Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

Lothar Feustel; Stephan Nakotte; Peter Dürre

doi:10.1128/AEM.70.2.798-803.2004

. 2004 Feb;70(2):798–803. doi: 10.1128/AEM.70.2.798-803.2004

Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

Lothar Feustel ¹, Stephan Nakotte ^1,^†, Peter Dürre ^1,^*

PMCID: PMC348925 PMID: 14766557

Abstract

The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding β-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in β-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression. adc (encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower). The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data). lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter.

The clostridia represent a large bacterial genus, comprising species of significant biotechnological as well as medical importance. The production of acetone and butanol, indigo dyeing, and flax retting by using clostridia were well-established industrial processes in the past, and much work is devoted to their economical reintroduction (2). In addition, clostridia are currently used as producers of enzymes. Members of this genus also produce a variety of potent toxins, including the most poisonous natural compounds known, tetanus and botulinum. However, even the latter compound has become an extremely valuable therapeutic and cosmetic agent (for recent reviews, see reference 2). An even larger biotechnological and medical potential of the clostridia still awaits elucidation and exploitation. However, for realization of this purpose it is of utmost importance to have tools at hand which allow for easy genetic modification and analysis.

Clostridium acetobutylicum and Clostridium perfringens have emerged as model organisms for apathogenic and pathogenic clostridia, respectively (1). Reporter gene systems are indispensable for monitoring of gene expression and characterization of promoter strength and regulation. Several systems have been proposed for the pathogenic species. The luxAB genes of Vibrio fischeri have been used to measure the activity of the promoter of the alpha-toxin (phospholipase C) of C. perfringens. Aeration of the anaerobic samples was required to initiate bioluminescence (21, 22). A similar system based on the luciferase gene (lucB) of the firefly Photinus pyralis was developed for Clostridium botulinum, but appeared not to be an ideal reporter (27; E. A. Johnson, personal communication). The chloramphenicol acetyltransferase gene (catP) of C. perfringens (28) has also been used to monitor expression at the transcriptional level, again mostly for the C. perfringens plc gene (5, 14). However, the use of catP is restricted, since some clostridia, such as Clostridium beijerinckii NCIMB 8052 (formerly C. acetobutylicum), carry natural chloramphenicol resistance (19). Another reporter tested was the gusA gene of Escherichia coli, encoding β-glucuronidase. This system has been used to measure expression from the cpe gene (encoding the enterotoxin) and the nanEA operon (encoding N-acetylmannosamine-6-phosphate epimerase and sialic acid lyase) promoters of C. perfringens (15, 33, 36). Finally, the above-mentioned luxAB genes and a lacZ gene from Thermoanaerobacterium thermosulfurigenes (encoding a β-galactosidase) (6) were tested for monitoring of expression from a botulinum toxin gene promoter in C. botulinum. Efficient detection was reliant on the presence in the plasmid of a transcriptional regulator gene (7).

With apathogenic clostridia, however, few such studies have been reported so far (31). The lacZ gene mentioned above was introduced into C. acetobutylicum (9, 29, 30), the gusA gene was successfully used in C. acetobutylicum and C. beijerinckii (12, 24), and for C. beijerinckii, usage of the eglA gene (encoding a β-1,4-endoglucanase) from Clostridium saccharobutylicum (formerly C. acetobutylicum) was proposed (23).

Here we report the successful use of the lacZ and lucB genes as reporters for the promoter activity of genes required for solventogenesis in C. acetobutylicum. In the case of lacZ, a sequence error in the original database entry was detected, which could have a significant influence on new reporter constructions.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and maintenance.

The strains and plasmids used in this study are listed in Table 1. Precultures of C. acetobutylicum DSM 792 were grown anaerobically in modified 2× YT medium (25 mM glucose, 1.6% [wt/vol] tryptone, 1.0% [wt/vol] yeast extract, 86 mM NaCl) (20). Growth experiments were performed in batch cultures with anaerobic, morpholineethanesulfonic acid-buffered minimal medium at 37°C (4). The pH was adjusted to 7.3 with 6 N NaOH before autoclaving. Cells harvested for β-galactosidase detection were grown in air-tight 1,000-ml Müller and Krempel bottles; growth for luciferase monitoring took place in 125-ml Müller and Krempel bottles. Anaerobic conditions were generated by N₂ sparging before autoclaving. Growth was monitored by measurement of the optical density at 600 nm (OD₆₀₀) as well as by measurement of the pH. Media for recombinant C. acetobutylicum strains were supplemented with 4 μg of clarithromycin (Abbott Laboratories, Queenborough, United Kingdom)/ml. Clostridial strains were preserved as spore suspensions (in modified 2× YT medium) at −70°C.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristic(s)^a	Source or reference
Strains
C. acetobutylicum
DSM 792	Wild type	DSMZ^b
E. coli
XL1-Blue	Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 [F′ proAB laql^qZΔM15 Tn10 (Tc^r)]	Stratagene GmbH, Heidelberg, Germany
ER2275	trp-31 his-1 tonA2 rpsL104 supE44 xyl-7 mtl-2 metB1 e14⁻ Δ(lac)U169 endA1 recA1 R(zbgZ10::Tn10)Tc^s Δ(mcr-hsd-mrr)114::1510 [F′ proAB laql^qZΔM15 zzf::mini Tn10(Km^r)]	E. T. Papoutsakis, Evanston, Ill.
N99	strA ΔlacZ λ⁻ F⁻gaK2 rpsL, IN(rrnD-rrnE)	6
Plasmids
pCT102	Ap^r, 2.7-kbp PstI fragment carrying lacZ from T. thermosulfurigenes cloned in pUC18	6
pAN1	Cm^r, φ3TI, p15A origin	14
pLacZF	Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lacZ	26
pLacZFT	Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lacZ, termination structure downstream of adc from C. acetobutylicum	This study
pLacZFT_adc	adc promoter, Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lacZ	This study
pLacZFT_bdhA	bdhA promoter, Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lacZ	This study
pLacZFT_bdhB	bdhB promoter, Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lacZ	This study
pLacZFT_sol	sol promoter, Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lacZ	This study
pLucBF	Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lucB	This study
pLucBF_ptb	ptb promoter, Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lucB	This study
pLucBF_sol	sol promoter, Ap^r MLS^r Em^r, repL gene, ColE1 origin, promoterless lucB	This study

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Abbreviations: Ap^r, ampicillin resistance gene; Cm^r, chloramphenicol resistance gene; φ3TI, Bacillus subtilis phage φ3TI methyltransferase gene; MLS^r, macrolide, lincosamide, and streptogramin B resistance gene; Em^r, erythromycin resistance gene; repL, gram-positive origin of replication from pIM13.

DSMZ, Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

E. coli strain XL1-Blue (Stratagene) was used as the host for cloning experiments. In vivo methylation of plasmids took place in E. coli strain ER2275 carrying the pAN1 plasmid (17). The strains were grown at 37°C aerobically on a rotary shaker in Luria-Bertani medium (25) supplemented with 100 μg of ampicillin/ml (XL1-Blue transformed with either pLacZFT or pLucBF) or 100 μg of ampicillin/ml and 34 μg of chloramphenicol/ml (ER2275). E. coli strains were preserved in Luria-Bertani medium containing 10% (vol/vol) glycerol at −70°C.

Plasmids.

Construction of the pLacZF plasmid used for transcriptional studies and carrying the lacZ gene was described earlier (29) (Fig. 1). pLacZF was also used for the construction of pLucBF. pLacZF is based on pIMP1 (16), which carries a replicon yielding a plasmid copy number of 6 to 8 (13). pBRluc, carrying the lucB gene, was generously made available by Damian Lynch, Department of Medical Microbiology and Hygiene, University of Ulm, Ulm, Germany.

pLacZFT, pLacZFT_adc, pLacZFT_sol, pLacZFT_bdhA, and pLacZFT_bdhB construction.

Plasmid pLacZF was modified by insertion of the transcriptional terminator between the adc and ctfB genes of C. acetobutylicum. This element was amplified as a 150-bp fragment by PCR with clostridial genomic DNA (primers Term_up and Term_down [Table 2 ]). The primers were designed to contain a PstI site, which was then used for ligation into pLacZF, yielding pLacZFT. The intergenic regions upstream of adc, bdhA, bdhB, and sol were amplified via PCR with C. acetobutylicum DNA and the primers listed in Table 2. After BamHI and SalI double digestion, the fragments were cloned in frame upstream of the lacZ gene of pLacZFT and were transformed into E. coli XL1-Blue, followed by ampicillin selection. Purified plasmids of positive clones were verified by restriction analysis and sequencing of the inserted fragment. Sequencing of both strands was performed with respective IRD800-labeled (5′) primers (MWG Biotech AG, Ebersberg, Germany) by using the SequiTherm EXCEL II Long Read sequencing kit LC (Biozym Diagnostik GmbH, Hess. Oldendorf, Germany) and a LiCor 4000 L DNA sequencer (MWG Biotech AG).

TABLE 2.

Primers used for PCR amplifications

Primer	Sequence (5′→3′)^a
AdhE-fus-up-Sal	aattcGTCGACtgttgtgactttcataaatatacac
p.adhE+act.bam	tattGGATCCattaattagggttatatatactag
padc_down	tttgtttGTCGACttcatcctttaacataaaag
padc_up	aaacctGGATCCtatttattttttgtattgg
pbdhB_down	taaatgcagaGGATCCtcttgag
pbdhb_up	tatGTCGACttcgaaatcaaccattttaacc
pbdhA_down	aaatggGGATCCttattgtaatc
pbdhA_up	ttggtGTCGACtaatcaaaacttagcatac
pptb_down	ttgttggGGATCCtatgaaggatacag
pptb_up	attaaaGTCGACaatcactggtcgtacac
pLucBF_down	acgtaGTCGACgaagacgccaaaaac
pLucBF_up	acctgtCTGCAGttacaatttggactttcc
Term_down	aatgaaCTGCAGcccatgg
Term_up	tttctaCTGCAGacattcttgc

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Bases representing restriction enzyme cutting sites are given as in capital letters.

pLucBF, pLucBF_sol, and pLucBF_ptb construction.

The lucB gene was amplified from pBRluc via PCR with the primers listed in Table 2. Because a translational fusion was desired, the start codon was exchanged with a SalI cutting site. The fragment obtained was cloned into purified, PstI- and SalI-digested pLacZFT, resulting in pLucBF (Fig. 1). The PstI/SalI digestion of pLacZFT completely removed the lacZ gene and the transcriptional terminator. Successful cloning was verified by PstI/SalI double digestion and sequencing. The procedures mentioned above were also used for the construction of pLucB_sol and pLucBF_ptb. Primer sequences are given in Table 2.

DNA isolation and manipulation and electroporation of C. acetobutylicum.

Plasmid DNA was isolated by using the Gfx-Plasmid-Prep kit (Amersham Pharmacia Biotech GmbH, Freiburg, Germany) according to the manufacturer's manual. The electroporation of DNA into C. acetobutylicum was performed as described previously (18). The total genomic DNA from C. acetobutylicum was isolated as described previously (3).

Restriction enzymes and T4 DNA ligase were obtained from MWG Biotech AG. Standard ligation conditions were incubation for 1 h at 22°C (with a preceding 5-min step at 55°C). PCRs were performed with DeepVent DNA polymerase (NEB) according to the manufacturer's manual. The reaction mixture contained 400 pM concentrations of each deoxynucleoside triphosphate, 100 pM concentrations of each primer, 1× reaction buffer (supplied by the manufacturer), 2 to 10 ng of template DNA, and 2 U of polymerase. Thirty cycles were performed in a thermal cycler (Biozym PTC-200; Biozym Diagnostik GmbH). Each cycle consisted of the following three steps: denaturation at 95°C for 30 s, annealing (depending on the melting temperatures of the primers; usually 45 to 55°C) for 30 s, and elongation at 72°C for 30 s up to 100 s (depending on the calculated fragment length). The primers usually contained either a SalI or BamHI cutting site (Table 2) for directed cloning.

DNA fragments were purified by use of 100-kDa Microcon (Millipore GmbH, Schwalbach, Germany) tubes. Digests or PCR amplifications (usually in 50-μl volumes) were mixed with 450 μl of sterile, deionized water and loaded onto the Microcon tube. They were then centrifuged at 2,000 × g at room temperature until only a small volume remained above the cellulose matrix (∼50 μl). The eluate was discarded, and the supernatant was centrifuged in a new collection tube. This method was also used to concentrate small amounts of DNA.

Analysis of fermentation products.

The concentrations of acetone and butanol in culture supernatants were determined by use of a Chrompack CP 9001 gas chromatograph equipped with a flame ionization detector and a Chromosorb 101 packed column. Isobutanol (110 mM) in 2 N H₂SO₄ was added before detection to 1 ml of the probe as an internal standard for the calculation of product concentrations (29).

β-Galactosidase assay.

The β-galactosidase assay was performed spectrophotometrically with o-nitrophenyl-β-d-galactopyranoside, as described previously (29).

Luciferase assay.

For luciferase assays, 2-ml culture samples were used. The cells were centrifuged (5 min, 4°C, 6,000 × g) and the pellet was suspended in 2 ml of potassium phosphate buffer (50 mM, pH 7), with vigorous shaking to saturate the sample with oxygen, and then kept on ice. The cell suspension (132.5 μl) and 37.5 μl of ATP solution (100 mM) were pipetted into a NUNC vial (Sarstedt AG & Co., Nürnbrecht, Germany), which was put into the luminescence reader. Upon starting detection, the machine automatically added 150 μl of 2× assay buffer (glycyl glycine, 62.5 mM; MgCl₂, 25 mM; pH 7.8) and 150 μl of luciferin solution (330 μM d-luciferin dissolved in potassium phosphate buffer [10 mM, pH 6.5]). Luminescence was measured in a Flash'n Glow luminescence reader (Berthold Detection Systems, Pforzheim, Germany) at 560 nm for 15 s. In order to plot the results over the run time, the quotient of measured relative light units (RLU) and the OD₆₀₀ was calculated.

Protein concentration.

Protein concentrations were determined by using the Pierce BCA protein assay kit (Perbio Science Deutschland GmbH, Bonn, Germany) according to the manufacturer's instructions.

RESULTS

Reexamination of the lacZ sequence.

During the course of early experiments aimed at analyzing the promoter of the adc operon of C. acetobutylicum (encoding acetoacetate decarboxylase) (9), it was observed that some constructions yielded only faint blue colonies of E. coli N99 on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates. Determination of the β-galactosidase activity also resulted in very low values. All respective constructs had been obtained by first digesting the pCT102 original vector carrying the lacZ gene from T. thermosulfurigenes EM1 (6) with HaeII and PstI, isolating the 2,713-bp PstI fragment with lacZ, and subcloning it into NsiI-digested (cuts within the adc gene) pUG67 (10). All resulting plasmids still showed a dark blue phenotype on X-Gal plates. For further constructions, the lacZ gene (with or without promoter) was cut out with various restriction enzymes at the 5′ end (depending on the promoter sequences used) and BstXI at the 3′ end. All such constructions showed only very low β-galactosidase activity. According to the published sequence, the BstXI site (CCANNNNNNTGG) should be located well after the lacZ gene (stop codon from nucleotides 2400 to 2402), between nucleotides 2457 and 2468. Since the data obtained could not be explained by the original sequence, the lacZ gene on pCT102 was resequenced. Indeed, an additional A was detected at position 2400 of the original sequence. This resulted in a lacZ gene product which is 31 amino acid residues longer than originally assumed (with a corrected stop codon from nucleotides 2493 to 2495). For all further constructions, the complete lacZ gene was used.

Determination of expression of genes involved in solventogenesis by using the lacZ reporter.

The promoters of several genes or operons that are required for solventogenesis (adc, bdhA, bdhB, and sol, encoding acetoacetate decarboxylase, butanol dehydrogenase A, butanol dehydrogenase B, and butyraldehyde/butanol dehydrogenase E as well as coenzyme A transferase, respectively) were examined in order to characterize the strength and pattern of their activities. The upstream regions, including the first three to five codons of the corresponding adc, bdhA, bdhB, and adhE genes, were cloned into reporter plasmid pLacZFT as described in Materials and Methods. All determinations with recombinant strains were done in triplicate. Maximal transcription from the adc promoter was reached late in the exponential-growth phase (Fig. 2B). Expression of the acetoacetate decarboxylase gene was turned off 2 h after reaching its maximum. The adc and bdhB promoters were active already during exponential growth, but maximal transcript concentrations were reached in the stationary phase. Similarly, the transcriptional activity of the sol operon increased during the exponential-growth phase. The highest promoter activity was reached when the culture entered the stationary phase and butanol production was initiated (Fig. 2C). Figure 2D shows the activity pattern of the bdhA promoter. For this promoter, maximal expression was observed before the pH shift. The expression levels of bdhA and sol were much lower than those of adc and bdhB.

FIG. 2. — In vivo determinations of promoter activities of genes required for solventogenesis by means of the *lacZ* reporter. (A) Relationship of OD and pH during growth. Symbols: •, pH of culture; ⧫, OD₆₀₀. (B) Specific β-galactosidase activity and pH for *adc* and *bdhB* promoter constructs. Symbols: ○, pH for *bdhB*; ▵, pH for *adc*; •, specific β-galactosidase activity obtained for the *bdhB* promoter; ▴, specific β-galactosidase activity obtained for the *adc* promoter. (C) Specific β-galactosidase activity and pH for the *sol* promoter construct. Symbols: ○, pH for *sol*; •, specific β-galactosidase activity obtained for the *sol* promoter. (D) Specific β-galactosidase activity and pH for the *bdhA* promoter construct. Symbols: ○, pH for *bdhA*; •, specific β-galactosidase activity obtained for the *bdhA* promoter.

Use of lucB as a reporter in C. acetobutylicum.

The lucB gene of the American firefly was amplified and used to replace the lacZ gene incorporated in pLacZFT, resulting in pLucBF. The ptb promoter was cloned upstream of the firefly luciferase gene to determine whether activity could be measured in C. acetobutylicum. After transformation of the plasmid and cultivation of the recombinant cells, expression could be detected for the pLucBF_ptb construct (Fig. 3). Transcription from the ptb promoter led to a constant, high level of about 100,000 RLU per OD unit. Beginning at the onset of solventogenesis, transcription was almost completely repressed.

FIG. 3. — In vivo determinations of *ptb* and *sol* operon promoter activities by means of the *lucB* reporter. (A) RLU per OD unit for the *ptb* promoter construct. Symbols: ○, pH of culture; •, RLU/OD unit obtained for the *ptb* promoter. (B) RLU per OD unit for the *sol* operon promoter construct. Symbols: ○, pH of culture; •, RLU/OD unit obtained for the *sol* operon promoter.

During the course of these experiments, it was found that the addition of 25 mM ATP to the reaction mixture reproducibly increased the luminescence up to 10-fold. Washing of the harvested C. acetobutylicum cells was also crucial. The extracellular pH dropped from 6.8 to about 5 at the end of growth. This acidification led to a shift of bioluminescence to red (shift from 560 to 617 nm) and therefore to inconsistent data (technical support of MoBiTec, Göttingen, Germany [distributor of d-luciferin sodium salt]). Washing of cells with potassium phosphate buffer (50 mM, pH 7) was necessary to avoid this unwanted effect.

In addition to the ptb promoter, the upstream region of the sol operon, as an example of a gene required for solventogenesis, was tested for luciferase activity (Fig. 3). Although the maximal activity of sol transcription was lower than that for ptb (1,771 RLU/OD unit), the data showed the same pattern of transcription as that obtained with the β-galactosidase assay (Fig. 2C). The pH shift induced transcription of the sol operon, which reached its highest activity in the middle of the shift phase. Transcription then declined rapidly before the cells entered the stationary phase.

The lucB reporter system proved to be extremely sensitive. Values with about 10,000-fold differences could be reproducibly measured. Therefore, exponential plots are shown in Fig. 3. The data clearly demonstrate high-level expression from the ptb promoter during exponential growth, followed by a drastic decrease when solvent production reached its maximum. In agreement with the lacZ reporter data, the sol operon was expressed at a much lower level.

The luciferase stability was also determined by keeping a sample on ice and measuring its luminescence over time. The initial luminescence decreased rapidly during the first 5 min (Fig. 4). The luminescence then stayed stable for about half an hour. Thus, even large amounts of samples can reliably be measured during this period.

FIG. 4. — Stability of luciferase activity. The *sol* operon promoter construct was used for these determinations. The initial RLU value obtained (18-h sample from Fig. 3) was set to 100%. The sample was kept on ice and measured for an additional 60 min.

DISCUSSION

As detailed in the introduction, the lacZ gene of T. thermosulfurigenes has become a valuable reporter for various clostridia. This is due to the fact that the G+C content of 31.5% of Thermoanaerobacterium (6) is almost as low as that typically found within clostridial species. Therefore, its codon usage is similar to that of clostridial species. In addition, species such as C. acetobutylicum ATCC 824 cleave the substrate lactose by means of a phospho-β-galactosidase and do not possess a β-galactosidase (35). This is because lactose is transported into the cell as lactose phosphate via a phosphotransferase system, as was shown for C. saccharobutylicum (formerly C. acetobutylicum P262) (8). Thus, in such strains there is no problem with background activity. However, this feature is strain dependent and has to be checked for every clostridial species prior to experimentation. Since the lacZ reporter has been successfully used in apathogenic and pathogenic clostridia, the sequence correction reported here is of significant importance to all groups working in this field. The very low level of activity obtained from BstXI (partial deletion) constructs points to an important role of the C terminus of the enzyme in catalytic functioning. A PstI digest allowed us to cut out the complete gene from the original vector, pCT102. Alternatively, PCR amplification from plasmid or chromosomal DNA with primers carrying additional suitable restriction sites is easily possible.

The reporter gene study reported here is the first to compare all promoters of the genes that are directly required for solventogenesis. The results obtained nicely match data from previous Northern blot experiments (11, 26). bdhA is expressed already during the exponential-growth phase and reaches a maximum as soon the pH starts to drop. At this time, almost no acetone and butanol can be detected in the medium, pointing to a role of BdhA in ethanol or very low-level butanol formation, in accordance with previous investigations (26, 32, 34). The enzyme thus might serve as an electron sink in the early, acidogenic fermentation. The sol operon is then induced (at about 13 h) until it reaches its maximal expression, and transcription declines after about 23 h. bdhB, on the other hand, is induced about 2 h later but keeps its maximal expression level longer than the sol operon (up to 30 h). This is exactly the pattern found with Northern blot experiments. These data confirm that BdhB is indeed the butanol dehydrogenase responsible for high-level butanol production. The reporter studies, however, also allowed an insight into promoter strength. The expression level of bdhB was more than 67-fold higher than that of the sol operon and about 17-fold higher than that of bdhA. A comparison of reporter studies, RNA analyses with the adc gene, and enzymatic determinations of acetoacetate decarboxylase revealed that all methods indicated very early induction, and at least the former ones revealed two expression peaks (11). However, the reporter transcript seems to be rather unstable and decreases rapidly after reaching its maximum. This is in contrast to both adc transcript and acetoacetate decarboxylase activity, which are stable throughout the solventogenic phase (11). On the other hand, it is a feature which stresses the excellent ability of lacZ as a reporter. The rapid decay of transcripts allows for sensitive measurements throughout growth. At least a 100-fold difference in activity can easily be measured. The strength of the adc promoter was found to be ∼1.3-fold higher than that of bdhB. In a previous study using lacZ as a reporter for adc, a promoter strength comparable to those of ptb-buk and thlA had been determined (30). The specific activities seemed to be higher in that report, but the unit definition relied on nanomoles instead of micromoles. Thus, the values reported here are about 10-fold higher. This might be due to construct variations, which would also explain the higher transcript stability found for the former report.

The second reporter analyzed for this study was based on the luciferase gene (lucB) of the firefly P. pyralis. The decisive advantage of this system is that it is not affected by background activity at all. Thus, it can also be used in clostridia that possess a β-galactosidase. A second major advantage is the speed of sampling and assaying, which comes close to online conditions. Previous experiments with luciferase from V. fischeri yielded different results. Whereas bioluminescence could be used as a quantitative reporter for the relatively aerotolerant C. perfringens (21, 22), the highest values for C. botulinum type B were only reached after exposition to the air for 3 h, which introduced an unacceptable degree of variability into the assay (7). Using C. botulinum type A, Schmidt et al. (27) also concluded that luciferase expression (this time from P. pyralis) is possible but that luciferase does not represent an ideal reporter due to its lack of high-level expression. Thus, both luciferase systems are functional in clostridia, but the best suited enzyme has to be determined for the strain or species under investigation. The light emission from LucB of P. pyralis can be increased up to 10-fold by the addition of ATP to the assay. Washing of cells also proved to be essential for obtaining reliable values. The data obtained for this study with representative promoters of acidogenic (ptb) and solventogenic (sol) growth phases revealed the high sensitivity of the assay (a 10,000-fold difference can easily be measured). The pattern of sol expression with the lucB reporter was identical to that with lacZ, indicating that the cloning of the transcription terminator was not essential for exclusion of downstream effects. ptb expression was about 113-fold higher than that of sol. Again, this is in accordance with lacZ reporter data. As shown in Fig. 2, adc expression was about 84-fold higher than that of sol. In addition, the data of Tummala et al. (30) revealed the same expression level for adc and ptb.

In conclusion, both lacZ and lucB represent valuable reporter gene systems for clostridia. Their use revealed that expression levels from promoters of genes required for solventogenesis vary up to ∼100-fold, with adc and bdhB being much more strongly expressed than bdhA and the sol operon.

Acknowledgments

We thank Eric A. Johnson for sharing his data on lucB expression in C. botulinum prior to publication, Damian Lynch for providing pBRluc carrying the lucB gene, and Abbott Laboratories for a gift of clarithromycin.

This work was supported by grants from the BMBF GenoMik project (Competence Network Göttingen) and the European Community (contract no. QLK3-CT-2001-01737).

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7.Davis, T. O., I. Henderson, J. K. Brehm, and N. P. Minton. 2000. Development of a transformation and gene reporter system for group II, non proteolytic Clostridium botulinum type B strains. J. Mol. Microbiol. Biotechnol. 2:59-69. [PubMed] [Google Scholar]
8.Diez-Gonzalez, F., and J. B. Russell. 1996. The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms. FEMS Microbiol. Lett. 136:123-127. [Google Scholar]
9.Dürre, P., R.-J. Fischer, A. Kuhn, K. Lorenz, W. Schreiber, B. Stürzenhofecker, S. Ullmann, K. Winzer, and U. Sauer. 1995. Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation. FEMS Microbiol. Rev. 17:251-262. [DOI] [PubMed] [Google Scholar]
10.Gerischer, U., and P. Dürre. 1990. Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region of Clostridium acetobutylicum. J. Bacteriol. 172:6907-6918. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Gerischer, U., and P. Dürre. 1992. mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J. Bacteriol. 174:426-433. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Girbal, L., I. Mortier-Barrière, F. Raynaud, C. Rouanet, C. Croux, and P. Soucaille. 2003. Development of a sensitive gene expression reporter system and an inducible promoter-repressor system for Clostridium acetobutylicum. Appl. Environ. Microbiol. 69:4985-4988. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Lee, S. Y., L. D. Mermelstein, and E. T. Papoutsakis. 1993. Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824. FEMS Microbiol. Lett. 108:319-324. [DOI] [PubMed] [Google Scholar]
14.Matsushita, C., O. Matsushita, M. Koyama, and A. Okabe. 1994. A Clostridium perfringens vector for the selection of promoters. Plasmid 31:317-319. [DOI] [PubMed] [Google Scholar]
15.Melville, S. B., R. Labbe, and A. L. Sonenshein. 1994. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect. Immun. 62:5550-5558. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Mermelstein, L. D., N. E. Welker, G. N. Bennett, and E. T. Papoutsakis. 1992. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Bio/Technology 10:190-195. [DOI] [PubMed] [Google Scholar]
17.Mermelstein, L. D., and E. T. Papoutsakis. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage Φ3TI to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl. Environ. Micobiol. 59:1077-1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Nakotte, S., S. Schaffer, M. Böhringer, and P. Dürre. 1998. Electroporation of, plasmid isolation from, and plasmid conservation in Clostridium acetobutylicum DSM 792. Appl. Microbiol. Biotechnol. 50:564-567. [DOI] [PubMed] [Google Scholar]
19.O'Brien, R. W., and J. G. Morris. 1971. The ferredoxin-dependent reduction of chloramphenicol by Clostridium acetobutylicum. J. Gen. Microbiol. 67:265-271. [DOI] [PubMed] [Google Scholar]
20.Oultram, J. D., M. Loughlin, T.-J. Swinfield, J. K. Brehm, D. E. Thompson, and N. P. Minton. 1988. Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation. FEMS Microbiol. Lett. 56:83-88. [Google Scholar]
21.Phillips-Jones, M. K. 1993. Bioluminescence (lux) expression in the anaerobe Clostridium perfringens. FEMS Microbiol. Lett. 106:265-270. [DOI] [PubMed] [Google Scholar]
22.Phillips-Jones, M. K. 2000. Use of a lux reporter system for monitoring rapid changes in α-toxin gene expression in Clostridium perfringens during growth. FEMS Microbiol. Lett. 188:29-33. [DOI] [PubMed] [Google Scholar]
23.Quixley, K. W. M., and S. J. Reid. 2000. Construction of a reporter gene vector for Clostridium beijerinckii using a Clostridium endoglucanase gene. J. Mol. Microbiol. Biotechnol. 2:53-57. [PubMed] [Google Scholar]
24.Ravagnani, A., K. C. B. Jennert, E. Steiner, R. Grünberg, J. R. Jefferies, S. R. Wilkinson, D. I. Young, E. C. Tidswell, D. P. Brown, P. Youngman, J. G. Morris, and M. Young. 2000. Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol. Microbiol. 37:1172-1185. [DOI] [PubMed] [Google Scholar]
25.Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
26.Sauer, U., and P. Dürre. 1995. Differential induction of genes related to solvent formation during the shift from acidogenesis to solventogenesis in continuous culture of Clostridium acetobutylicum. FEMS Microbiol. Lett. 125:115-120. [Google Scholar]
27.Schmidt, J. A., S. S. Dineen, M. Bradshaw, and E. A. Johnson. 1999. Evaluation of a luciferase reporter system for the study of neurotoxin gene expression in Clostridium botulinum type A strain 62A, p. 14-16. In 1999 annual report. Food Research Institute, Madison, Wisc. [Online.] http://www.wisc.edu/fri/annrpt/bot99.pdf.
28.Steffen, C., and H. Matzura. 1989. Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens. Gene 75:349-354. [DOI] [PubMed] [Google Scholar]
29.Thormann, K., L. Feustel, K. Lorenz, S. Nakotte, and P. Dürre. 2002. Control of butanol formation in Clostridium acetobutylicum by transcriptional activation. J. Bacteriol. 187:1966-1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Tummala, S. B., N. E. Welker, and E. T. Papoutsakis. 1999. Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 65:3793-3799. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Tummala, S. B., C. Tomas, L. M. Harris, N. E. Welker, F. B. Rudolph, G. N. Bennett, and E. T. Papoutsakis. 2001. Genetic tools for solventogenic clostridia, p. 105-123. In H. Bahl and P. Dürre (ed.), Clostridia. Biotechnology and medical applications. Wiley-VCH Verlag GmbH, Weinheim, Germany.
32.Walter, K. A., G. N. Bennett, and E. T. Papoutsakis. 1992. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J. Bacteriol. 174:7149-7158. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Walters, D. M., V. L. Stirewalt, and S. B. Melville. 1999. Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens. J. Bacteriol. 181:4526-4532. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Welch, R. W., F. B. Rudolph, and E. T. Papoutsakis. 1989. Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824). Arch. Biochem. Biophys. 273:309-318. [DOI] [PubMed] [Google Scholar]
35.Yu, P.-L., J. B. Smart, and B. M. Ennis. 1987. Differential induction of β-galactosidase and phospho-β-galactosidase activities in the fermentation of whey permeate by Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 26:254-257. [Google Scholar]
36.Zhao, Y., and S. B. Melville. 1998. Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens. J. Bacteriol. 180:136-142. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r1] 1.Bahl, H., and P. Dürre. 1993. Clostridia, p. 285-323. In H. Sahm (ed.), Biotechnology, 2nd ed., vol. 1. VCH-Verlag Gmbh, Weinheim, Germany.

[r2] 2.Bahl, H., and P. Dürre. 2001. Clostridia. Biotechnology and medical applications. Wiley-VCH Verlag GmbH, Weinheim, Germany.

[r3] 3.Bertram, J., and P. Dürre. 1989. Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch. Microbiol. 151:551-557. [Google Scholar]

[r4] 4.Bertram, J., A. Kuhn, and P. Dürre. 1990. Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation. Arch. Microbiol. 153:373-377. [Google Scholar]

[r5] 5.Bullifent, H. L., A. Moir, and R. W. Titball. 1995. The construction of a reporter system and use for the investigation of Clostridium perfringens gene expression. FEMS Microbiol. Lett. 131:99-105. [DOI] [PubMed] [Google Scholar]

[r6] 6.Burchhardt, G., and H. Bahl. 1991. Cloning and analysis of the β-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1. Gene 106:13-19. [DOI] [PubMed] [Google Scholar]

[r7] 7.Davis, T. O., I. Henderson, J. K. Brehm, and N. P. Minton. 2000. Development of a transformation and gene reporter system for group II, non proteolytic Clostridium botulinum type B strains. J. Mol. Microbiol. Biotechnol. 2:59-69. [PubMed] [Google Scholar]

[r8] 8.Diez-Gonzalez, F., and J. B. Russell. 1996. The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms. FEMS Microbiol. Lett. 136:123-127. [Google Scholar]

[r9] 9.Dürre, P., R.-J. Fischer, A. Kuhn, K. Lorenz, W. Schreiber, B. Stürzenhofecker, S. Ullmann, K. Winzer, and U. Sauer. 1995. Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation. FEMS Microbiol. Rev. 17:251-262. [DOI] [PubMed] [Google Scholar]

[r10] 10.Gerischer, U., and P. Dürre. 1990. Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region of Clostridium acetobutylicum. J. Bacteriol. 172:6907-6918. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.Gerischer, U., and P. Dürre. 1992. mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J. Bacteriol. 174:426-433. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Girbal, L., I. Mortier-Barrière, F. Raynaud, C. Rouanet, C. Croux, and P. Soucaille. 2003. Development of a sensitive gene expression reporter system and an inducible promoter-repressor system for Clostridium acetobutylicum. Appl. Environ. Microbiol. 69:4985-4988. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r13] 13.Lee, S. Y., L. D. Mermelstein, and E. T. Papoutsakis. 1993. Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824. FEMS Microbiol. Lett. 108:319-324. [DOI] [PubMed] [Google Scholar]

[r14] 14.Matsushita, C., O. Matsushita, M. Koyama, and A. Okabe. 1994. A Clostridium perfringens vector for the selection of promoters. Plasmid 31:317-319. [DOI] [PubMed] [Google Scholar]

[r15] 15.Melville, S. B., R. Labbe, and A. L. Sonenshein. 1994. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect. Immun. 62:5550-5558. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r16] 16.Mermelstein, L. D., N. E. Welker, G. N. Bennett, and E. T. Papoutsakis. 1992. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Bio/Technology 10:190-195. [DOI] [PubMed] [Google Scholar]

[r17] 17.Mermelstein, L. D., and E. T. Papoutsakis. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage Φ3TI to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl. Environ. Micobiol. 59:1077-1081. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r18] 18.Nakotte, S., S. Schaffer, M. Böhringer, and P. Dürre. 1998. Electroporation of, plasmid isolation from, and plasmid conservation in Clostridium acetobutylicum DSM 792. Appl. Microbiol. Biotechnol. 50:564-567. [DOI] [PubMed] [Google Scholar]

[r19] 19.O'Brien, R. W., and J. G. Morris. 1971. The ferredoxin-dependent reduction of chloramphenicol by Clostridium acetobutylicum. J. Gen. Microbiol. 67:265-271. [DOI] [PubMed] [Google Scholar]

[r20] 20.Oultram, J. D., M. Loughlin, T.-J. Swinfield, J. K. Brehm, D. E. Thompson, and N. P. Minton. 1988. Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation. FEMS Microbiol. Lett. 56:83-88. [Google Scholar]

[r21] 21.Phillips-Jones, M. K. 1993. Bioluminescence (lux) expression in the anaerobe Clostridium perfringens. FEMS Microbiol. Lett. 106:265-270. [DOI] [PubMed] [Google Scholar]

[r22] 22.Phillips-Jones, M. K. 2000. Use of a lux reporter system for monitoring rapid changes in α-toxin gene expression in Clostridium perfringens during growth. FEMS Microbiol. Lett. 188:29-33. [DOI] [PubMed] [Google Scholar]

[r23] 23.Quixley, K. W. M., and S. J. Reid. 2000. Construction of a reporter gene vector for Clostridium beijerinckii using a Clostridium endoglucanase gene. J. Mol. Microbiol. Biotechnol. 2:53-57. [PubMed] [Google Scholar]

[r24] 24.Ravagnani, A., K. C. B. Jennert, E. Steiner, R. Grünberg, J. R. Jefferies, S. R. Wilkinson, D. I. Young, E. C. Tidswell, D. P. Brown, P. Youngman, J. G. Morris, and M. Young. 2000. Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol. Microbiol. 37:1172-1185. [DOI] [PubMed] [Google Scholar]

[r25] 25.Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[r26] 26.Sauer, U., and P. Dürre. 1995. Differential induction of genes related to solvent formation during the shift from acidogenesis to solventogenesis in continuous culture of Clostridium acetobutylicum. FEMS Microbiol. Lett. 125:115-120. [Google Scholar]

[r27] 27.Schmidt, J. A., S. S. Dineen, M. Bradshaw, and E. A. Johnson. 1999. Evaluation of a luciferase reporter system for the study of neurotoxin gene expression in Clostridium botulinum type A strain 62A, p. 14-16. In 1999 annual report. Food Research Institute, Madison, Wisc. [Online.] http://www.wisc.edu/fri/annrpt/bot99.pdf.

[r28] 28.Steffen, C., and H. Matzura. 1989. Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens. Gene 75:349-354. [DOI] [PubMed] [Google Scholar]

[r29] 29.Thormann, K., L. Feustel, K. Lorenz, S. Nakotte, and P. Dürre. 2002. Control of butanol formation in Clostridium acetobutylicum by transcriptional activation. J. Bacteriol. 187:1966-1973. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r30] 30.Tummala, S. B., N. E. Welker, and E. T. Papoutsakis. 1999. Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 65:3793-3799. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r31] 31.Tummala, S. B., C. Tomas, L. M. Harris, N. E. Welker, F. B. Rudolph, G. N. Bennett, and E. T. Papoutsakis. 2001. Genetic tools for solventogenic clostridia, p. 105-123. In H. Bahl and P. Dürre (ed.), Clostridia. Biotechnology and medical applications. Wiley-VCH Verlag GmbH, Weinheim, Germany.

[r32] 32.Walter, K. A., G. N. Bennett, and E. T. Papoutsakis. 1992. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J. Bacteriol. 174:7149-7158. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r33] 33.Walters, D. M., V. L. Stirewalt, and S. B. Melville. 1999. Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens. J. Bacteriol. 181:4526-4532. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r34] 34.Welch, R. W., F. B. Rudolph, and E. T. Papoutsakis. 1989. Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824). Arch. Biochem. Biophys. 273:309-318. [DOI] [PubMed] [Google Scholar]

[r35] 35.Yu, P.-L., J. B. Smart, and B. M. Ennis. 1987. Differential induction of β-galactosidase and phospho-β-galactosidase activities in the fermentation of whey permeate by Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 26:254-257. [Google Scholar]

[r36] 36.Zhao, Y., and S. B. Melville. 1998. Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens. J. Bacteriol. 180:136-142. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

Lothar Feustel

Stephan Nakotte

Peter Dürre

Abstract

MATERIALS AND METHODS