacuoles us-
15
16
J.D. Parry et al. / FEMS Microbiology Ecology 35 (2001) 11^17
were equivalent to those using current surrogate prey particles. The use of £uorimetry reduced the potential for
operator error but led to an initial `lag' period of 5 h
before any discernible decrease in £uorescence could be
detected. It was considered as tedious as microscopic
counts, thus the development of an automated method
of analysis, using a £uorescence microtitre plate reader is
currently underway.
Acknowledgements
This study was funded by a NERC grant (GR9/3804)
awarded to J.D.P. However, it is acknowledged that the
initial idea of employing GFP-expressing E. coli as a surrogate prey originated from H.L.J.J. Extensive pumppriming experiments carried out by H.L.J.J. (Silwood
Park), J.D.P. and J.D. (Lancaster University, Leverhulme
grant F/185/X), formed the basis of the NERC grant. All
authors would like to thank the following researchers (PG
and UG) for their help in developing the method and
providing valuable feedback : Stephanie Hodge and
Harashran Ghotra (Silwood Park), and Dr. Amelia
Hunt, Katie Harper, Joanna English, Ruth Baldry, Clare
Boam, Kerstin Boese, Kate Dodge, Lisa Goodall and
Joe Latimer (Lancaster University). The authors would
also like to thank the useful comments made by the anonymous referees.
References
[1] Francey, R.J. (1998) The global carbon budget; recent advances.
Environ. Conserv. 25, 81^82.
[2] Nakano, S. (1996) Bacterial response to extracellular dissolved organic carbon released from healthy and senescent Fragilaria crotonensis
(Bacillariophyceae) in experimental systems. Hydrobiologia 339, 47^
55.
[3] Hunt, A.P., Parry, J. and Hamilton-Taylor, J. (2000) Further evidence of elemental composition as an indicator of the bioavailability
of humic substances to bacteria. Limnol. Oceanogr. 45, 237^241.
[4] Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A. and
Thingstad, T.F. (1983) The ecological role of water column microbes
in the sea. Mar. Ecol. Prog. Ser. 10, 257^263.
[5] Sherr, E.B. and Sherr, B.F. (1987) High rates of consumption of
bacteria by pelagic ciliates. Nature 325, 710^711.
[6] McManus, G.B. and Fuhrman, J.A. (1986) Bacterivory in seawater
studied with the use of inert £uorescent particles. Limnol. Oceanogr.
31, 420^426.
[7] Wilkner, J., Andersson, A., Normark, S. and Hagstrom, A. (1986)
Use of genetically marked minicells as a probe in measurement of
predation of bacteria in aquatic environments. Appl. Environ. Microbiol. 52, 4^8.
[8] Sherr, B.F., Sherr, E.B. and Fallon, R.D. (1987) Use of monodispersed, £uorescently labelled bacteria to estimate in situ protozoan
bacterivory. Appl. Environ. Microbiol. 53, 958^965.
[9] Landry, M.R., Lehner-Fournier, J.M., Sunderstrom, J.A., Fagerness,
V.L. and Selph, K.E. (1991) Discrimination between living and heatkilled prey by a marine zoo£agellate, Paraphysomonas vestita
(Stokes). J. Exp. Mar. Biol. Ecol. 146, 139^151.
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
5 min of formation and £uorescence loss could be solely
due to this acidi¢cation [17].
Monitoring the concentration of `free' DTAF-stained
E. coli over time via epi£uorescence microscopy showed
a linear decrease enabling calculation of ingestion rate
(Table 2). There was no signi¢cant di¡erence between
the ingestion rate values obtained by this and the direct
method (P s 0.05). This indirect method may have overestimated ingestion rate since it assumed that any loss of
FLB £uorescence in the presence of a protozoan was solely due to ingestion, if the processing of FLB was equivalent to that of unstained bacteria [8,29,39]. However, the
method does not consider that the processing of stained
and unstained prey might not be equivalent in some species of protozoa and that some losses could be due to
attachment of the prey to container walls [33] or even to
the protozoan cells themselves. With regards to GFP-expressing E. coli cells, the loss of £uorescence (and hence
reduction in prey concentration) can only be due to enclosure of the prey-cell within a protozoan food vacuole as
loss through attachment would not be associated with
acidi¢cation. This was con¢rmed by a lack of discernible
change in RF ml31 within the live E. coli control systems.
Acidi¢cation was shown to have no e¡ect on the RF
ml31 of DTAF-stained E. coli but the RF ml31 of the test
cultures (prey in the presence of the ciliate) unexpectedly
increased over time. The majority of this increased £uorescence (75%) was associated with the solution (data not
shown) and appeared to be due to the ciliate excreting the
stain, as it did not accumulate within the cytoplasm. This
could have only been due to damage/digestion of the prey
within food vacuoles as this loss of stain has not been
observed with FLB in the absence of a predator ([8], this
study) or at pH 6 7 (this study). Interestingly, the T. pyriformis population did not increase in abundance when fed
with this prey state, as it had with the live-£uorescing
E. coli, which yielded a speci¢c growth rate of 0.10 h31
and a gross growth e¤ciency of 68%. It is thought that the
process of re-ingestion was, for some yet undetermined
reason, blocked in this experiment. Although this does
not occur in all experiments using this combination of
predator and prey state, here, faecal pellets (ca. 5 Wm in
diameter) comprising faded DTAF-stained prey and
DAPI-stained indigenous prey were abundant in the medium surrounding the protozoan. These pellets amassed to
form aggregates (ca. 20 Wm in diameter) which may have
been too large for the ciliate to re-ingest. No such pellets/
aggregates were detected with DTAF-stained E. coli in the
absence of the ciliate (control) or in those systems containing live unstained E. coli, after DAPI staining. Thus, this
experiment inadvertently supported that of Schlimme et al.
[38] who found that as much as 90% of ingested bacteria
prey may require a second passage through a T. pyriformis
cell, before complete digestion occurs.
In conclusion, the use of a live GFP-expressing E. coli
yielded estimates of ingestion rate for T. pyriformis which
J.D. Parry et al. / FEMS Microbiology Ecology 35 (2001) 11^17
[25] Chrzanowski, T.H. and Simek, T.H. (1990) Prey-size selection by
freshwater £agellated protozoa. Limnol. Oceanogr. 35, 1429^1436.
[26] Monger, B.C. and Landry, M.R. (1991) Dependency of prey size on
grazing by free-living marine zoo£agellates. Mar. Ecol. Prog. Ser. 74,
239^248.
[27] Monger, B.C. and Landry, M.R. (1992) Size-selective grazing by
heterotrophic £agellates : an analysis using live-stained bacteria and
dual-beam £ow cytometry. Arch. Hydrobiol. Beih. 37, 173^185.
[28] Pelegri, S.P., Christaki, U., Dolan, J. and Rassoulzadegan, F. (1999)
Particulate and dissolved organic carbon production by the heterotrophic nano£agellate Pteridomonas danica Patterson and Fenchel.
Microb. Ecol. 37, 276^284.
[29] Gonzalez, J.M., Sherr, E.B. and Sherr, B.F. (1993) Di¡erential feeding by marine £agellates on growing versus starving, and on motile
versus non-motile, bacterial prey. Mar. Ecol. Prog. Ser. 102, 257^267.
[30] Bokman, S.H. and Ward, W.W. (1981) Renaturation of Aequoria
green-£uorescent protein. Biochem. Biophys. Res. Commun. 101,
1372^1380.
[31] Mehlis, I.K., Hausmann, K. and Wecke, J. (1990) Rapid digestion of
Staphylococcus aureus by Paramecium : an evaluation of the role of
bacterial autolytic enzymes. Eur. J. Protistol. 26, 103^109.
[32] Scott, K.P., Mercer, D.K., Glover, L.A. and Flint, H.J. (1998) The
green £uorescent protein as a visible marker for lactic acid bacteria in
complex ecosystems. FEMS Microbiol. Ecol. 26, 219^230.
[33] Sleigh, M.A. and Zubkov, M.V. (1998) Methods for estimating bacterivory by protozoa. Eur. J. Protistol. 34, 273^280.
[34] Capriulo, G.M. and Degnan, C. (1991) E¡ect of food concentration
on digestion and vacuole passage time in the heterotrichous marine
ciliate Fibrea salina. Mar. Biol. 110, 199^202.
[35] Dolan, J.R. and Coats, D.W. (1991) Preliminary prey digestion in a
predacious estuarine ciliate and the use of digestion data to estimate
ingestion. Limnol. Oceanogr. 36, 558^565.
[36] Dolan, J.R. and Simek, K. (1997) Processing of ingested matter in
Strombidium sulcatum, a marine ciliate (Oligotrichida). Limnol. Oceanogr. 43, 393^397.
[37] Dolan, J.R. and Simek, K. (1998) Ingestion and digestion of an
autotrophic picoplankter, Synechococcus, by a heterotrophic nano£agellate, Bodo saltans. Limnol. Oceanogr. 43, 1740^1746.
[38] Schlimme, W., Marchiani, M., Hanselmann, K. and Jenni, B. (1997)
Gene transfer between bacteria within digestive vacuoles of protozoa.
FEMS Micobiol. Ecol. 23, 239^247.
[39] Sherr, B.F., Sherr, E.B. and Rassoulzadegan, F. (1988) Rates of
digestion of bacteria by marine phagotrophic protozoa: temperature
dependence. Appl. Environ. Microbiol. 54, 1091^1095.
FEMSEC 1198 27-2-01
Downloaded from http://femsec.oxfordjournals.org/ by guest on March 5, 2016
[10] Zubkov, M.V. and Sleigh, M.A. (1995) Ingestion and assimilation by
marine protists fed on bacteria labelled with radioactive thymidine
and leucine estimated without separating predator and prey. Microb.
Ecol. 30, 157^170.
[11] Shimomura, O. (1979) Structure of the chromophore of Aequoria
green £uorescent protein. FEBS Lett. 104, 220^222.
[12] Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. and
Cormier, M.J. (1992) Primary structure of the Aequoria victoria
green-£uorescent protein. Gene 111, 229^233.
[13] Chal¢e, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher, D.C.
(1994) Green £uorescent protein as a marker for gene expression.
Science 263, 802^805.
[14] Heim, R., Prasher, D.C. and Tsein, R.Y. (1994) Wavelength mutations and post-translational autoxidation of green £uorescent protein.
Proc. Natl. Acad. Sci. USA 91, 12501^12504.
[15] Prosser, J.I., Kilham, K., Glover, L.A. and Rattray, E.A.S. (1996)
Luminescence-based systems for detection of bacteria in the environment. CRC Crit. Rev. Biotechnol. 16, 157^183.
[16] Casey, W.M. and Nguyen, N.T. (1996) The use of the green £uorescent protein to rapidly assess viability of E. coli in preserved solutions. PDA J. Pharm. Sci. Technol. 50, 353^355.
[17] Fok, A.K., Lee, Y. and Allen, R.D. (1982) The correlation of digestive vacuole pH and size with the digestive cycle in Paramecium
caudatum. J. Protozool. 29, 409^414.
[18] Nilsson, J.R. (1977) On food vacuole formation in Tetrahymena pyriformis GL. J. Protozool. 24, 502^507.
[19] Crameri, A., Whitehorn, E.A., Tate, E. and Stemmer, W.P.C. (1996)
Improved green £uorescent protein by molecular evolution using
DNA shu¥ing. Nat. Biotechnol. 14, 315^319.
[20] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
[21] Needham, J.G., Lutz, F.E., Galtso¡, P.S. and Welch, P.L. (1937)
Culture Methods for Invertebrate Animals. Constock Publishing
Co., Ithaca, New York (reprinted in 1957 by Dover Press, New
York).
[22] Porter, K. and Feig, Y.S. (1980) The use of DAPI for identifying and
counting aquatic micro£ora. Limnol. Oceanogr. 25, 943^948.
[23] Frost, B.W. (1972) E¡ects of size and concentration of food particles
on the feeding behaviour of the marine planktonic copepod Valanus
padi¢cus. Limnol. Oceanogr. 20, 805^815.
[24] Heinbokel, J.F. (1978) Studies on the functional role of tintinnids in
the Southern California Bight. III. Grazing impact of natural assemblages. Mar. Biol. 52, 23^32.
17