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Appl Environ Microbiol. Sep 1999; 65(9): 3793–3799.
PMCID: PMC99702

Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

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 β-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 β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-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 β-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 β-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 β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.

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 β-galactosidase activity (21), a reporter system with this lacZ gene as a reporter gene would be more sensitive to promoter activity. The β-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 Table1.1.

TABLE 1
Bacterial strains and plasmids

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 (A600) 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).

β-Galactosidase stability and time course studies.

The inocula used for time course and β-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 β-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 β-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 β-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 β-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 β-galactosidase activity, strain ATCC 824 static flask cultures were examined to determine whether there was any β-galactosidase activity. In all phases of growth in static flask cultures, the β-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. (Fig.1),1), was introduced into C. acetobutylicum ATCC 824, and the β-galactosidase activities in static flask cultures of this strain were determined. The maximum β-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.

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, ...

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 Figure11 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.

β-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 β-galactosidase specific activities. The time courses of typical β-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. Fig.22 and and3.3. 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 β-galactosidase activity of control strain ATCC 824(pHT3) was negligible compared to the β-galactosidase activities of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA).

FIG. 2
Time course profiles of β-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 ...
FIG. 3
Time course profiles of β-galactosidase specific activities of ATCC 824(pHT4) (A), ATCC 824(pHT5) (B), and ATCC 824(pHTA) (C) in static flask cultures. Symbols: ×, A600; [filled lozenge], flask 1 specific activity; ■, flask 2 specific ...

Furthermore, Fig. Fig.33 shows that the β-galactosidase specific activity profiles of ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) were different. The β-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) β-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 β-galactosidase specific activity was approximately 80% greater than the early-exponential-phase activity. The β-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).

β-Galactosidase stability analysis.

In most of the experiments performed with the new reporter system, there was a decrease in β-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 β-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 β-galactosidase in strains ATCC 824(pHT4) and ATCC 824(pHT5).

The total and specific β-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. Fig.4.4. The total-protein levels increased in untreated samples throughout growth and leveled off by hour 25, reflecting the total-biomass profile (as determined by A600). 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 β-galactosidase activity in Fig. Fig.44 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. Fig.4).4). These observations suggest that adding chloramphenicol did not completely inhibit β-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.

FIG. 4
Time course profiles of total protein (A), total β-galactosidase activity (B), and β-galactosidase specific activity (C) in a chloramphenicol treatment experiment performed with a static flask culture of ATCC 824(pHT4). Symbols: [filled triangle], ...

The specific activity profiles for both untreated and chloramphenicol-treated samples from the same culture are shown in Fig. Fig.4C.4C. The complexity of the data required a semiquantitative interpretation in which we used a transient mass balance for β-galactosidase (1):

equation M1
1

where [p] is the β-galactosidase concentration on a total-protein basis (i.e., the specific activity), kp is the rate constant for β-galactosidase production, ξ is the translational efficiency for β-galactosidase, [mRNA] is the lacZ mRNA concentration on a total-protein basis, ke is the rate constant for β-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 β-galactosidase. The production term refers to β-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 β-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. (Fig.3C)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. (Fig.4C)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:

equation M2
2

where M indicates that the values are the mean concentrations for the treated sample over time interval Δt. The dilution term (last term on the right side of equation 1) is negligible because there is negligible protein and biomass synthesis (Fig. (Fig.4A)4A) after chloramphenicol is added, and thus the corresponding specific growth rate (μ′) is almost zero. The (kpξ)′ 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 β-galactosidase synthesis and that β-galactosidase degradation was negligible even compared to chloramphenicol-inhibited β-galactosidase synthesis. In the last three samples (late exponential to stationary phase), [p]utr was equal to or greater than [p]tr, suggesting that β-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. (Fig.44).

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 Figure55 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 β-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.

FIG. 5
Time course profiles of total protein (A), total β-galactosidase activity (B), and β-galactosidase specific activity (C) in a chloramphenicol treatment experiment performed with a static flask culture of ATCC 824(pHT5). Symbols: [filled triangle], ...

The specific activity profile obtained in the β-galactosidase stability experiment performed with ATCC 824(pHT5) is also shown in Fig. Fig.5.5. 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. (Fig.5).5). As in the ATCC 824(pHT4) β-galactosidase stability experiment, this suggests that β-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 β-galactosidase was degraded, as shown in the ATCC 824(pHT4) β-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) β-galactosidase stability experiment.

β-Galactosidase activities in controlled-pH batch fermentations.

We also examined the ability of the reporter system to reflect endogenous promoter activity. The kinetics of β-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. Fig.6.6. 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 Figure66 shows that both the PTB and β-galactosidase specific activities increased in the early exponential phase. The PTB specific activity then continued to increase until the late exponential phase, while the β-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.

FIG. 6
Specific activity profiles of β-galactosidase and PTB in a controlled pH ≥5.0 fermentation of C. acetobutylicum ATCC 824(pHT4). Symbols: ×, A600; □, pH; [filled lozenge], PTB specific activity; [filled triangle], β-galactosidase ...

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 β-galactosidase that could be assayed in C. acetobutylicum ATCC 824. Detection of β-galactosidase specific activity after this reporter gene was introduced on a plasmid indicated that the β-galactosidase produced by the new reporter system was functional. This was confirmed by the 1,000-fold increases in β-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 β-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 β-galactosidase specific activity profiles. This demonstrated that the reporter system can adequately distinguish between different promoters. Also, the nearly constant levels of β-galactosidase specific activity observed in the stationary phase with strain ATCC 824(pHTA) indicated that β-galactosidase production by strain ATCC 824(pHTA) continues into the stationary phase at a rate that most likely balances the rate of β-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 β-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 β-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 β-galactosidase stability analysis of ATCC 824(pHT4) (Fig. (Fig.4)4) showed that β-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 β-galactosidase specific activity profile of ATCC 824(pHT4) (Fig. (Fig.3).3). In the early phases of growth, β-galactosidase specific activity increased until the late exponential phase, at which point the specific activity began to decrease. Both the β-galactosidase specific activity profile of ATCC 824(pHT4) and the results of the β-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 β-galactosidase stability analysis of ATCC 824(pHT5) (Fig. (Fig.5)5) showed that β-galactosidase production is greatest in the early and mid-exponential phases of growth. As growth continues, β-galactosidase is still produced, but at a decreased rate, until the stationary phase, in which the rate of β-galactosidase production is less than the β-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 β-galactosidase stability experiment results suggest that the thl promoter is stronger than the ptb promoter, because the β-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.

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