FEMS Microbiology Ecology 15 (1994) 193-206
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
193
FEMSEC 00575
Specific monitoring by PCR amplification and
bioluminescence of firefly luciferase gene-tagged
bacteria added to environmental samples
Annelie M611er a, Kersti Gustafsson b and Janet K. Jansson ,,a
a Department of Biochemistry, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden and
b Swedish National Chemicals Inspectorate, P.O. Box 1384, S-171 27 Solna, Sweden
(Received 14 February 1994; revision received 7 June 1994; accepted 22 June 1994)
Abstract: The firefly luciferase gene, luc, was demonstrated to hold promise as a specific marker for monitoring of genetically
modified bacteria in the environment, PCR amplification and bioluminescence procedures were modified and compared for
environmental monitoring of luc-tagged bacteria, using Escherichia coli as a model. The methods were used to track luc-tagged
bacterial cells added to intact sediment core microcosms. Detection limits for the luc-tagged cells were the following, expressed as
ceils per 0.5 g of sediment: 102, by PCR amplification; 103, by whole cell luminescence; and 103-104, by measurement of
luminescence in cell extracts.
Key words: Firefly luciferase marker gene; Bioluminescence; PCR amplification; Microcosms; Genetically modified microorganisms
Introduction
Genetically modified microorganisms (GMMs)
hold great promise for many applications including environmental, industrial and agricultural
uses. The advantages of these specially designed
microorganisms are offset in part by public fears
as to the potential risk of hazard to the environment or to human health after release. Therefore
it should be a responsibility of the creator of the
engineered microorganism to prove by appropriate testing that risks are minimal.
* Corresponding author. Tel.: (46) (8) 162469. Fax: (46) (8)
153679. E-mail: [email protected].
SSDI 0 1 6 8 - 6 4 9 6 ( 9 4 ) 0 0 0 5 8 - 1
The key to test procedures is a reliable detection method that is specific for the GMM, a n d / o r
engineered DNA sequence. In addition the detection methods need to be extremely sensitive in
order to study the fate of the GMM a n d / o r
engineered DNA in environmental samples or
field tests.
Since many GMMs have been modified by
addition of a defined segment of DNA, it is often
useful to track that particular segment by DNA
probe methods [1]. Alternatively, the GMM may
be tagged with a marker gene which may then be
traced based on the phenotype expressed by the
added marker. Various genes have been used as
markers which are advantageous to different' degrees [2] including antibiotic resistance genes and
194
the metabolic genes; lacZ, encoding /3-galactosidase [3] and uidA, encoding /3-glucuronidase
[4]. While useful, these markers have limited applications due to the potential background of
indigenous strains from which they were initially
isolated [4,5]. Another marker which has recently
increased in prominence is the bacterial luciferase gene cassette; i.e., luxAB [6-15], which
emits light as a product of the luciferase reaction.
This marker is advantageous because it is very
sensitive and specific for marked strains released
in terrestrial environments. However, the lux
genes were isolated from Vibrio spp. limiting the
specificity and usefulness of lux as a marker in
marine environments since luminous bacteria are
easily detected using lux as a hybridization probe
[16], and even some non-luminescent Vibrio cells
have D N A homologous to the bacterial luciferase
gene [17].
An entirely different class of luciferase enzyme
has been isolated from the firefly, Photinus pyralis
and related species [18,19]. The firefly luciferase
catalyzes the following reaction:
Luciferin + A T P + 0 2 ~ Oxyluciferin + AMP
+ PPi + C02 + hv (562 nm)
Lampinen et al. [9] have recently compared sensitivities of the bacterial and eukaryotic luciferase
genes cloned into Bacillus spp. and found that
the eukaryotic enzyme was approximately 10times more sensitive. The firefly luciferase gene,
luc, has been demonstrated to be useful as a
marker for detection of Rhizobium meliloti in
bacteroids [20,21], and in soil [20] based on detection of bioluminescence in engineered strains.
The eukaryotic luciferase gene is an ideal
marker to detect by PCR amplification since it is
not naturally present in the microbial population.
The use of luc as a marker should therefore
alleviate the problem of false positive results.
PCR in combination with D N A probes has been
shown to be able to detect 1 cell in 100 ml water
when the sample was first concentrated by filtration [22], 1 cell per gram of sediment [23] and
1-10 cfu per gram of soil [24]. Therefore, this is
one of the most sensitive detection methods
demonstrated at present.
In this study we have developed and directly
compared bioluminescence and PCR methods for
detection of the firefly luciferase gene marker
used to tag a model GMM. First, the methods
were developed and detection limits determined
using pure cultures of cells. The model GMMs
were then monitored using both methods in intact sediment core microcosms.
Materials and Methods
Strains, plasmids and growth conditions
Escherichia coli JM109 (recA1 supE44 endA1
hsdR17 thi leu rpsL lacY galK galTara tonA thr txs
D (lac-proAB) F'(traD36 proAB + lacI q IacZDM
15) was used as recipient strain for recombinant
plasmids.
A derivative of the plasmid vector, pGEM-luc
(Promega), 4933 bp, was constructed. The tac
promoter was excised as a 97 bp B a m H I / H i n d l l I
fragment from plasmid pRD540 (Pharmacia) and
purified on a polyacrylamide gel. The tac promoter was cloned immediately before the luc
gene on pGEM-luc and the resulting construct
with a size of 5030 bp is referred to as p J J303.
E. coli was grown in Luria Broth (LB) at 37°C
in a shaking incubator. Plasmid p J J303 transformed cells were grown in the presence of ampicillin (50 Izg ml-1). An E. coli JM109 (pJJ303)
transformant was used as a model GMM for
development of the methodologies described below.
For studies of maximal luminescence in pure
culture, isopropylthiogalactoside (IPTG; 125 tzg
m1-1) was added during the last hour of culture
incubation to induce the tac promoter regulating
transcription of the luc gene, since the E. coli
host strain, JM109, expresses the lacI repressor.
Although the luc gene was expressed at a high
level without I P T G due to the strength of the tac
promoter, the intensity of bioluminescence in
whole cells was 10-times greater after induction
over the entire growth cycle.
Original cell concentrations were determined
by colony counts on LB agar medium with 50/xg
ml-~ amplicillin and by direct microscopic counting of acridine orange stained cells in a Zeiss
Axiophot epifluorescence microscope.
195
Bioluminescence measurements in pure culture
For whole cell measurements, 1 ml aliquots
were centrifuged at 10000 × g for 10 rain, and
the cell pellets were resuspended in 100 tzl whole
cell buffer consisting of 1 mM beetle luciferin
(Promega) in 100 mM citrate buffer, pH 5.0 [25].
The suspensions were incubated 5 rain at 37°C to
allow time for the luciferin substrate to enter the
cells and then directly monitored for light production as quanta s-1 in a luminometer.
The luminometer used was equipped with a
sensitive photomultiplier tube, Model R268, Hamamatsu TV Co., Tokyo, Japan and built by Bo
H6jer (Department of Biochemistry, Stockholm
University). The sample and photomultiplier detector were enclosed in a light-tight box. The
limit of resolution of the luminometer was determined to be 1.5 × 10 -7 pmol pure firefly luciferase (Sigma). The background noise of the
luminometer averaged 150-200 quanta s-1. Specific background values were taken for each reading and subtracted from the sample reading. Only
those measurements consistently at least 30
quanta s- 1 above background were considered as
positive for light production.
Cell extracts were prepared using the Luciferase Assay System (Promega) according to the
manufacturer's instructions. Samples (1 ml) were
centrifuged 10 rain at 10000 × g and cell pellets
were resuspended in 10 /zl buffer (0.1 M
K2HPO 4, pH 7.8; 2 mM EDTA). The cells were
frozen in liquid nitrogen, thawed at room temperature and lysed by the addition of 30 /xl of
1 X cell lysis reagent (Promega) plus lysozyme
(Sigma), 2 mg m1-1 and bovine serum albumin
(Sigma), 3 mg m1-1. After incubation at room
temperature for at least 10 min, a 10/~1 aliquot of
the cell extract was added to 100 /.d luciferase
assay reagent (Promega) and immediately assayed
for luciferase activity, by quantitation of light
output in the luminometer.
For specific activity measurements of whole
cell and cell extract bioluminescence, protein
concentration was determined using the method
developed by Lowry [26]. Bovine serum albumin
was used as a protein standard.
Bioluminescent colonies were detected after
growth on LB (+ampicillin) by adsorption of
colonies onto nitrocellulose membrane filters
(0.45 ~m pore size, Schleicher and Schuell). The
filters were transferred to LB (+IPTG, 125 /xg
ml-a) and incubated 1 hr at 37°C. The membrane
with colonies facing upwards was then transferred to the lid of a petri plate which had the
surface covered with 0.8 ml whole cell buffer. The
plate was covered with plastic to keep the bacteria moist and after 30-60 minutes further incubation at 37°C biolumineseent colonies could be
visualized in a dark room. For a permanent
record, and aid in quantitation of colonies, X-ray
film (Fuji RX100) was placed on top of the filter
for 5 rain exposure. Blackened areas, corresponding to exposure of the X-ray film by the light
emitting colonies, could be counted after film
development [25].
PCR amplification
The following primers were designed for amplification of the luc gene segment using published sequence information [18]:
Outer primers: 5'-CTG GTT GCT GGA ACA ATI"
GC (70 bases downstream of the start codon)
5'-CGG TAA GAC CTF TCG GTA CT
(102 bases upstream of the stop codon)
Inner primers: 5'-ACT TGA CTG GCC ACG TAA
TC (135 bases downstream of the start codon)
5'-CAT ATC GAG GTG AAC ATC ACG
(127 bases upstream of the stop codon)
The predicted product size was 1480 bp after one
round and 1390 bp after two rounds of amplification.
A pure culture of E. coli JM109 (pJJ303) was
diluted in T 1/10 E (10 mM Tris-HC1 (pH 8.0),
0.1 mM EDTA (pH 8.0)). Samples (1 ml) were
centrifuged for 10 min at 10000 × g and the
pellets resuspended in 35/zl T1/10 E. The cells
were lysed by boiling for 10 rain followed by a
quick vortex and spin to bring down condensation. The template DNA was added to a premade
10 × PCR Mastermix to a total volume of 50 /zl.
The 10 x Mastermix contained KCI, 500 mM;
Tris-HCl (pH 8.3), 100 mM; gelatin, 0.1%; dNTPs,
2 mM each; outer primers, 1 /.~M each and 15
196
mM MgC12. To minimize evaporation a layer of
mineral oil (80 #1) was placed on top of each
sample. We routinely used 'Hot start', 5 min at
98°C, to avoid primer dimers and unspecific binding of the primers. The program was put on hold
while 2.5 Units Taq-polymerase (Boehringer
Mannheim) were added. Then the amplification
was initiated in a Perkin Elmer model 480 thermal cycler; using a 3-step program with denaturation at 95°C for 1 min; annealing at 50°C for 1.5
min; and extension at 72°C for 2 min and a total
of 35 cycles. A negative control consisting of all
ingredients except template D N A was routinely
run with each PCR amplification.
When the PCR program was completed, 10/zl
of the reaction mix was examined by d e c trophoresis on a 1% agarose gel, 12 V cm -1 for
30 min. If no DNA was visible on an agarose gel
after the first round of amplification the reaction
mix was subjected to a second round of amplification of 25 cycles using inner primers and fresh
mastermix, and again screened on an agarose gel
for bands of the predicted size.
Fractionation of bacterial cells from sediment
Total sediment bacteria were fractionated from
sediment using a modified version of the procedure described by Wagner-D6bler et al. [27]. The
sediment, 0.5 g wet weight, was suspended in 1 ml
0.1 M sodium phosphate buffer (pH 6.5) for bioluminescence measurements or in 1 ml PBS buffer
plus 5 mg acid washed polyvinylpolypyrrolidone
(PVPP) for subsequent PCR amplification. The
use of PVPP has earlier been shown to remove
humic substances [1], which can interfere with the
PCR reaction by chelation of Mg 2÷ [28,29] an
essential requirement for Taq polymerase activity. The suspensions were vortexed vigorously for
1 min and centrifuged in a microcentrifuge 6 min
at 2200 × g. The supernatant was decanted to a
new tube and centrifuged at 1 0 0 0 0 × g for 10
min to pellet the bacteria and the bacterial pellet
was saved. A fresh 1 ml aliquot of buffer was
added to the sediment pellet in the original tube
and the low speed centrifugation procedure was
repeated twice. Each resultant supernatant from
the low-speed centrifugations was added to the
microcentrifuge tube containing the bacterial pel-
let and recentrifuged at 10000 × g for 10 min
until all three supernatant fractions were combined and the bacterial fraction was pelleted in
one tube. The total time required for isolation of
bacteria from sediment was approximately 2 h.
Microcosms
Sediment core samples consisting of sediment
and overlaying water were collected in July 1992
from two sites: freshwater Lake M~ilaren at a
water depth of 38 m and from a coastal station of
the Baltic proper at a water depth of 52 m. This
part of the Baltic Sea has a salinity of 7-8 %0.
Both stations sampled are oxic soft bottoms, rich
in organic matter in the upper layers and more
argillaceous beneath with similar macrobenthic
fauna communities [30-32]. Therefore, the basic
difference between the two stations refers to
salinity. The sediments had moisture contents of
78% or 61% for Lake M~ilaren or the Baltic Sea,
respectively.
The sediment cores were collected with a modified Kajak gravity corer [33]. By using Plexiglas
coring tubes with an internal diameter of 80 mm
at least the upper 50 mm of the sediment is
unaffected by core shortening [34]. Upon sampling, the cores were sealed at top and bottom by
means of adapted stoppers [33]. During the transport to the laboratory the cores stood vertically
and were chilled in dark boxes. At the laboratory,
the upper stoppers were removed and the cores
were gently bubbled with air.
Inoculation of microcosms
The microcosms were inoculated with E. coli
(p J J303). An overnight culture was centrifuged at
6000 × g for 10 min, the cells were washed in
PBS buffer, pH 7.15 (g 1-1: N a 2 H P O 4 . 1 2 H 2 0 ,
3.56; N a H 2 P O a . 2 H 2 0 , 0.52; NaCI, 8.5), and recentrifuged. The cell pellet was resuspended in
the original volume with PBS, and incubated while
stirring overnight at 4°C to starve and preadapt
the cells to that temperature. The cell suspension
was recentrifuged and the pellets resuspended in
1 / 1 0 the original volume of PBS and added to
the cores in 1 ml aliquots to a total of 10 additions, one every half hour, achieving a final density of approximately 5 × 10 7 cells ml-1 overlying
197
water as determined by direct microscopic counts
and plate counts on selective agar medium containing ampicillin.
After inoculation, samples of water and sediment were taken for subsequent analyses and the
microcosms were further incubated at 4°C in a
temperature controlled room in the dark.
Sampling of microcosms
The water phase was sampled with a 10 ml
pipette before sampling the sediment to prevent
contamination. For luminescence measurements,
bacteria were collected by centrifugation of 1 ml
aliquots of water at 10000 x g for 10 min in
microcentrifuge tubes, or for PCR amplification,
100/zl samples were taken.
Sediment samples were taken as minicores
through the vertical length of the sediment core
using a long glass tube fitted to a pipette bulb.
The pipette bulb was compressed until entering
the sediment, after which pressure was released
and the sediment was gradually suctioned into
the tube while pushing the tube down through
the sediment profile. The sample, which also
contained the interface between water and sediment, was mixed well before taking aliquots for
analyses. Total bacteria were extracted from sediment using the procedure described above and
further processed for PCR amplification. For bioluminescence assays, the pellet was resuspended
in PBS buffer and divided into two equal portions
which were subsequently centrifuged in microcentrifuge tubes.
Monitoring GMM by bioluminescence
For whole cell bioluminescence measurements,
cells extracted from sediment were directly
brought up in 100 /zl whole cell buffer and assayed as previously described.
For cell extract bioluminescence measurements, the cells pelleted by centrifugation were
resuspended in 100 ~1 buffer (0.1 M K2HPO 4,
pH 7.8; 2 mM EDTA), quickly frozen and thawed,
lysed by the addition of 300 /zl of 1 X cell lysis
reagent containing lysozyme and BSA and 10 ~1
aliquots were assayed for light production as previously described.
Background noise from the luminometer was
always subtracted from the final measurement.
Importantly, there was no measurable background luminescence in the environmental samples.
Monitoring of GMM by PCR
For the water samples there was no need for
purification or dilution of the cell pellet before
cell lysis and PCR amplification. By contrast, the
bacterial pellet extracted from sediment required
additional purification for PCR amplification. The
cells fractionated from sediment were resuspended in 100/zl of sterile distilled water; 50/zl
were processed for PCR reactions and the remainder was serially diluted and used for selective plating. The 50 /.tl cell suspension was lysed
by boiling for 10 min followed by vortexing and a
short centrifugation to bring down condensation.
DNA was purified from the lysate using the Magic
DNA Clean-up System (Promega). The columns
were used according to the manufacturers specifications except for the eluting-solution in which 50
/zl T 1/10 E was substituted. The 50/zl of eluate
were diluted 1:100 and a 27 ~1 aliquot of the
diluted purified sediment extract containing template DNA was then added to the first round
mastermix. To increase specificity, the PCR amplification program had an increased annealing
temperature of 62°C for DNA samples isolated
from sediment.
Determination of luminescence detection limits and
efficiencies in sediment samples
For testing of the various methods for detection of luc-tagged ceils in sediment, sediment was
obtained from intact sediment cores. The cores
were dismantled, the sediment portion was isolated, thoroughly mixed, aliquoted into 50 ml
sterile plastic tubes, and stored at 4°C until use.
At the initiation of each experiment, a suspension
of E. coli JM109 (pJJ303), with a known cell
density, was added to 0.5 g samples of sediment
in microcentrifuge tubes.
Limits of detection of luc-tagged cells in sediment samples by bioluminescence and PCR amplification methodologies were determined using
the procedures described above for monitoring in
microcosms, except one change for the whole cell
198
luminescence measurements. The bacterial pellet
fractionated from sediment was washed with 500
/xl 0.1 M citric acid (pH 5.0) and repelleted by
centrifugation before addition of whole cell
buffer. This extra washing step resulted in a 10fold increase in luciferase activity.
a
2
Results
I
Detection of luc-tagged cells in pure culture by
bioluminescence
Both whole cell and cell extract bioluminescence measurements were quantitative methods
for detection of E. coli cells tagged with the luc
gene. The amount of bioluminescence intensity
increased as a function of cell density for both
types of measurements (Fig. 1) and was linear
over five orders of magnitude. However, at cell
concentrations lower than 102 ml-1, the response
was not linear even though quanta were measured over background levels. The sensitivity of
the whole cell assay was greater than the cell
extract assay for late log phase cells; whereas the
situation seemed to be the reverse in stationary
phase cultures (Fig. 1).
In order to determine any relative differences
in the two types of bioluminescence measurements, the specific activities of the luciferase enzyme reaction in both whole cells and cell extracts were determined for different cell growth
phases; i.e. early log, log, late log and stationary
phases (Fig. 2). On a protein basis, the light
response maintained higher levels at all growth
stages in cell extracts. Whole cells had a significantly lower luciferase activity at stationary phase
compared to log phase as determined by A N O V A ;
F = 15.35 ( P < 1%). The difference was also significant for cell extracts, but not as great; F =
6.81(P = 4%).
C~
I
I
I
I
I
I
7
0
5
4
3
0
I
3
4
2
1
1
2
5
6
7
8
Log cell number • ml-1
---I--
--O--
Fig. 1. W h o l e cell and cell extract b i o l u m i n e s c e n c e m e a s u r e m e n t s as a function of cell density with initial cell concentrations of 3 x l0 s cells ml- 1(Late log phase) (a) or 1 x 10 9 ml - 1
(Early stationary phase) (b). Line equations were the following: Whole cells - I - (y = 1.1339+0.66601x, R = 0.9826); Cell
extracts -o- (y = 0.63223+0.7898x, R = 0.99366) (a); Whole
cells - I - (y=-0.13668+0.83832x, R=0.98474); Cell extracts -o- (y = 0.58173+0.78149x, R = 0.98512) (b). Data
represent the average values of duplicate samples taken from
broth cultures serially diluted in buffer.
amplified template corresponding to a higher
number of cells added to the reaction mixture
until saturation was reached at concentrations of
50 cells ml-1 and above (Fig. 3).
Detection limits in pure culture
Detection of luc-tagged cells in pure culture by PCR
After P C R amplification the luc gene was detected in extracts from cells by agarose gel electrophoresis (Fig. 3). Both outer and inner sets of
primers produced amplified template of the predicted sizes of 1480 or 1390 bp, respectively.
There was a visible increase in the amount of
Whole cell and cell extract bioluminescence
assays and PCR amplification were compared as
to their sensitivity of detection of luc-tagged cells
(Table 1). In pure culture, all three methods were
very sensitive. For the whole cell assay, the detection limit was 3 cells m l - t at late log phase (Fig.
la), whereas at stationary phase the detection
199
9
Table 1
Limit of detection of E. coli (pJJ303) in sediment by different
methods
.E
O
I
o
~7
Detection method
Detection
limit in
pure culture
(cells ml - 1)
Detection
limit in
sediment
(cells/0.5 g
sediment)
Cell extract luciferase activity
Whole cell luciferase activity
PCR
10 x
10° -10 t
10°
103-104
103
102
'E6
o"
o~s
4
Early10g
Log
Late 10g
Stationary
Fig. 2. Comparison of cell extract (tO) and whole cell (11)
luciferase enzyme specific activities relative to cellular protein
concentrations for different growth stages of E. coli JM109
(pJJ303). Results represent the average of 4 replicate samples
taken from 250 ml liquid cultures. Error bars represent one
standard error.
limit was a p p r o x i m a t e l y 25 cells m1-1 (Fig. lb).
T h e cell extract assay consistently h a d d e t e c t i o n
limits in t h e r a n g e o f 10 cells m1-1 o f culture,
r e g a r d l e s s o f age o f t h e cells. Since only a q u a r t e r
1 2
3 4
5 6 7
8
9 10 11
Results represent the average of duplicate 0.5 g sediment
samples from Lake Miilaren. Sediment samples were spiked
with dilutions of log phase cells, previously induced with
IPTG for the bioluminescence measurements.
of t h e original s a m p l e was u s e d in t h e b i o l u m i n e s c e n c e assay (0.25 ml), t h e light o u t p u t f r o m
a p p r o x i m a t e l y 5 cells was actually m e a s u r e d (Fig.
ib).
Cell dilutions f r o m t h e s a m e s t a t i o n a r y p h a s e
c u l t u r e d e s c r i b e d above (Fig. l b ) w e r e u s e d for
P C R a m p l i f i c a t i o n for d i r e c t c o m p a r i s o n of t h e
two m e t h o d s . A f t e r o n e r o u n d of P C R amplification, t h e d e t e c t i o n limit was a p p r o x i m a t e l y 103
cells m1-1. W h e n aliquots f r o m t h e first r o u n d
w e r e f u r t h e r a m p l i f i e d using i n n e r p r i m e r s t h e
luc g e n e c o u l d b e d e t e c t e d f r o m extracts of a cell
dilution c o n t a i n i n g as few as 1 cell m l - 1 (Fig. 3,
l a n e 9). I n fact t h e luc g e n e c o u l d even b e det e c t e d in a h i g h e r d i l u t i o n (Fig. 3, l a n e 10), p e r h a p s d u e to c a r r y - o v e r of p l a s m i d m o l e c u l e s f r o m
lysed cells.
Limit of detection of luc-tagged cells in sediment
Fig. 3. PCR product after 2 rounds of amplification from a
serial dilution of E. coli JM109 (pJJ303) with an original cell
concentration of i x 109 cells m l - i (early stationary phase). 10
p.l of PCR amplified material from the 50 /zl first round
reaction was added to fresh Mastermix for initiation of the
second round of amplification using inner primers with an
extended annealing time of 1.5 min. Lane 1, A HindlIl
molecular weight markers; Dilutions: Lane 2, 10-5; Lane 3,
10-6; Lane 4, 10-7; Lane 5, 5 x l 0 - S ; Lane 6, 10-8; Lane 7,
5x10-9; Lane 8, 2.5×10-9; Lane 9, 10-9; Lane 10, 10-1°;
Lane 11, Negative control consisting of Mastermix run through
two rounds of amplification without addition of template.
T h e sensitivity o f d e t e c t i o n o f cells in s e d i m e n t
was g r e a t l y r e d u c e d w h e n c o m p a r e d to p u r e cult u r e d e t e r m i n a t i o n s ( T a b l e 1). P C R a m p l i f i c a t i o n
was t h e m o s t sensitive m e t h o d for d e t e c t i o n of
t h e luc-gene in s e d i m e n t with a d e t e c t i o n limit o f
10 2 cells 0.5 g - 1 ( T a b l e 1). U s i n g l u m i n e s c e n c e
m e a s u r e m e n t s to d e t e c t luc-tagged cells in sedim e n t , t h e d e t e c t i o n limits w e r e h i g h e r by o n e o r
two o r d e r s o f m a g n i t u d e ( T a b l e 1).
Reduction of relative light yield from cells isolated
from sediment
By selective p l a t e c o u n t i n g we d e t e r m i n e d t h a t
o n t h e a v e r a g e a p p r o x i m a t e l y 66% ( L a k e M~ila-
200
8
B
~
7
0
2
3
2
_J_
b
3
~
2
I
C
5
~8
0
4
i
0
I
1
I
9
3
16
DAYS
•
Lake sediment
[]
Baltic sediment
[]
Lake water
[]
Baltic water
201
Table 2
Percent of known E. coli JM109 (pJJ303) colony forming
units (cfu) and initial luciferase activity (quanta s -1) measured after fractionation of cells from sediment compared to
the known amount of cfu and quanta s-1 initially added to
sediment
Sediment
source
Selective
plate
%a
Wholecell
luciferase
activity % b
Cell extract
luciferase
activity% b
Lake M~ilaren
Baltic Sea
66+ 18
86+ 6
7.66 + 2.69
7.05+1.11
1.52__.0.54
0.18+0.10
Sediment samples were spiked with dilutions of E. coli JM109
(pJJ303) broth cultures.
a % of total cfu added that were reisolated from sediment.
b % = [quanta s- 1 measured in bacterial pellet isolated from
sediment/quanta s -1 of cells initially added to sediment]×
100.
Values are the mean of four independent determinations.
ren) to 86% (Baltic Sea) of the cells added to
sediment samples were directly reisolated from
sediment (Table 2). Ampicillin (50 ~ g m1-1) was
sufficiently selective against indigenous bacteria
at the higher dilutions for sediment samples from
Lake M~ilaren and for all dilutions from Baltic
Sea sediment. At lower dilutions there was some
background growth on the ampicillin agar plates
plated from Lake Mhlaren samples, so only bioluminescent colonies were counted as being E. coli
JM109 (pJJ303).
The light yield was significantly lower for cells
directly extracted from sediment by either bioluminescence assay when compared to the known
luciferase activity of the cells initially added to
the samples (Table 2). The whole cell luciferase
activities in Lake M~ilaren and Baltic Sea sediments, were reduced to 8% and 7% of the initial
values, respectively. There were greater reductions of light output in cell extracts to 1.5% and
0.2% of initial values added for Lake M~ilaren
and Baltic Sea sediments, respectively. The approximate 10-fold difference in the % reduction
of cell extract bioluminescence for the two types
of sediments was seen consistently in repeated
experiments.
Monitoring o f luc-tagged bacteria in microcosms
The microcosm set-up was used for monitoring
survival of the model luc-tagged G M M in a complex environment under laboratory controlled
conditions. Detection of the luc-tagged bacteria
by whole cell and cell extract bioluminescence
measurements and selective plating over the sampling period are shown in Fig. 4. Unlike the pure
culture studies, the cell extract measurements
were diluted 10 fold, in order to dilute some of
the inhibitory material present in the samples.
Therefore, only 1 / 4 0 of the bacterial extract was
actually measured in the reaction mix (Fig. 4c).
Light intensity and cfu were both highest at
the beginning of the experiment, and in general
higher in the water samples than in the sediments. After 3 days of incubation all measurements indicated a decline in cell number. At the
end of the sampling period light intensity measurements were highest in sediments compared to
overlaying water for both Baltic Sea and lake
samples. This finding correlates to the higher
number of culturable ceils isolated on selective
medium from sediments for the later sampling
dates (Fig. 4a).
The luc gene marker was also detected in
sediment and water samples from the microcosms
by PCR amplification over the incubation period.
Very strong P C R signals were obtained for the
first three sampling days for both types of water
samples. For this experiment, P C R amplification
had a high degree of precision as demonstrated
by the similar intensities of the amplified template from the three replicate cores (Fig. 5). After
the first 3 days of incubation, the amount of PCR
amplified product decreased so that by day 9 it
was sometimes necessary to run the water sam-
Fig. 4. Detection of E. coli (pJJ303) in sediment/water microcosmsover time as determined by selective plate counts and by whole
cell and cell extract bioluminescence measurements. Number of E. coli (pJJ303) isolated from microcosmsculturable on LB agar
plates containing ampicillin (50/~g ml-i) (a); whole cell light intensity measurements (b); cell extract light intensity measurements
(e). Data represent the average values of samples taken from three replicate microcosms; except for * and ** where cfu were
counted from only one or two microcosms,respectively. Error bars represent one standard error.
202
a
123
b
4 5 6 7 2 3 4 5 6 7
Fig. 5. PCR amplification of the luc gene template in cells
isolated from microcosm water samples after 0 (a) and 1 (h)
days incubation with added E. coli (pJJ303). Results are
shown for triplicate microcosms: Lake M~ilaren (lanes 2, 3, 4);
and the Baltic Sea (lanes 5,6,7). After PCR amplification 10
/*1 of reaction mixture was electrophoresed on an ethidium
bromide stained agarose gel for visualization. Lane 1, HindIII
digested A DNA as a size marker.
ples through a second round of amplification
using the nested primers to see a band on an
agarose gel. A final sampling was done after 22
days incubation. At that date, a weakly positive
P C R response was observed for the Lake M~ilaren
water 0nly, but no cells were detected by selective
plating or bioluminescence measurements.
Routinely, two rounds of amplification were
conducted for the sediment samples. P C R products on the agarose gel were weak with sediment
samples at the beginning of the experiment. However, the P C R amplified product from sediments
increased over the course of the experiment with
highest yields at 9 days incubation.
No P C R amplification product was observed
with uninoculated sediment or water sample controls.
Discussion
We developed and compared bioluminescence
and P C R amplification procedures for detection
of the firefly luciferase marker gene in environmental samples. Since the same target gene, luc,
was assayed in both cases, detection of the gene
by P C R amplification could be used to complement activity m e a s u r e m e n t s of the gene product.
We optimized sensitivity of detection of luc-
tagged cells by using a multicopy n u m b e r plasmid
(300-400 copies) as the vector for luc and a
strong promoter (tac) in the bacterium E. coil,
known to express the luc gene [27]. Our purpose
was to demonstrate the potential for measurement of the luc gene in complex environmental
samples and to develop the required methods
and extraction procedures using a model GMM.
In pure culture, the bioluminescence assays
and P C R amplification were all found to be very
sensitive and specific for detection of the luc-gene
marker (Table 1, Figs. 1 and 3). The detection
limits for both P C R amplification and whole cell
bioluminescence methods were fewer than 10 cells
m l - ~ and one order of magnitude higher for the
cell extract measurements. Other investigators
have found that the detection limits of bioluminescence measured in cells tagged with the bacterial luciferase genes were in the range of 100 to
102 dependent on conditions used [8,11]. Single
/ux-tagged Anabaena cells could be observed by
microscopy [12]. Reported detection limits of PCR
amplification in pure cultures are variable, but
detection limits have been reported similar to
those we found [27,35].
Using cell extract luminescence as a detection
method, Cebolla et al. [20] recently reported a
detection limit of 3.4 x 10 4 cells for a pure culture of R. meliloti cells tagged with the luc-gene
integrated into the chromosome [20]. This large
difference in sensitivity, compared to our results,
is presumably due to the lower copy number of
the luc gene, i.e. one chromosomal copy per cell,
compared to our studies with luc on a multicopy
number plasmid. A 10-fold increase in expression
of the luc gene in R. meliloti was observed when
the gene was on a plasmid contained in 7.5 to 15
copies per cell [20].
The different methods we investigated have
different quantitative values. The best method for
quantitation of gene dosage (luciferase gene
product) is the cell extract bioluminescence
method. Since A T P and substrate are directly
added to the cell extract the amount of luciferase
present in the cells is directly measured and the
cellular reserves of A T P are not critical (Fig. 2).
By contrast, although whole cell measurements
are also quantitative, the relative light output
203
diminishes with age of the cells (Fig. 2), as implicated by the higher limit of detection we found
for stationary phase cells compared to late log
phase cells (Fig. 1).
Detection limits in sediment were poorer than
in pure culture (Table 1). In sediment, the whole
cell luciferase assay was more sensitive compared
to the cell extract assay. This may be due to the
higher degree of inhibition in sediment we determined for luciferase activity in cell lysates, compared to whole cells (Table 2). The firefly luciferase enzyme is known to be inhibited by chloride ions and is strongly inhibited at salt concentrations greater than 0.1 mM [36]. This may partly
explain the higher light yield in samples from
Lake M~ilaren compared to Baltic Sea samples
having salinity values in the range of 0.17 mM
and 10.3 mM, respectively (Table 2).
Until recently, detection of bioluminescent
cells in the environment has focused on the bacterial luciferase system. Depending on the assay
used, different detection limits in soil or sediment
have been cited for cells marked with lux genes.
For example, 200 to 6000 /ux-tagged cells, dependent on the E. coli strain used, could be
directly detected by luminometry in sterile soil
samples [11], showing the potential for non-extractive measurements of bioluminescence. In another study, a 5 cell g- 1 inoculum of bioluminescent Xanthomonas campestris added to soil could
be detected after growth in medium, compared to
5 x 103 cfu g-1 without pregrowth. [14]. By charge
coupled device (CCD) enhanced microscopy, single cells could be visualized in soil samples [13],
showing the promise of this type of methodology.
There have been various efforts by other investigators to optimize PCR for detection of low
numbers of cells in environmental samples. However, results vary dependent on many factors.
These include the following: (1) number of target
gene copies; (2) number of cycles of amplification; (3) use of nested primers; (4) use of DNA
probes; (5) concentration of cells from water samples [22]; (6) complexity of abiotic and biotic
components, and (7) extraction and purification
of DNA from sediment and soil, which is often
the most difficult step [24,28,29,37,38].
The procedure we used for isolation and pu-
rification of DNA is quick and easy and should
be easily adaptable for routine monitoring of
luc-tagged GMMs in the environment. It was
simple to amplify DNA in natural water samples,
whereas the sediment samples required more extensive processing to reduce humic material and
salts that otherwise inhibit Taq polymerase. We
chose to amplify large target sequences (1480 and
1390 bp using outer and inner primers, respectively) to increase the amount of ethidium bromide bound on a molar basis, compared to smaller
templates, in order to optimize our detection
sensitivity on agarose gels. Since fragments larger
than approx. 350 bp are difficult to amplify if the
DNA is isolated by direct extraction due to shearing effects [29,37] we isolated intact bacteria from
sediments rather than by direct DNA extraction.
There were different PCR results obtained
from the two different sampling sites (i.e. fresh
water compared to brackish water). We generally
had a greater PCR response in the lake M~ilaren
samples. Although we do not know the reason for
this discrepancy it could be due to differences in
salt, or humic acid concentrations.
The limit of detection in sediment by PCR was
60-70 cells 0.5 g-1. Since only a fraction of the
total DNA isolated from sediment was added to
the PCR mixture, this corresponds to approximately 50 copies of the luc gene template that
could be detected. These results are comparable
to previously published results in sterile sediment
[29], and non-sterile soil with the addition of T4
gene 32 protein [28]. Even greater sensitivity and
specificity may be obtained by using a specific
DNA probe after PCR amplification [23].
We successfully monitored E. coli in the microcosms using the various methods developed.
Surprisingly, the E. coli cells were viable and
healthy at least 16 days in the microcosms at 4°C.
This was demonstrated by their growth on selective medium and whole cell bioluminescence
which requires a cellular reserve of ATP.
The results obtained from the microcosms by
bioluminescence measurements and plate counts
were similar. However, at the later sampling dates
there was a relatively higher number of cells
isolated on selective medium compared to the
amount of bioluminescence measured (Fig. 4).
204
This d i s c r e p a n c y c o u l d be d u e to a d e c r e a s e in
p l a s m i d copy n u m b e r d u r i n g t h e p r o l o n g e d incub a t i o n u n d e r o l i g o t r o p h i c c o n d i t i o n s in t h e microcosms. This p h e n o m e n o n has b e e n r e p o r t e d
for b a c t e r i a t a g g e d with the b a c t e r i a l lux g e n e s
on a p l a s m i d [6]. By c o m p a r i s o n , w h e n t h e lux
g e n e s w e r e stably i n t e g r a t e d into the c h r o m o s o m e t h e light o u t p u t was m o r e s t a b l e on a p e r
cell basis [6].
O f t e n , E. coli a n d o t h e r l a b o r a t o r y strains
r e a c h a n o n - c u l t u r a b l e state a f t e r p r o l o n g e d incub a t i o n u n d e r n u t r i e n t - l i m i t e d c o n d i t i o n s f o u n d in
n a t u r e . D e t e c t i o n o f b i o l u m i n e s c e n t cells, o r d e t e c t i o n o f the g e n e by P C R a r e a t t r a c t i v e o p t i o n s
t h a t do n o t rely on cultivation o f t h e o r g a n i s m s .
F u r t h e r m o r e , for b a c t e r i a r e l e a s e d into t h e envi-
r o n m e n t , a n t i b i o t i c r e s i s t a n c e g e n e s s h o u l d not
be u s e d as m a r k e r s d u e to the risk o f s p r e a d o f
a n t i b i o t i c resistance.
Selective p l a t i n g is still a v a l u a b l e p r o c e d u r e
for q u a n t i t a t i o n o f c u l t u r a b l e cells, a n d we u s e d
p l a t e c o u n t i n g with a n t i b i o t i c s e l e c t i o n as a verific a t i o n o f cell n u m b e r . C e b o l l a et al. (1993)
d e m o n s t r a t e d t h e utility o f d i s t i n c t i o n o f l u m i n e s c e n t luc-tagged R. meliloti cells against a backg r o u n d o f i n d i g e n o u s b a c t e r i a on a g a r m e d i u m
w i t h o u t a n t i b i o t i c selection [20].
W e n o t only d e v e l o p e d w h o l e cell a n d cell
extract b i o l u m i n e s e n c e a n d P C R m e t h o d o l o g i e s
for d e t e c t i o n of luc-tagged b a c t e r i a in environm e n t a l samples, b u t for t h e first t i m e directly
compared the methods. Advantages and disad-
Table 3
Advantages and disadvantages of different methods for detection of the luc-marker gene
Assay for detection of
luc-marker gene
Advantages
Disadvantages
Whole cell
bioluminescence
In situ measurements are possible
Single cell detection by microscopy
Detection of non-culturable,
but viable cells
Assay is very rapid and simple
Useful for screening of
luminescent colonies
No background luminescence
in our samples
Best method for quantitation
of gene dosage
Commercial assay kit is available
Assay is rapid
Useful for detection of nonculturable
cells, viable (and nonviable?)
No background luminescence
in our samples
Light yield is not dependent
on cellular ATP levels
Most sensitive method in sediment
Detection of non-culturable,
viable and non-viable cells
No background hybridization to
the luc gene in the natural
microbial populations tested
May be possible to use as a
quantitative method with
future modifications
Light yield is dependent on
ATP reserves
Components extracted from
sediment inhibit light yield
Cell extract
bioluminescence
PCR amplification
Components extracted from sediment
strongly inhibit light yield
Contamination risks are high
Quantitation is difficult
Purification of samples from
sediment is necessary
205
vantages of the different methods are summarized in Table 3. The bioluminescence measurements hold promise for routine monitoring of
GMMs in the field, due to the simplicity and
speed of the assays. We are currently improving
purification of the cell extracts from sediment to
remove inhibitory compounds interfering with luminescence.
We propose the firefly luciferase gene to be an
ideal marker for GMMs released into the environment due to the following reasons: (1) the
detection assays are very sensitive; (2) the lucgene is very specific for the tagged bacteria; (3)
there is no phenotype in the absence of luciferin;
(4) the bioluminescence assays are extremely fast
and easy; (5) the assays are relatively inexpensive;
and (6) luc-tagged ceils can be detected by a
variety of complementary methods. The possiblity
of using more than one method to detect the
same marker gene provides one with flexibility in
choosing the appropriate method for specific applications.
4
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6
7
8
9
10
Acknowledgements
We would like to thank Sven Blomqvist (Department of Systems Ecology, Stockholm University) for donating sediment core samples for our
use and Bo H6ijer (Department of Biochemistry,
Stockholm University) for construction and calibration of the luminometer used in these studies.
This work was supported by the Swedish Environmental Protection Agency, the Swedish National
Chemicals Inspectorate and the Carl Tryggers
Foundation.
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