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Applied and Environmental Microbiology, September 1999, p. 3793-3799, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development and Characterization of a Gene
Expression Reporter System for Clostridium acetobutylicum
ATCC 824
Seshu B.
Tummala,1
Neil E.
Welker,2 and
Eleftherios T.
Papoutsakis1,*
Department of Chemical
Engineering1 and Department of
Biochemistry, Molecular Biology, and Cell
Biology,2 Northwestern University, Evanston,
Illinois 60208
Received 19 January 1999/Accepted 9 June 1999
 |
ABSTRACT |
A gene expression reporter system (pHT3) for Clostridium
acetobutylicum ATCC 824 was developed by using the
lacZ gene from Thermoanaerobacterium
thermosulfurogenes EM1 as the reporter gene. In order to test the
reporter system, promoters of three key metabolic pathway genes,
ptb (coding for phosphotransbutyrylase), thl
(coding for thiolase), and adc (coding for acetoacetate
decarboxylase), were cloned upstream of the reporter gene in pHT3 in
order to construct vectors pHT4, pHT5, and pHTA, respectively.
Detection of 
-galactosidase activity in time course studies
performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC
824(pHTA) demonstrated that the reporter gene produced a functional
-galactosidase in C. acetobutylicum. In addition, time
course studies revealed differences in the -galactosidase specific
activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC
824(pHTA), suggesting that the reporter system developed in this study
is able to effectively distinguish between different promoters. The
stability of the -galactosidase produced by the reporter gene was
also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using
chloramphenicol treatment to inhibit protein synthesis. The data
indicated that the -galactosidase produced by the lacZ
gene from T. thermosulfurogenes EM1 was stable in the
exponential phase of growth. In pH-controlled fermentations of ATCC
824(pHT4), the kinetics of -galactosidase formation from the
ptb promoter and phosphotransbutyrylase formation from its
own autologous promoter were found to be similar.
| |
INTRODUCTION |
Clostridium
acetobutylicum ATCC 824 is a gram-positive, spore-forming,
obligate anaerobe that is able to ferment various sugars to form the
commercial solvents acetone and butanol. The acetone-butanol fermentation ceased to be economical in the late 1950s due to the
development of more efficient petrochemical processes, but recent
developments in the molecular genetics of solventogenic clostridia have
revived interest in this fermentation process. In the last few years,
there have been several advances in the metabolic engineering of
C. acetobutylicum aimed at generating superproducing
strains. In 1993, Mermelstein and Papoutsakis developed an
electroporation protocol for introducing plasmid DNA into C. acetobutylicum ATCC 824 (11). This allowed carbon and
energy fluxes to be redirected by overexpression of solventogenic genes carried on a plasmid (12). Another major advance involved
the use of nonreplicative integrational plasmids to knock out specific clostridial genes by homologous recombination (6). More
recently, Desai and Papoutsakis showed that antisense RNA can be used
in C. acetobutylicum to downregulate production of specific
enzymes (4). Antisense RNA strategies can be used for
repression of specific metabolic enzymes in order to redirect carbon
fluxes toward desirable pathways.
Nevertheless, several necessary genetic tools are still missing, and
this fact hinders progress in this field. One of the missing critical
tools is a gene expression reporter system. A reporter system would
allow workers to study expression of both autologous and heterologous
promoters in solventogenic clostridia and to understand the regulation
of these promoters. An understanding of promoter strength and
regulation could lead to more effective clostridial expression vectors.
Such vectors eventually could be used to augment expression of
solventogenic pathway genes in order to enhance solvent production.
Moreover, an understanding of promoter strength and regulation could be
coupled with antisense RNA strategies and knockout mutations to develop
more complex metabolic engineering strategies for increasing solvent production.
Since no effective gene expression reporter systems for C. acetobutylicum have been available, no clostridial promoters have been characterized yet. Since Escherichia coli genes are
poorly expressed in C. acetobutylicum, traditional E. coli reporter genes, such as lacZ, cannot be used in
C. acetobutylicum. The green fluorescent protein gene has
been used as a reporter gene in many bacterial systems. However, the
green fluorescent protein gene does not appear to be a good candidate
for use as a reporter gene for C. acetobutylicum because of
its requirement for oxygen, which is necessary for the development of
the chromophore responsible for fluorescence (8). A
chloramphenicol acetyltransferase gene, catP, from
Clostridium perfringens has also been considered for potential use as a reporter gene for C. acetobutylicum.
Although adequate as a reporter gene for C. perfringens
(2, 10), catP may not be well-suited for use as a
reporter gene for C. acetobutylicum, because C. acetobutylicum contains high levels of nonspecific coenzyme A
transferases which might interfere with the chloramphenicol acetyltransferase assay.
Burchhardt and Bahl (3) cloned and analyzed the
lacZ gene from Clostridium thermosulfurogenes EM1
(later classified as Thermoanaerobacterium
thermosulfurogenes EM1 [5]). These authors also
proposed that this gene would be an excellent candidate as a reporter
gene for C. acetobutylicum (3). Since C. acetobutylicum ATCC 824 lacks endogenous -galactosidase
activity (21), a reporter system with this lacZ
gene as a reporter gene would be more sensitive to promoter activity.
The -galactosidase assay has also been well-documented, is
relatively easy to perform, and is sensitive to low levels of activity.
In this study, we developed a gene expression reporter system (pHT3) in
which the lacZ gene from T. thermosulfurogenes
EM1 is the reporter gene for C. acetobutylicum ATCC 824. Several experiments were performed to characterize the reporter system
and to assess its potential for use in promoter characterization
studies. One set of experiments was performed to evaluate the
functionality of the reporter gene's product and its ability to
discriminate between different promoters. In addition, we investigated
the stability of the reporter gene product. Another set of experiments was performed to examine whether the reporter system with the ptb promoter can reflect the promoter activity of the
endogenous ptb gene.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Table
1.
Growth conditions and maintenance of strains.
E. coli
strains were grown aerobically at 37°C in L broth. For recombinant
strains, antibiotics were added to the medium at the following final
concentrations: 50 µg/ml for ampicillin and 35 µg/ml for
chloramphenicol. Both recombinant and wild-type E. coli
strains were stored at 
85°C in L broth supplemented with 10% glycerol.
For growth in liquid medium,
C. acetobutylicum ATCC 824 isolates were grown anaerobically in 10 ml of
Clostridium
growth medium
(CGM) at 37°C (
19). In all experiments
growth in liquid medium
was monitored by measuring the absorbance at
600 nm (A
600) of
appropriate dilutions with a model DU
series 60 spectrophotometer
(Beckman, Fullerton, Calif.). For growth on
solid medium,
C. acetobutylicum ATCC 824 was grown
anaerobically at 37°C on 2× YTG (pH 5.8) agar
plates. For
recombinant strains, erythromycin was added to liquid
and solid media
at final concentrations of 100 and 40 µg/ml, respectively.
Both
recombinant and wild-type
C. acetobutylicum ATCC 824 isolates
were stored at
85°C in CGM supplemented with 20%
glycerol.
Plasmid DNA isolation and manipulation and cell
transformation.
The alkaline lysis method of Lee and Rasheed was
used for plasmid isolation in E. coli (9). To
isolate plasmids from recombinant isolates of C. acetobutylicum ATCC 824, an alkaline lysis method developed by
Harris was used (7). Briefly, recombinant isolates of
C. acetobutylicum were grown anaerobically in screw-cap
tubes to the late exponential phase (A600, 1.0 to 2.0). The
cells were collected from 3 to 6 ml of samples of a culture and washed
twice in 500 µl of 0.5 M KCl-0.1 M EDTA-0.05 M Tris-HCl and once in 500 µl of SET buffer (25% sucrose, 0.05 M Tris-HCl, 0.05 M EDTA). The cell pellet was resuspended in 450 µl of SET buffer containing lysozyme (5 mg/ml) and then incubated at 37°C for 10 min. After incubation, 350 µl of alkaline sodium dodecyl sulfate was added, and
then 350 µl of ice-cold 3 M K+-5 M acetate was added.
The plasmid DNA was further purified by using a standard
phenol-chloroform extraction procedure.
All of the commercial enzymes utilized in this study (i.e., restriction
enzymes, T4 DNA ligase, calf intestinal alkaline phosphatase,
T4 DNA
polymerase, and the Klenow fragment of DNA polymerase)
were used under
the conditions recommended by the supplier. DNA
fragments were isolated
from agarose gels by electrophoresis onto
DEAE-cellulose membranes
(
15). All plasmids were constructed
in
E. coli
first and then transformed into
C. acetobutylicum.
E. coli and
C. acetobutylicum were electrotransformed by
using
previously described methods (
11,
15).
-Galactosidase stability and time course studies.
The
inocula used for time course and -galactosidase stability
experiments were prepared in the same way. For each inoculum, 10-ml
portions of CGM in screw-cap tubes were inoculated with colonies of
recombinant strains that had been grown on solid medium and heat
shocked for 10 min at 70 to 80°C. After the medium cooled to room
temperature, erythromycin was added to the appropriate final
concentration. The 10-ml cultures were then grown anaerobically at
37°C. At the early exponential phase (A600, 0.4), a 5-ml
sample of each culture was used to inoculate 50 ml of CGM containing erythromycin. When the culture reached the early exponential phase (A600, ca. 0.4), 7.5 ml of the culture was used to
inoculate each of two culture flasks containing 750 ml of CGM
supplemented with erythromycin. The first sample was removed from each
culture at the early exponential phase (A600, 0.1 to 0.4).
After this, samples were removed from both cultures every 3 or 4 h
until the cultures reached the stationary phase (A600, 4.0 to 6.0). The exponential phases in static flask culture experiments
tend to vary, but in general, a culture is in the exponential phase
when the A600 is between 0.1 and 2.0 (the A600
in the mid-exponential phase is between 0.4 and 1.5, and the
A600 in the late exponential phase is between 1.5 and 2.0).
Between the exponential phase and the stationary phase
(A600, 2.0 to 4.0), the culture is considered to be in
transition from the exponential phase to the stationary phase; this
stage is referred to below as the late exponential to early stationary phase.
For time course studies, a 50-ml sample was taken from each flask, and
the cells were collected by centrifugation at 10,000
×
g for 10 min at 4°C; the pellet was then frozen at
20°C. For
-galactosidase stability experiments, a 25-ml sample
was taken
from each flask, and the cells were collected by
centrifugation
at 10,000 ×
g for 10 min at 4°C; the
pellet was also frozen at
20°C. Moreover, a second 25-ml sample was
transferred from each
flask to a 50-ml centrifuge tube. Chloramphenicol
was added to
each tube to a final concentration of 200 µg/ml. The
tubes were
then incubated anaerobically for an additional 2.5 h at
37°C (
18).
Then cells were collected from the
chloramphenicol-treated samples
in the same way that the untreated
sample cells were
collected.
Controlled pH fermentations.
Batch fermentation cultures of
C. acetobutylicum ATCC 824 recombinants were grown in a
2.0-liter Biostat M fermentor (B. Braun, Allentown, Pa.) and a
5.0-liter BioFlo II bioreactor (New Brunswick Scientific, Edison, N.J.)
with working volumes of 1.5 and 4.0 liters, respectively. All
fermentations were performed as described by Desai and Papoutsakis, by
using CGM supplemented with 75 µg of clarithromycin per ml instead of
erythromycin (4). After the pH values of the fermentation
preparations dropped to 5.0, low-end pH control was implemented by
adding 6 N ammonium hydroxide periodically to maintain the pH at 
5.0.
For the batch fermentations used in this study, a culture was in the
exponential phase when the A600 ranged from 0.1 to 2.0 and
a culture was in the stationary phase when the A600 ranged
from 4.0 to 5.5; these values were approximately the same as the values
observed in the static flask culture experiments. Also, the
A600 values for different exponential phases of a culture
were roughly similar to the values in static flask culture experiments
(i.e., the A600 in the early exponential phase was 0.1 to
0.4, the A600 in the mid-exponential phase was 0.4 and 1.5, the A600 in the late exponential phase was 1.5 to 2.0, and
the A600 in the late exponential-early stationary phase was
2.0 to 5.5).
Enzyme assays.
Frozen cells were thawed and suspended to an
A600 of ~10 to 20 in at least 1 ml of Z buffer which
contained (per liter) 16.1 g of
Na2HPO4 · 7H2O, 5.5 g
of NaH2PO4 · H2O, 0.75 g of KCl, 0.246 g of MgSO4 · 7H2O, and
2.7 ml of -mercaptoethanol. A 1-ml aliquot of each sample was then
sonicated, and crude extracts were harvested as described by Desai and
Papoutsakis (4). For the -galactosidase assay, crude
extracts were further processed as described by Burchhardt and Bahl,
with a slight modification (3). The modification was used to
remove heat-labile clostridial proteins. This was done by taking a
portion of each crude lysate and heat treating it at 60°C for 30 min.
The precipitate (denatured protein) was then removed by centrifugation
at 16,000 × g in a microcentrifuge for 30 min at
4°C. The supernatant fluids were stored at 4°C.
The
-galactosidase assay was performed with the heat-treated crude
lysates as described by Miller, except that 60°C was used
as the
assay temperature (
14). One unit of activity was defined
as
the amount of enzyme that catalyzed the formation of 1 nmol
of
orthonitrophenol per min at 60°C (extinction coefficient, 0.0045
µM
1cm
1). Phosphotransbutyrylase (PTB)
activity was measured in the butyryl
phosphate-forming direction by
monitoring the amount of liberated
coenzyme A as a complex with
5,5'-dithio-bis(2-nitrobenzoic acid)
at 412 nm, as previously described
(
20). One unit of activity
was defined as the amount of
enzyme that catalyzed the formation
of 1 µmol of coenzyme
A-5,5'-dithio-bis(2-nitrobenzoic acid) complex
per min at room
temperature (extinction coefficient, 13.6 mM
1cm
1). The Bio-Rad protein assay (Bio-Rad
Laboratories, Hercules,
Calif.) based on the Bradford method was used
to measure the total
protein concentrations in all crude
lysates.
| |
RESULTS |
Development of the gene expression reporter system.
To ensure
that C. acetobutylicum ATCC 824 has no endogenous
-galactosidase activity, strain ATCC 824 static flask cultures were
examined to determine whether there was any -galactosidase activity.
In all phases of growth in static flask cultures, the -galactosidase
specific activities of C. acetobutylicum ATCC 824 were
insignificant (data not shown). The ability of lacZ from T. thermosulfurogenes EM1 to express a functional gene
product in C. acetobutylicum ATCC 824 was then examined. A
2.5-kb HhaI-PstI fragment of pCT102, which
contained the lacZ structural gene and a putative ribosome
binding site (including 44 bp upstream of the ribosome binding site and
315 bp downstream of the lacZ stop codon), was treated with
both T4 DNA polymerase and the Klenow fragment and then inserted into
the PstI site of pIMP1, which was also treated with T4 DNA
polymerase. The resulting plasmid, pHT3 (Fig.
1), was introduced into C. acetobutylicum ATCC 824, and the -galactosidase activities in
static flask cultures of this strain were determined. The maximum
-galactosidase specific activity detected was 16 U/mg, which was
detected in the early exponential phase of growth. This is a very low
level of activity.
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FIG. 1.
Construction of the reporter system (pHT3) and three
test vectors (pHT4, pHT5, and pHTA). For each plasmid, the locations
and directions of transcription of relevant genes are indicated
(arrows). Relevant restriction sites are shown. Abbreviations: lacZ,
lacZ gene from T. thermosulfurogenes EM1; AmpR,
ampicillin resistance genes; ColE1, Col E1 origin of replication; MLSr,
macrolide-lincosamide-streptogramin B resistance gene; repL, pIM13
origin of replication.
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The putative promoter regions from three key metabolic pathway genes of
C. acetobutylicum ATCC 824,
ptb,
thl,
and
adc, were
cloned upstream of
lacZ in pHT3 in
order to test the newly developed
reporter system. Figure
1 summarizes
the construction of the reporter
system and the three test vectors
developed in this study. A 445-bp
SacI-
BamHI
fragment of pSOS94 which contained the putative
ptb promoter
region was treated with both T4 DNA polymerase and the
Klenow fragment
to form a blunt-ended DNA fragment. This blunt-ended
fragment was then
inserted into the single
SmaI site of pHT3 to
create vector
pHT4. Similarly, a 440-bp
SacI-
BamHI fragment of
pSOS95 yielded the desired putative
thl promoter region.
This
fragment was also treated with both T4 DNA polymerase and the
Klenow fragment to form blunt-ended DNA. In order to construct
pHT5,
the blunt-ended DNA fragment was then inserted into the
single
SmaI site of pHT3. Similar to the construction of pHT4
and
pHT5, a 318-bp
KpnI-
PpuMI fragment of pFNK6 which
contained
the
adc putative promoter region was isolated and
treated with
both T4 DNA polymerase and the Klenow fragment and then
inserted
into the single
SmaI site of pHT3 to create vector
pHTA.
-Galactosidase activities in static flask cultures without pH
control.
To show that clostridial promoters function in the
reporter system, static flask cultures containing strains ATCC
824(pHT3), ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) were
analyzed to determine their -galactosidase specific activities. The
time courses of typical -galactosidase specific activity profiles for static flask cultures of ATCC 824(pHT3), ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) are shown in Fig.
2 and 3.
Strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) exhibited
significantly higher levels of activity than the control strain, ATCC
824(pHT3) [the maximum levels for ATCC 824(pHT3), ATCC 824(pHT4), ATCC
824(pHT5), and ATCC 824(pHTA) were approximately 16, 1,200, 1,450, and
1,250, U/mg, respectively]. Thus, the -galactosidase activity of
control strain ATCC 824(pHT3) was negligible compared to the
-galactosidase activities of strains ATCC 824(pHT4), ATCC 824(pHT5),
and ATCC 824(pHTA).
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FIG. 2.
Time course profiles of -galactosidase specific
activity of ATCC 824(pHT3) in static flask cultures. Symbols: ×,
A600;  , specific activity. Two flasks (flasks 1 and 2)
were used in this experiment, but only the flask 2 specific activity
profile is shown because the flask 1 and flask 2 data were identical.
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FIG. 3.
Time course profiles of -galactosidase specific
activities of ATCC 824(pHT4) (A), ATCC 824(pHT5) (B), and ATCC
824(pHTA) (C) in static flask cultures. Symbols: ×, A600;
 , flask 1 specific activity; , flask 2 specific activity.
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|
Furthermore, Fig.
3 shows that the
-galactosidase specific activity
profiles of ATCC 824(pHT4), ATCC 824(pHT5), and ATCC
824(pHTA) were
different. The
-galactosidase specific activity
of strain ATCC
824(pHT4) increased approximately 40% from the
early exponential phase
to the late exponential phase and then
decreased rapidly during the
late exponential to early stationary
phase to a level that was ca. 15%
below the level in the early
exponential phase. In contrast, the strain
ATCC 824(pHT5)
-galactosidase
specific activity increased 110% from
the early exponential phase
to the mid-exponential phase, when the
activity peaked. The activity
remained steady until the late
exponential to early stationary
phase. Then the activity decreased
slowly until the final
-galactosidase
specific activity was
approximately 80% greater than the early-exponential-phase
activity.
The
-galactosidase specific activity of strain ATCC
824(pHTA)
increased approximately 180% from the early exponential
phase to the
early stationary phase and remained at that level
throughout the rest
of the experiment. When the strains were compared
to each other, strain
ATCC 824(pHT5) had the highest maximum level
of activity (ca. 1,450 U/mg), while strain ATCC 824(pHTA) had
the highest final specific
activity (ca. 1,250 U/mg).
-Galactosidase stability analysis.
In most of the
experiments performed with the new reporter system, there was a
decrease in -galactosidase specific activity during the late
exponential to early stationary phase. This decrease may have been due
to differences in promoter activity at different stages of growth, or
it may have been due to -galactosidase instability as a result of
protein degradation. Welch et al. examined the in vivo stability of an
enzyme by using chloramphenicol to inhibit protein synthesis
(18). We used this method to examine the stability of
-galactosidase in strains ATCC 824(pHT4) and ATCC 824(pHT5).
The total and specific
-galactosidase activities, as well as
total-protein profiles for untreated and chloramphenicol-treated
samples of a static flask culture of ATCC 824(pHT4), are shown
in Fig.
4. The total-protein levels increased in
untreated samples
throughout growth and leveled off by hour 25, reflecting the total-biomass
profile (as determined by
A
600). There were no significant differences
in the
total-protein levels between the chloramphenicol-treated
samples and
the untreated samples throughout the exponential phase
of growth,
suggesting that chloramphenicol inhibited total-protein
synthesis, as
expected. In the stationary phase, the total-protein
levels in
chloramphenicol-treated samples were approximately 10%
lower than the
total-protein levels in untreated samples, reflecting
the fact that
there was a certain amount of protein degradation.
The profiles of
total
-galactosidase activity in Fig.
4 show
that the total activity
of the chloramphenicol-treated sample
in the early exponential phase
(first sample) was approximately
equal to the total activity of the
untreated sample in the early
exponential phase. In the early
exponential to late exponential
growth phase (next two samples), the
total activities of the chloramphenicol-treated
samples were higher
than the total activities of the untreated
samples. The differences
between the chloramphenicol-treated and
untreated samples, however,
were significantly less than the increases
in total activity of the
untreated samples that would have been
harvested at the same time as
the treated samples (the activities
of these untreated samples could be
interpolated from the untreated-sample
activity profile by drawing a
vertical line starting from the
point representing the treated-sample
activity, as shown in Fig.
4). These observations suggest that adding
chloramphenicol did
not completely inhibit
-galactosidase synthesis
during these
stages of active growth. In the late exponential to early
stationary
growth phase (fourth samples), there was not a significant
difference
between the total activities of chloramphenicol-treated and
untreated
samples. However, in the stationary phase (last two samples),
the specific activities of chloramphenicol-treated samples were
approximately 15% lower than the specific activities of untreated
samples.
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FIG. 4.
Time course profiles of total protein (A), total
-galactosidase activity (B), and -galactosidase specific activity
(C) in a chloramphenicol treatment experiment performed with a static
flask culture of ATCC 824(pHT4). Symbols:  , untreated samples;  ,
chloramphenicol-treated samples; ×, A600.
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The specific activity profiles for both untreated and
chloramphenicol-treated samples from the same culture are shown in Fig.
4C. The complexity of the data required a semiquantitative
interpretation
in which we used a transient mass balance for
-galactosidase
(
1):
![d[p]/dt = k<SUB>p</SUB>&xgr;[<UP>mRNA</UP>]<UP> − </UP>k<SUB>e</SUB>[p] − &mgr;[p]](/content/vol65/issue9/fulltext/3793/img001.gif)
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(1)
|
where [
p] is the
-galactosidase concentration on a
total-protein basis (i.e., the specific activity),
kp is the rate constant
for
-galactosidase
production,
is the translational efficiency
for
-galactosidase,
[mRNA] is the
lacZ mRNA concentration on
a total-protein
basis,
ke is the rate constant for
-galactosidase
degradation, and µ is the specific growth rate
(
1). The three
terms on the right side of equation 1 represent the rates of production,
degradation, and dilution,
respectively, for
-galactosidase.
The production term refers to
-galactosidase synthesis from mRNA.
Degradation effects are caused
by protein degradation that may
be due to endogenous proteases. The
dilution term results from
an increase in total-protein amount due to
biomass expansion.
[mRNA] reflects the activity of the cloned
promoter and
-galactosidase
mRNA stability and can be represented by
its own mass balance
equation (
1). As described above, we
found that it was useful
to compare the specific activity of the
chloramphenicol-treated
sample ([
p]
tr,1 for
sample 1) (Fig.
3C) with the specific activity
of the corresponding
untreated sample ([
p]
utr,1 for sample 1),
as
well as with the specific activity of the untreated sample
([
p]
utr',1) (Fig.
4C) that would have been
harvested at
the same time as the treated sample. For the second,
third, fourth,
etc. sets of samples, the subscripts used were 2, 3, 4, etc. In
order to facilitate interpretation of the experimental data, we
rewrote equation 1 in its finite difference form as follows:
![[p]<SUB><UP>tr,1</UP></SUB> − [p]<SUB><UP>utr,1</UP></SUB> = {(k<SUB>p</SUB>&xgr;)′[<UP>mRNA</UP>]<SUB>M</SUB> − k<SUB>e</SUB>[p]<SUB>M</SUB>}&Dgr;t](/content/vol65/issue9/fulltext/3793/img002.gif)
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(2)
|
where
M indicates that the values are the mean
concentrations for the treated sample over time interval
t. The dilution
term (last term on the right side of
equation 1) is negligible
because there is negligible protein and
biomass synthesis (Fig.
4A) after chloramphenicol is added, and thus
the corresponding
specific growth rate (µ') is almost zero. The
(
kp)' values are
modified constants due to
chloramphenicol inhibition of protein
synthesis.
In the first sample (early exponential phase),
[
p]
tr,1 was only slightly greater than
[
p]
utr,1. In the next two samples (mid-
to
late exponential phase), however, [
p]
tr was
considerably greater
than [
p]
utr, which,
according to equation 2, suggests that chloramphenicol
did not
completely inhibit
-galactosidase synthesis and that
-galactosidase degradation was negligible even compared to
chloramphenicol-inhibited
-galactosidase synthesis. In the last
three samples (late exponential
to stationary phase),
[
p]
utr was equal to or greater than
[
p]
tr,
suggesting that
-galactosidase
degradation did take place, albeit
at low levels, in the nonactive
growth phase of the culture in
which the
ptb promoter
activity was substantially reduced (Fig.
4).
We also briefly compared the chloramphenicol-treated samples with the
untreated samples that would have been harvested at
the same time as
the treated sample. Most interesting were the
second and third samples,
in which [
p]
tr was equal to or greater
than
[
p]
utr'. Our interpretation, based on an
equation that
was derived from equations 1 and 2, is that this finding
can be
explained by the dilution effect (represented by the third term
on the right side of equation 1). This dilution effect was observed
only in the untreated culture and could become dominant during
the
active exponential growth
phase.
A protein stability analysis was also performed with strain ATCC
824(pHT5). Figure
5 shows the
total-protein, total-activity,
and specific activity profiles of both
chloramphenicol-treated
and untreated samples from a static flask
culture of ATCC 824(pHT5).
Similar to the results of the protein
stability analysis performed
with strain ATCC 824(pHT4), the
total-protein profile obtained
for ATCC 824(pHT5) shows that there were
not significant differences
in the total-protein profiles of
chloramphenicol-treated and untreated
samples in the exponential phases
of growth (samples 1 to 4).
In the later stages (the last two samples),
however, the total-protein
levels in the chloramphenicol-treated
samples were lower than
the total-protein levels in the untreated
samples, which indicated
that
-galactosidase degradation occurred.
The total-activity
profiles show that in the early exponential phase
the total activities
of untreated and chloramphenicol-treated samples
were roughly
the same. As growth continued from the early exponential
phase
to the late exponential-early stationary phase, the total
activity
of the chloramphenicol-treated samples was higher than the
total
activity of the untreated samples. In contrast, toward the end
of
the culture, the total activity in the untreated samples was
approximately 20% greater than the total activity in the
chloramphenicol-treated
samples.
View larger version (18K):
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|
FIG. 5.
Time course profiles of total protein (A), total
-galactosidase activity (B), and -galactosidase specific activity
(C) in a chloramphenicol treatment experiment performed with a static
flask culture of ATCC 824(pHT5). Symbols: , untreated samples; ,
chloramphenicol-treated samples ×, A600.
|
|
The specific activity profile obtained in the
-galactosidase
stability experiment performed with ATCC 824(pHT5) is also shown
in
Fig.
5. The specific activities of untreated and
chloramphenicol-treated
samples were approximately equal in the early
exponential phase,
as shown by the finding that
[
p]
tr,1 was approximately equal to
[
p]
utr,1. For the next three samples, the
specific activities
of the chloramphenicol-treated samples were greater
than the specific
activities of the untreated samples, as shown by the
fact that
[
p]
tr was greater than
[
p]
utr (Fig.
5). As in the ATCC 824(pHT4)
-galactosidase stability experiment, this suggests that
-galactosidase
is stable in the exponential phase. In the late
exponential to
stationary phase (the last two samples),
[
p]
tr was less than
[
p]
utr,
suggesting that
-galactosidase was
degraded, as shown in the
ATCC 824(pHT4)
-galactosidase stability
experiment. However,
since [
p]
tr was greater
than [
p]
utr' for samples 2 to 4,
the dilution
effect in conjunction with degradation seemed to
be the cause of the
decreases in specific activity in untreated
samples. Similar results
were obtained in the ATCC 824(pHT4)
-galactosidase
stability
experiment.
-Galactosidase activities in controlled-pH batch
fermentations.
We also examined the ability of the reporter system
to reflect endogenous promoter activity. The kinetics of
-galactosidase formation due to the ptb promoter was
compared to PTB activity due to the natural promoter in controlled-pH
fermentations (pH 5.0) of strain ATCC 824(pHT4). The results of a
typical experiment are shown in Fig. 6.
Similar but not identical profiles were expected for the two proteins
due to potentially different in vivo mRNA and protein stabilities, as
well as different codon usage. Figure 6 shows that both the PTB and
-galactosidase specific activities increased in the early
exponential phase. The PTB specific activity then continued to increase
until the late exponential phase, while the -galactosidase specific
activity remained essentially constant. However, from the late
exponential phase to stationary phase, the two specific activities
decreased at nearly the same rate to final levels that were ca. 75% of
the peak values.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6.
Specific activity profiles of -galactosidase and PTB
in a controlled pH 5.0 fermentation of C. acetobutylicum
ATCC 824(pHT4). Symbols: ×, A600;  , pH; , PTB
specific activity; , -galactosidase specific activity.
|
|
| |
DISCUSSION |
The goal of this study was to develop an effective gene expression
reporter system for C. acetobutylicum ATCC 824. We
investigated the lacZ gene from T. thermosulfurogenes EM1 to determine whether it could be used as a
reporter gene. Thus, the first task in this study was to show that a
reporter system in which this lacZ gene was used could
produce a functional -galactosidase that could be assayed in
C. acetobutylicum ATCC 824. Detection of -galactosidase specific activity after this reporter gene was introduced on a plasmid
indicated that the -galactosidase produced by the new reporter
system was functional. This was confirmed by the 1,000-fold increases
in -galactosidase specific activities in strains containing the
ptb, thl, or adc promoters upstream of
the reporter gene. The substantial differences in specific activity
between the reporter systems with and without clostridial promoters
also suggest that the sensitivity of the reporter system is sufficient
to monitor even weak promoters.
Furthermore, the effectiveness of the reporter system was demonstrated
by the -galactosidase specific activity profiles obtained in several
experiments. Time course studies in which static flask culture
experiments were performed with the ptb, thl, and
adc promoters cloned upstream of the reporter gene resulted
in different -galactosidase specific activity profiles. This
demonstrated that the reporter system can adequately distinguish
between different promoters. Also, the nearly constant levels of
-galactosidase specific activity observed in the stationary phase
with strain ATCC 824(pHTA) indicated that -galactosidase production
by strain ATCC 824(pHTA) continues into the stationary phase at a rate
that most likely balances the rate of -galactosidase degradation
that has been shown to occur in the stationary phase by protein
stability experiments performed with ATCC 824(pHT4) and ATCC 824(pHT5). Thus, the experiment in which strain ATCC 824(pHTA) was used showed that the gene expression system developed in this study can detect promoter activity in the stationary phase. In controlled pH 5.0 fermentations with ATCC 824(pHT4), the ability to express
ptb from the endogenous ptb promoter and
lacZ from the cloned ptb promoter in pHT4 was
examined by using the -galactosidase and PTB enzyme assays. The
results showed that expression of lacZ and expression of
ptb occurred in similar but not identical fashions. The
possible explanations for the observed differences between the
-galactosidase and PTB specific activity profiles include different
mRNA stabilities, different translation efficiencies, and different
protein stabilities.
A comparison of the chloramphenicol-treated and untreated samples from
the -galactosidase stability analysis of ATCC 824(pHT4) (Fig. 4)
showed that -galactosidase production is significantly greater in
the exponential phase of growth than in the later stages of growth,
which suggests that the ptb promoter is an
early-growth-associated promoter. This finding is similar to what was
observed with the -galactosidase specific activity profile of ATCC
824(pHT4) (Fig. 3). In the early phases of growth, -galactosidase
specific activity increased until the late exponential phase, at which
point the specific activity began to decrease. Both the
-galactosidase specific activity profile of ATCC 824(pHT4) and the
results of the -galactosidase stability experiment performed with
ATCC 824(pHT4) are consistent with the results of previous studies of
PTB in which it was suggested that the ptb promoter is
associated with the early growth phase (17).
A similar comparison of the chloramphenicol-treated and untreated
samples used for the -galactosidase stability analysis of ATCC
824(pHT5) (Fig. 5) showed that -galactosidase production is greatest
in the early and mid-exponential phases of growth. As growth continues,
-galactosidase is still produced, but at a decreased rate, until the
stationary phase, in which the rate of -galactosidase production is
less than the -galactosidase degradation rate. This suggests that
the thl promoter is on throughout the exponential phase of
growth and, like the ptb promoter, is also an
early-growth-associated promoter. In addition, the static flask culture
and -galactosidase stability experiment results suggest that the
thl promoter is stronger than the ptb promoter, because the -galactosidase specific activities in these experiments were higher with strain ATCC 824(pHT5) than with ATCC 824(pHT4). Also,
experiments performed with ATCC 824(pHTA) indicated that the
adc promoter, which is assumed to be active predominantly during the solventogenic phase, is also active in the acidogenic phase
of growth.
In conclusion, a reporter system in which the lacZ gene from
T. thermosulfurogenes EM1 is used was developed and analyzed in this study for use in C. acetobutylicum ATCC 824. We hope
that this system will facilitate gene expression and promoter
characterization studies in this organism and other solventogenic clostridia.
| |
ACKNOWLEDGMENTS |
This work was supported by National Science Foundation grant
BES-9632217.
We thank Phillipe Soucaille for providing plasmids pSOS94 and pSOS95
and Hubert Bahl for donating plasmid pCT102 for this study to our
collaborator George Bennett of Rice University. We also thank Abbott
Laboratories for donating clarithromycin.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemical Engineering, Northwestern University, 2145 Sheridan Rd.,
Evanston, IL 60208. Phone: (847) 491-7455. Fax: (847) 491-3728. E-mail: e-paps{at}nwu.edu.
| |
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0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
