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Journal of Bacteriology, January 1999, p. 319-330, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor

Ramesh V. Nair,1,dagger Edward M. Green,2 David E. Watson,2 George N. Bennett,2 and Eleftherios T. Papoutsakis1,*

Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208,1 and Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 772512

Received 4 June 1998/Accepted 28 October 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871-885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes. Inactivation of solR in C. acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Several solventogenic genes (aad [42] or adhE [22], bdhA and bdhB [50, 65], adc [23, 48, 49], and ctfA and ctfB [22, 49]) have recently been cloned and sequenced from Clostridium acetobutylicum and another solventogenic Clostridium species (29) (adh1 [74]). These genes share a common induction pattern in that they are all expressed only at the onset of solventogenesis during the late exponential growth stage. Speculation abounds as to the factors that are responsible for triggering solventogenesis. Some of these are believed to be pH, threshold butyrate concentration (62), and nutrient limitations (51). A Spo0A-mediated regulation of events during stationary-phase metabolism is implicated in a Clostridium beijerinckii strain (68). A repressor protein (similar to the LacI family of repressors) encoded by regA from a solventogenic Clostridium species (formerly C. acetobutylicum P262 [29]) is believed to be involved in the regulation of starch degradation (15, 70). To date, no gene encoding a regulatory protein that modulates solvent formation genes has been cloned from C. acetobutylicum. Recently the sigA product (57) from strain DSM 792 (which is grouped with the type strain ATCC 824 [29, 30]) was identified. However, so far sigma factors involved in transcription of solventogenic genes have not been found in C. acetobutylicum (20, 41).

Clustering of genes involving both mono- and polycistronic operons in C. acetobutylicum has been reported elsewhere (6, 9, 22, 42, 49, 65). The only polycistronic operon that has so far been cloned from C. acetobutylicum involving solvent pathway genes is that of aad-ctfA-ctfB (42) (a virtually identical system in C. acetobutylicum DSM 792 is the sol operon involving the genes adhE and ctfA amd ctfB [22]). This operon contains genes involved in both butanol and acetone formation, the two predominant solvents produced by C. acetobutylicum. We have recently reported that in C. acetobutylicum ATCC 824, this operon and the adc gene are located on a large 210-kb plasmid (pSOL1) and not on the chromosome (13). Understanding of the regulation of these solventogenic genes is crucial for metabolically engineering (37) this organism to improve production of butanol and acetone. In a search for regulatory proteins of this polycistronic operon, sequencing further upstream of aad was initiated, which resulted in the discovery of a 957-bp open reading frame (ORF) (orf1) 663 bp upstream of aad on the same DNA strand (42). The proximal location on pSOL1 of orf1 (now designated solR) to aad and the size of the protein (~37 kDa) that may be encoded by this gene suggested that this may be a regulatory protein. The present study examines the possible regulatory role played by this solR-coded product.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains and plasmids. All bacterial strains and plasmids used in this study are shown in Table 1.

                              
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  		TABLE 1.   Bacterial strains and plasmids used in this study

Growth conditions and maintenance. All Escherichia coli strains were grown aerobically at 37°C in Luria-Bertani (LB) medium. For recombinant strains, media were appropriately supplemented with ampicillin (50 to 60 µg/ml), erythromycin (ERM) (200 µg/ml), and chloramphenicol (32 µg/ml). Both recombinant and wild-type strains were stored at -85°C in 15% (vol/vol) glycerol (53).

C. acetobutylicum ATCC 824 was maintained as spores in corn mash glucose medium (CMG) (51) at 4°C under nitrogen. Spores were activated by heating at 70 to 80°C for 10 min. Recombinant clostridial strains were stored frozen in 15% (vol/vol) glycerol at -85°C or as single colonies on reinforced clostridial agar (RCA; Difco Laboratories, Detroit, Mich.), pH 6.8. In 10 ml-tube cultures, C. acetobutylicum was grown under anaerobic conditions at 37°C in 2× YTG (45), reinforced clostridial medium (RCM; Difco Laboratories), or clostridium growth medium (CGM) (51). Recombinant C. acetobutylicum cells (carrying macrolide-, lincosamide-, and streptogramin B-resistant [MLSr] plasmids) were cultured in the above media supplemented with 40 µg of ERM per ml on plates and 100 µg of ERM per ml in liquid culture.

Controlled-pH fermentor experiments. Large-scale batch fermentations (5.5 liters) of various C. acetobutylicum strains were performed in a BioFlo II fermentor (New Brunswick Scientific, Edison, N.J.) with a culture volume of 5 liters (CGM with 80 g of glucose per liter instead of 50 g/liter), as previously described (42).

Glucose and fermentation product analysis. Residual glucose concentration in culture supernatants was measured with a Select biochemistry analyzer (model 2700; YSI, Yellow Springs, Ohio) and a YSI dextrose membrane according to the manufacturer's instructions. The concentrations of butanol, acetone, ethanol, butyrate, and acetate were determined with a Varian Vista 6000 gas chromatograph (Varian, Walnut Creek, Calif.) (36).

DNA isolation, transformation, and manipulation. Plasmid isolation from E. coli was done by the method of Birnboim and Doly (7), with the additional steps of the procedure of Wu and Welker (71) when the DNA was to be sequenced. Large-scale plasmid isolation was undertaken with a QIAGEN Plasmid Maxi Kit (QIAGEN, Chatsworth, Calif.). Plasmid DNA was desalted and concentrated using Microcon-100 microconcentrators (Amicon, Beverly, Mass.). Bacterial DNA was prepared from 10 ml of exponential-phase C. acetobutylicum cells (optical density at 600 nm of ~0.8) with a Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn.). Previously published methods were used for electrotransformation of E. coli (19) and C. acetobutylicum (39). Prior to transformation of C. acetobutylicum, plasmids pCO1 and pO1X were methylated in E. coli(pAN1) by the B. subtilis phage phi 3TI methyltransferase, which protects the plasmid DNA from restriction by the clostridial endonuclease Cac824I (38). Approximately 15 µg of methylated nonreplicating plasmid pO1X DNA was used to transform C. acetobutylicum.

Southern hybridization. Plasmid (pO1X) and bacterial (wild-type, mutant B, and mutant H) DNAs were digested to completion with either EcoRI or ScaI. The DNA was transferred from an agarose gel to a HYBOND-N+ nylon membrane (Amersham Life Science, Arlington Heights, Ill.) by capillary blotting (60) and then probed with a radiolabeled solR gene fragment, isolated from pO1X. The gene fragment was labeled with [alpha -32P]dATP using a random priming DECAprime II DNA Labeling Kit (Ambion, Austin, Tex.), and unincorporated radionucleotides were removed by exclusion chromatography on Sephadex G-50. The prehybridization, hybridization, and washing steps were performed at 42°C in accordance with the membrane manufacturer's instructions, and the radioactive membranes were visualized after exposure to X-ray film.

Primer extension. Total RNA was isolated from C. acetobutylicum as previously described (65). Primer extension reactions were performed as previously described (24) with Moloney murine leukemia virus reverse transcriptase (Amersham) using 20 µg of total RNA, unless otherwise stated. To determine the 5' end of solR mRNA, an end-labeled oligonucleotide, BORFU-PE (5'-CGCAATAGGTATGACATATG-3') complementary to the N-terminal region of solR was used in a primer extension reaction. Oligonucleotide primers were end labeled with [gamma -32P]ATP (NEN Research Products) as previously described (41). The precise transcriptional start site of solR mRNA was determined by sequencing of plasmid pCO1 around the N-terminal end of solR by the Sanger dideoxy chain-termination method (54), as previously described (42), using the same synthetic oligonucleotide (20-mer) primer (BORFU-PE). RNA for solR primer extension studies was isolated from wild-type and recombinant C. acetobutylicum [strain ATCC 824(pCO1)] cells collected during the acidogenic (early exponential growth phase, stage A, 5 h) and early solventogenic (late exponential growth phase, stage B, 10 h) stages in batch fermentations with a controlled pH (pH >=  4.5). RNA for the time course primer extension experiments was isolated from ATCC 824(pCO1) and mutant B (ATCC 824 solR::pO1X) cells isolated during the early exponential (stage A, 5 h), late exponential (stage B, 10 h), early stationary (stage C, 25 h), and late stationary (stage D, 50 h) stages in batch fermentations with a controlled pH (pH >=  4.5). The presence of mRNA corresponding to solR, aad, and adc genes in each of the above four stages was verified by performing primer extension reactions using end-labeled 20-mer synthetic oligonucleotides BORFU-PE, BYDH-PE (5'-TTTACTGTTGTGACTTTCAT-3'), and N-ADC (5'-TTCATCCTTTAACATAAAAG-3') that are complementary to the N-terminal ends of the respective genes.

Northern analysis. The 0.9-kb clostridial EcoRI fragment from pO1X was labeled with [alpha -32P]dATP (NEN) using the random-priming Prime-It II Kit (Stratagene, La Jolla, Calif.) per the manufacturer's instructions. Unincorporated radionucleotides were removed by using NucTrap probe purification columns (Stratagene). Northern blotting was performed as previously described (64) with the following modifications. Transfer of the RNA to 0.2-µm-pore-size Maximum-Strength Nytran Plus nylon membrane (Schleicher & Schuell, Keene, N.H.) was done by using a TurboBlotter Nytran System (Schleicher & Schuell) according to the manufacturer's instructions. Prehybridization and hybridization steps were carried out for 24 h at 29°C, while all washes were performed at 44°C. The air-dried filter was exposed at -85°C to XOMAT-AR film for 5 days with a DuPont Cronex Xtra Life Lightning-Plus Intensifying Screen (E. I. DuPont de Nemours, Wilmington, Del.) used according to the manufacturer's instructions to amplify the signal.

Construction of plasmids. (i) pSOLR. The DNA fragment containing solR and the promoter region of aad was amplified by PCR, as described earlier (42), with plasmid (pHXS5) used as the template DNA. The upstream primer DAP-UP (5'-ATGGTCGGCGTGAATTCGTGAACAATTG-3') was generated by substituting a G for a T at nucleotide position 220 (42) and a T for a A at nucleotide position 224 to provide an internal EcoRI site (underlined). The downstream primer DAP-DN (5'-TGCTGCCATTGCTGCAGTTCTAAAGATT-3') was generated by substituting a G for a T at nucleotide position 2171 (42) on the complementary strand to provide an internal PstI site (underlined). The 1,979-bp amplified DNA fragment was digested sequentially with EcoRI (cuts above the engineered site) and PvuII (cuts at nucleotide position 1768 [42]) to generate a 1,550-bp fragment containing solR, its natural promoter, and the two putative rho-independent terminators downstream of solR (42). This 1.55-kb DNA fragment was ligated into EcoRI-SmaI-digested pUC19 vector to yield the ~4.2-kb plasmid pSOLR (Fig. 1a).


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  		FIG. 1.   Schematic representations of the plasmids pSOLR (a), pCO1 (b), and pO1X (c). P is the solR promoter, and T1 and T2 are transcriptional terminators identified previously (42) downstream of solR. solR' is the 0.9-kb internal fragment of solR (see construction of pO1X in Materials and Methods).

(ii) pCO1. pSOLR digested with XbaI and plasmid pIM13 (35) digested with HindIII were then treated with DNA polymerase I large (Klenow) fragment (New England Biolabs), according to the manufacturer's instructions, in order to generate blunt-ended termini. The B. subtilis plasmid pIM13 provides MLSr and a gram-positive bacterial origin of replication (35). The two blunt-ended fragments (linearized pSOLR and the larger, ~2.0-kb HindIII fragment from pIM13) were then ligated to yield the ~6.2-kb plasmid pCO1 (Fig. 1b).

(iii) pO1X. A 890-bp internal DNA fragment of solR was amplified by PCR with plasmid (pSOLR) DNA as the template (42). The upstream primer ORFX-UP (5'-TGCGATATGTAGAATTCTTCCAATATTT-3') was generated by substituting a G for a T at nucleotide position 491 (42) and a T for a A at nucleotide position 495 to provide an internal EcoRI site (underlined). The downstream primer ORFX-DN (5'-TTTTTATCATCGAATTCTATGCCTAAAT-3') was generated by substituting a A for a T at nucleotide position 1357 (42) on the complementary strand to provide an internal EcoRI site (underlined). The amplified DNA fragment was digested with EcoRI to generate a 0.9-kb fragment corresponding to bp 492 to 1358 (42) and ligated into EcoRI-digested pJC4 vector (33) to yield the ~6.2-kb plasmid pO1X (Fig. 1c). DNA sequence analysis showed that the insert in pO1X is derived from the solR gene and confirmed the sequence of this segment of the solR region reported by Fischer et al. (22).

PCR experiments. PCR primers were designed to correspond to the regions of the solR gene (see Fig. 4a). The solR453 forward primer (5'-GAGTTGAATTTAGCATGAATTTATTA-3'; bp 428 to 453) (42), the solR1361 reverse primer (5'-AATTTTCCGTTAAGTATTTTTTTATCAT-3'; bp 1361 to 1388) (42), primer Tc239 (5'-CATAGAAATTGCATCAACGCATA-3'; bp 239 to 261) (61) for the tetracycline resistance gene of pO1X, and primer Em373 (5'-CAATTGTTTTATTCTTTGGTTGAGTAC-3'; bp 373 to 399) (63) for the erythromycin resistance (MLSr) gene of pO1X were synthesized by Genosys (The Woodlands, TX).

The primers were used with C. acetobutylicum DNA isolated from ATCC 824, solR mutant B, and solR mutant H to probe the solR region and inserted sequences (if any) in these strains. The PCR conditions used with primer pairs solR453-Tc239 and Em373-solR1361 were as follows: 1× PCR optimization buffer D (Invitrogen, Carlsbad, Calif.), 0.4 µM (each) primer, 250 µM final concentrations of each deoxynucleoside triphosphate, 1 µl of template (~0.8 µg of either ATCC 824, SolR-B, or SolR-H DNA), and 1 U of Taq polymerase in a 50-µl reaction mixture volume. The PCR cycling conditions used were an initial denaturation step (2 min at 94°C) followed by 35 cycles, with each cycle consisting of 45 s at 94°C for denaturation and 1 min at 72°C for annealing-extension, and a final extension step (5 min at 72°C). When primers solR453 and solR1361 were used, Perkin-Elmer's (Foster City, Calif.) XL (extralong) PCR kit was used. Following the kit instructions, an optimal magnesium acetate level of 1.1 mM and the following cycling times were used: an initial denaturation step (2 min at 94°C), 16 cycles with each cycle consisting of 15 s at 94°C for denaturation and 5 min at 62°C for annealing and extension, then 12 additional cycles in which the denaturation conditions remained 94°C for 15 s and the annealing-extension time was increased successively by 15 s from the corresponding time of the previous cycle, and at the end a final extension step of 10 min at 72°C. PCR products were analyzed by gel electrophoresis on agarose gels and staining with ethidium bromide.

Computer programs. The Wisconsin Genetics Computer Group (17) sequence analysis software package (version 9.1, September 1997) was used for programs BestFit, PeptideStructure, PlotStructure, and FindPatterns.

Homology searches using BLAST (release 2.0, September 1997) (1) were done on the WWW BLAST Server (www.ncbi.nlm.nih.gov). The BLASTP program was used to search the nr peptide sequence database (all nonredundant GenBank CDS translations, PDB, SwissProt, and PIR).

Additional homology searches were done with the Blocks WWW Server (www.blocks.fhcrc.org) to look for the most highly conserved regions in groups of proteins. The database searched was BLOCKS (version 10.1, April 1998) (26). Wherever presented, consensus patterns are from the patterns section of PROSITE (release 14.0, November 1997) (4) obtained with the ScanProsite tool.

Homology searches were also done on the PRINTS (release 18.0, March 1998) protein motif fingerprint database (www.biochem.ucl.ac.uk) with the FingerPRINTScan tool to look for conserved motifs characteristic of protein families (3).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sequence analysis. Upon sequencing upstream of aad on plasmid pHXS5, a 957-bp ORF (orf1, now solR) (42) was located, which based on homology searches of protein databases and experimental evidence (this report), appears to encode a putative repressor protein (SolR) involved in negative regulation of solvent formation genes. A putative ribosomal binding site (5'-GGAAAGAG-3'), similar in sequence and spacing to those of other C. acetobutylicum genes (47), was found 11 bp upstream of the solR start codon. Two inverted repeat segments (42) were identified in the region of DNA between solR and aad (Delta G = -20.0 kcal/mol [75], positions 1399 to 1439; Delta G = -19.4 kcal/mol, positions 1617 to 1657). Northern blot analysis of the identical gene in C. acetobutylicum DSM 792 coding for ORF5 (22) showed two transcripts 1.3 and 1.0 kb long, indicating that both terminator structures are utilized. The solR gene is terminated by a single (UAA) stop codon.

Homology searches. The solR gene codes for a protein (SolR) containing 319 amino acid (aa) residues. The calculated molecular mass of the SolR protein is 36,916 Da.

The highest scoring fingerprint obtained via the FingerPRINTScan performed on the SolR sequence was that of HOMEOBOX. Most proteins containing homeobox domains are known to be sequence-specific DNA-binding transcription factors. The domain binds DNA through a helix-turn-helix (HTH) structure (59). HOMEOBOX is a three-element fingerprint that provides a signature for the homeobox domain. The three elements identified by FingerPRINTScan within SolR are NAYITRERIYFY (starts at residue 66), LGEPERALKYF (starts at residue 112), and KFKELIAKTK (starts at residue 286).

Proteins containing HTH DNA-binding motifs are characteristic of the cyclic AMP (cAMP) receptor protein (CRP)-fumarate and nitrogen regulatory protein (FNR) family of regulatory proteins, as will be discussed below. Overall, at the amino acid level, SolR exhibits a 20.5% identity (44.9% similarity) with the 210-aa CRP (46) from E. coli, a 17.2% identity (43.7% similarity) with the 250-aa FNR (46) from E. coli, and a 19.0% identity (46.0% similarity) with the 219-aa FNR-like protein (FLP) (28) from Lactobacillus casei, which is the first discovered member of the CRP-FNR family in a gram-positive organism. Figure 2 shows an alignment of alpha -helical DNA recognition sequences (HTH motif) of 33 DNA-binding proteins. The PROSITE (4) consensus pattern [LIVM]-[STAG]-[RHW]-X2-[ LI ]-[ GA ]-X-[ LIVMFYA ]-[ LIVS ]-G-X-[ STAC ]-X2-[MT]-X-[GST]-R-X-[LIVMF]-X2-[LIVMF], where the letters within the brackets represent the different possible amino acid residues at each position and the subscript numbers represent the number of occurrences of the indicated residue(s), has been presented for the HTH DNA-binding motif (within the GTR motif) of several repressor proteins. The corresponding putative region in SolR is presented in Fig. 2.


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  		FIG. 2.   Amino acid alignment of the DNA-binding HTH domains of the CRP-FNR family of regulatory proteins. Hydrophobic residues (h) and conserved residues in three different positions (boxed) are indicated at the top. CRP_ECOLI, CRP (catabolite gene activator protein) from E. coli (46); CAP_KLEAE, CAP-like protein from Klebsiella aerogenes (44); CAP_SHIFL and CAP_SALTY, CAP from Shigella flexneri and Salmonella typhimurium, respectively (14); CRP_HAEIN, CRP from Haemophilus influenzae (10); CLP_XANCA, CAP-like protein from Xanthomonas campestris (16); CRP_SALTY, CRP from S. typhimurium (58); BifA_ANABA, CRP-like protein from Anabaena sp. (67); CysR_SYNEC, regulatory protein from Synechococcus sp. (31); Hin, product of DNA inversion gene (46); TnpR_Tn3, Tn3 resolvase (46); Resolvase_gamma delta , resolvase from transposon gamma delta (46); AraC_ECOLI, arabinose regulatory protein from E. coli (46); ftz_DROME, product of segmentation gene fushi tarazu from Drosophila melanogaster (32); smox-2_SCHMA, Schistosoma mansoni homeobox-containing DNA-binding protein (66); MATa1_YEAST and MATalpha 2_YEAST, Saccharomyces cerevisiae proteins that specify a and alpha  diploid functions including sporulation (32); Cro_434, regulatory protein from bacteriophage 434 (46); C2_P22, repressor from bacteriophage P22 (56); CI_phi 80 and Gene30_phi 80, DNA-binding proteins from bacteriophage phi 80 (43); CII_lambda , regulatory protein from bacteriophage lambda  (46); LacR_ECOLI, lactose repressor from E. coli (46); GalR_ECOLI, galactose repressor from E. coli (46); CI_lambda , repressor from bacteriophage lambda  (55); Cro_lambda , regulatory protein from bacteriophage lambda  (46); FNR_ECOLI, FNR from E. coli (46); HlyX_ACTPL, regulatory protein from Actinobacillus pleuropneumoniae (34); AadR_RHOPA, FNR-CRP member from Rhodopseudomonas palustris (18); FixK_RHILE, FNR-like protein from Rhizobium leguminosarum (12); FixK_RHIME, FNR-CRP member from Rhizobium meliloti (5); FixK_BRAJA, FNR-like protein from Bradyrhizobium japonicum (2); FLP_LACCA, FNR-like protein from Lactobacillus casei (28); SolR_CLOAC, putative repressor protein from Clostridium acetobutylicum. Numbers preceding sequences represent amino acid positions within each protein. Previously reported (32, 46) consensus sequence alignments were used.

SolR protein secondary structure. The Wisconsin Genetics Computer Group sequence analysis software package was used to predict the SolR secondary structure by the method of Chou and Fasman (11) using programs PeptideStructure and PlotStructure. The putative DNA-binding site of SolR presented in Fig. 2 spans amino acid residues 164 to 187. The Chou-Fasman method predicts alpha -helical regions at the extreme ends of this 24-aa region with the conserved glycine residue (Gly-173) lying outside the turn region. Figure 2 clearly shows that DNA-binding regions (HTH motifs) within the CRP-FNR family of regulatory proteins lie predominantly in the C-terminal regions of the proteins. The putative DNA-binding region within SolR is present in the C-terminal half of the protein close to the central region.

Isolation and characterization of solR mutants B and H. The suicide plasmid pO1X was introduced into C. acetobutylicum ATCC 824 by electroporation. The resulting transformants (selected on ERM-containing plates) were grown for 48 to 72 h in CGM tube cultures and then analyzed for product concentrations. In these tube cultures, two such solR mutants, ATCC 824 solR::pO1X (designated mutants B and H), compared to the wild-type strain, produced ca. threefold-more butanol and acetone, ca. four- to fivefold-more ethanol, but only ca. 0.3- to 0.6-fold-more butyrate and acetate. Mutant B produced the most solvents and hence was used in further fermentation studies.

Total cellular DNA from the integrants (mutants B and H) and parental strain (ATCC 824) was characterized by Southern hybridization. DNA was digested with either ScaI or EcoRI and was then probed with the labeled 0.9-kb EcoRI fragment from pO1X (containing an internal fragment of solR). ScaI was initially chosen because this enzyme cuts at a single site in the backbone of the vector pJC4 but not in the clostridial solR insert. If a single copy of pO1X integrated into the bacterial DNA, digestion with ScaI should generate two solR-hybridizing fragments, whose combined size equals the combined size of the integrational plasmid (pO1X) and the ScaI fragment on the parental DNA that contains the homologous gene. EcoRI bacterial DNA fragments were similarly analyzed, and Fig. 3 combines all observations regarding mutants B and H and the parental ATCC 824 strain. The physical map of the clostridial insert in plasmid pHXS5 (42) earlier showed an EcoRI site less than 1 kb upstream of solR between the XbaI and ScaI sites. More-recent restriction analysis of this plasmid showed that this EcoRI site is actually absent in this region and that the EcoRI restriction site observed earlier in this vicinity was actually the site in the pUC19 polylinker adjacent to this end of the insert. Also, sequencing of the solR' region in pO1X revealed two errors in the sequence reported earlier (42) between nucleotides 220 to 239 in the solR structural gene. Consequently, a BglII site predicted by the earlier (42) sequence of solR in this region is actually absent. These sequence errors and a few others have been corrected in the GenBank entry (accession no. L14817) published previously. The actual nucleotide positions mentioned throughout this article are based on the previously published sequence (42) and may vary slightly from those based on the revised GenBank sequence entry.


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  		FIG. 3.   Southern analysis. Hybridization of a 0.9-kb solR fragment to ScaI- or EcoRI-digested bacterial DNA from C. acetobutylicum mutant B (lanes c), mutant H (lanes d), wild-type (lanes b), and plasmid pO1X (lanes a and e). Size markers (in kilobase pairs) are BstEII-digested lambda  DNA.

In the ScaI digestions shown in Fig. 3A, the probe hybridized to the expected 6.2-kb band generated by integrational plasmid pO1X (lanes a and e), a single fragment from the parental strain (with a migration position of 7.6 kb [lane b]), and two fragments (with migration positions of 6.4 and 7.4 kb) each from mutant B (lane c) and mutant H (lane d). Since the combined size of the ScaI fragments from the two mutants, B and H, equals the size of the integrational plasmid and the solR fragment on the bacterial DNA, it appears that a single copy of pO1X was integrated into the bacterial DNA via single-crossover homologous recombination. However, the similarity of the sizes of the bands in mutants B and H (~7.4 and 6.4 kb) to those of the parent strain (~7.6 kb) and plasmid pO1X (~6.2 kb) limits the ability to precisely map or distinguish the presence of an additional band which would have resulted from an additional in-tandem insertion of pO1X. It is possible that the retardation of bands may be due to an artifact; therefore, EcoRI digestion and PCR analysis were also conducted to better analyze the position of insertion of pO1X.

Analysis of EcoRI digests also shows disruption of the native fragment containing solR. In the EcoRI digestions shown in Fig. 3B, the probe hybridized to the expected ~0.9-kb fragment from the integrational plasmid pO1X (lane a), the expected one ~9 kb from the parental strain (lane b), three fragments from mutant B (0.9, 3, and 7 kb [lane c], and two fragments from mutant H (3 and 7 kb [lane d]. Since the internal EcoRI fragment of pO1X is ~0.9 kb, these findings suggest that mutant B contains at least an additional copy of pO1X inserted tandemly. Such additional inserts are frequently found in these types of recombination events (40) and are due to a tandem insertion.

The DNA isolated from solR mutants H and B was also analyzed by PCR amplification using primers designed to amplify the junction between the vector part of pO1X and the ends of the solR gene not present on the plasmid. First, the use of the solR453 and solR1361 primers gave, as expected, a ~0.9-kb fragment with C. acetobutylicum ATCC 824 DNA as the template; however, small amounts of this fragment were found with DNA from mutants H and B also (Fig. 4b, gel A). The low intensity of the ~0.9-kb fragment with templates prepared from mutants H and B may result from amplification of a small contamination or the presence of a small population of revertant cells in the sample which have lost the plasmid by excision which occurs at a low frequency. Another possibility is that the integration events occurred in only a fraction of the pSOL1 plasmids in a cell; the copy number of pSOL1, which carries the sol locus, is not yet known. A more intense fragment of ~7 kb was also found with mutant H, consistent with one insert of the pO1X plasmid into mutant H (Fig. 4b, gel A). In the case of mutant B, the large fragment (of at least 13 kb, which would be expected if one additional copy of pO1X was tandemly inserted) was not found. Apparently, due to its size it was not produced well in the PCR amplification even when extralong PCR conditions were used. Second, the junction fragments of solR with the integrated vector, as indicated by positive amplification of the expected-size fragment with the solR453-Tc239 primer pair and the Em373-solR1361 primer pair, were the same when DNA from both mutant, B and H was used as the template (Fig. 4b, gels B and C). No amplification was observed when these primer pairs were used with ATCC 824 DNA. Additional evidence for a tandem insertion was found when the Em373 and Tc239 primer pairs were used with mutant B DNA. In this case, a fragment of 2.2 kb was obtained (data not shown), indicating that these genes are oriented as they would be in a tandem insertion. Such a fragment was not found with mutant H (data not shown), the expected result for a single integration mutant. These combined results show that the pO1X insertions in mutants B and H were within the solR gene and the solR gene would be expected to be nonfunctional as a result of these insertions. Of course, we cannot rule out the possible presence of alterations in other genes which were not investigated within the derived strains.


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  		FIG. 4.   Schematic representations (a) and results of PCR analysis (b) on wild-type (WT) C. acetobutylicum ATCC 824 and solR mutants B and H using primers (a) designed to amplify the junction between the vector portion of pO1X and the solR gene. For each gel in panel b, lane 1 contains HindIII-digested lambda marker, lane 2 contains the WT genomic template, lane 3 contains the solR mutant B template, and lane 4 contains the solR mutant H as the template. (Gel A) Extralong PCR using primers solR453 and solR1361 designed to amplify the complete insert. In lane 2, WT DNA shows an expected ~0.9-kb band. This band is also seen, but much weaker, in both lanes 3 and 4. In addition, lane 4 contains a band with an apparent size of ~7 kb (marked with an arrow), consistent with the presence of one insert of pO1X into solR of mutant H. (Gel B) PCR results using primers solR453 and Tc238. A band of ~1.2 kb can be seen in lanes 3 and 4 with no product in lane 2. (Gel C) PCR results using primers Em373 and solR1361. A band of ~2.1 kb is visible in lanes 3 and 4. Again, no product was observed with WT DNA (lane 2).

Batch fermentation studies. Batch fermentations of C. acetobutylicum ATCC 824(pCO1) and mutant B were performed at a pH of >= 4.5 (Fig. 5), and the final product concentrations were compared to those obtained earlier with fermentations of wild-type and recombinant (carrying plasmid pCCL) strains (42) at the same pH (Table 2). Plasmid pCCL carries a truncated form of aad, and ATCC 824(pCCL) fermentations produce higher solvent titers than those of the wild-type strain for reasons which are still unknown. These data show that overexpression of solR in strain ATCC 824 [strain ATCC 824(pCO1)] results in loss of butanol and acetone, while ethanol production was reduced [five- to sevenfold from that of the control strains [ATCC 824 and ATCC 824(pCCL)]. As a result, considerably larger amounts of butyrate and acetate [2.5- to 15.0-fold and 1.5- to 6.4-fold increase, respectively, over the final levels produced by the control strains ATCC 824 and ATCC 824(pCCL)] accumulated.


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  		FIG. 5.   Product concentration and optical density (OD) profiles for controlled-pH (pH >=  4.5) batch fermentations with C. acetobutylicum strain ATCC 824(pCO1) (a) and mutant B (b). Zero time indicates the time at which the bioreactor was inoculated with a 1/10 (vol/vol) preculture. Symbols: box-plus , glucose; bullet , ethanol; black-triangle, acetone; , butanol; open circle , acetate; , butyrate; triangle , optical density at 600 nm; odot , pH.

                              
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  		TABLE 2.   Final product levels in C. acetobutylicum fermentor experiments at pH 4.5

Inactivation of solR (mutant B) led to higher butanol and acetone levels. There was a 3.2-fold and a 1.7-fold increase in the final butanol concentration over those of the wild-type and ATCC 824(pCCL) strains, respectively. There was a 2.1-fold and 1.5-fold increase in the corresponding levels of acetone. While butyrate and acetate levels were only half that of the wild-type strain, there was a 3.7-fold increase in the final levels of butyrate over that of strain ATCC 824(pCCL). In the mutant B fermentation, earlier induction of the AAD protein (results not shown) resulted in an increased flux towards butanol formation and a decreased flux towards acid accumulation early on, and consequently, acid uptake did not contribute as significantly to solvent formation. Final solvent titers were 17.8 g of butanol per liter, 8.1 g of acetone per liter, and 1.0 g of ethanol per liter for a net solvent titer of ~27.0 g/liter. Mutants B and H were also tested in several other fermentations which produced similar high solvent titers.

Earlier induction of solventogenic genes also leads to a more efficient conversion of glucose to solvents. The molar yields of butanol and acetone on glucose (final concentration [millimolar] of solvent/initial glucose concentration [millimolar]), for mutant B were 0.54 and 0.32, respectively, while those for the wild-type strain were only 0.17 and 0.15 and those for strain ATCC 824(pCCL) were 0.31 and 0.21, respectively. Strain ATCC 824(pCO1) produced no detectable butanol or acetone.

Transcriptional start site of solR. Primer extension analysis using primer BORFU-PE had a two-fold objective: (i) to identify the precise 5' end of the solR transcript and (ii) to compare transcriptional levels of solR in the wild-type (ATCC 824) strain and the recombinant [ATCC 824(pCO1)] strain that overexpresses solR. Results with mRNA from both wild-type and ATCC 824(pCO1) cells obtained from early exponential (stage A, 5 h) and late exponential (stage B, 10 h) growth stages (Fig. 6) show that solR mRNA is present at very low levels in the wild-type strain with bands in lanes 1 and 2 barely visible despite starting with twice the standard amount of total RNA (40 µg).


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  		FIG. 6.   Primer extension analysis. Primer extension products made with primer BORFU-PE complementary to the N-terminal end of solR are shown. RNA for these experiments was obtained from C. acetobutylicum ATCC 824 cells (lanes 1 and 2) and from C. acetobutylicum ATCC 824(pCO1) cells (lanes 7 and 8). Cell samples were collected during early exponential (stage A, 5 h) and late exponential (stage B, 10 h) growth phases. The late exponential growth phase is also the solventogenic phase of ATCC 824 cells. Regions of the plasmid pCO1 were sequenced with the same primer, and the resulting DNA sequences are shown in lanes 3 to 6. The corresponding 5'-to-3' DNA sequence of the complementary (coding) strand is indicated to the right of the gel (sequence continues onto a second line), wherein the boxed nucleotide is the transcriptional start site. The arrow indicates the position of the nearly invisible bands in lanes 1 and 2. The total RNA (20 µg) used for lanes 7 and 8 corresponding to strain ATCC 824(pCO1) was the same as that loaded in primer extension reactions performed previously (42) to map the transcriptional start site of aad. However, low transcriptional levels are characteristic of regulatory proteins, since they are present in low copy numbers and hence, on this suspicion, twice the standard amount of total RNA (40 µg) was used for lanes 1 and 2 (corresponding to wild-type strain ATCC 824) in order to obtain visible bands.

The solR transcriptional start site is shown in Fig. 6 at nucleotide position 407 (42), which is 35 nucleotides upstream from the initiation codon with C as the first transcribed nucleotide. Looking further upstream of this start site, a putative promoter structure TCGATA(17 bp)TATTAT was identified with 7 bp separating the -10 region of the promoter structure and the transcriptional start site. The -10 and -35 sequences are similar to those identified previously (73) as part of a consensus clostridial promoter with differences in only 3 of 12 positions. The location of the solR transcriptional start site and promoter region are in agreement with the results obtained for an identical gene coding for ORF5 in C. acetobutylicum DSM 792 (22).

Overexpression of solR in strain ATCC 824(pCO1) is clearly evident in lanes 7 and 8 (Fig. 6). Based on relative band intensities, it appears that solR is transcribed more actively in acidogenic cells (lane 7) than in solventogenic cells (lane 8). The low transcriptional levels of solR in wild-type cells make such a direct comparison based on relative band intensities more difficult. However, careful examination of the faint bands in lanes 1 and 2 (Fig. 6) appears to indicate that this could also be true in wild-type cells.

Expression of aad, ctfA, ctfB, and adc in a strain overproducing SolR. Primer extension analysis (Fig. 7) was used to examine the effects of SolR overproduction on the transcript levels of the solventogenic genes aad, ctfA, ctfB, and adc (sol locus). Since aad, ctfA, and ctfB form a polycistronic operon (22), the transcriptional levels of aad and ctfAB are identical. Based on the locations of the transcriptional start sites of solR (this study), aad (42), and adc (24) and the positions of primers BORFU-PE, BYDH-PE, and N-ADC, the primer extension products for solR and adc are 77 and 105 bp, respectively, while those of aad are 103 and 263 bp, respectively (the shorter product predominates as seen earlier [42]). Figure 7 shows the transcript levels corresponding to solR (lanes 1 to 4), aad (lanes 5 to 8), and adc (lanes 9 to 12) at four different stages of the ATCC 824(pCO1) fermentation. solR appears to be strongly induced in the early exponential growth phase (Fig. 7, lane 1) with continually decreasing transcription levels during the rest of the fermentation (Fig. 7, lanes 2 to 4). The primer extension gel (shown all the way to the top up to the loading wells in Fig. 7) shows that aad and adc (which are normally induced strongly at the end of the exponential growth phase, which corresponds to the onset of solvent formation [24, 41]) are not transcribed (Fig. 7, lanes 5 to 12). This appears to be a direct result of overexpression of solR. While homology searches suggested that SolR was perhaps a regulatory protein, this experiment offers the first tentative link between SolR overproduction and transcriptional shutdown of solventogenic genes. This suggests that SolR is a putative repressor protein that regulates the transcription of butanol and acetone formation genes of the sol locus (aad, ctfA, ctfB, and adc genes).


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  		FIG. 7.   Time course primer extension analysis of ATCC 824(pCO1) cells. RNA for the time course primer extension experiments was isolated from ATCC 824(pCO1) cells isolated during the early exponential growth (stage A, 5 h), late exponential growth (stage B, 10 h), early stationary (stage C, 25 h), and late stationary (stage D, 50 h) stages in a batch fermentation with a controlled pH (pH 4.5). The presence of mRNA corresponding to solR (lanes 1 to 4) aad (lanes 5 to 8) and adc (lanes 9 to 12) genes in each of the above four stages was verified by performing primer extension reactions with 20 µg of total RNA, using end-labeled 20-mer synthetic oligonucleotides BORFU-PE, BYDH-PE, and N-ADC that are complementary to the N-terminal ends of the respective genes.

Expression of aad and adc in the solR-inactivated mutant B. Samples from stages A, B, C, and D of a fermentation of C. acetobutylicum mutant B were used for primer extension analysis (Fig. 8) to examine the effects of solR inactivation on the transcript levels of aad and adc. Faint bands corresponding to a 77-bp primer extension product are barely visible in lane 1 (wild type) and in lanes 2 to 5 (mutant B). This implies that a transcript corresponding to solR exists in mutant B, but a Northern analysis would be needed to determine the altered size of this transcript due to insertional inactivation of solR. Strong expression of aad (Fig. 8, lanes 7 to 9) and adc (Fig. 8, lanes 11 and 12) is apparent. The expected 103- and 263-bp bands for aad and the 105-bp adc band are clearly visible (Fig. 8). Apparently, solR inactivation elevates predominantly the aad transcriptional levels from the proximal strong (42) promoter (103-bp band). The intensity of the 263-bp band corresponding to transcription from the distal promoter is probably identical to that in the wild-type strain (42).


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  		FIG. 8.   Time course primer extension analysis of mutant B cells. RNA for the time course primer extension experiments was isolated from mutant B cells sampled from the early exponential growth (stage A, 5 h), late exponential growth (stage B, 10 h), early stationary (stage C, 25 h), and late stationary (stage D, 50 h) stages in a batch fermentation with a controlled pH (pH >=  4.5). An early exponential growth phase (stage A) sample from an ATCC 824 pH 4.5 fermentation was examined for the solR transcript (lane 1). The presence of mRNA corresponding to solR (lanes 2 to 5), aad (lanes 6 to 9), and adc (lanes 10 to 13) genes in each of the above four stages was verified by performing primer extension reactions with 20 µg of total RNA, using end-labeled 20-mer synthetic oligonucleotides BORFU-PE, BYDH-PE, and N-ADC that are complementary to the N-terminal ends of the respective genes.

Alteration of solR transcription in mutant B. Northern analysis (Fig. 9) was used to compare solR transcript sizes from wild-type and mutant B cells, since the presence of a transcript in mutant B at levels comparable to that in the wild-type cells was evident (Fig. 8, lanes 1 to 5). Based on the location of the mapped solR transcriptional start site and the locations of the two terminator structures (42), both of which structures are used (22), the expected solR transcript sizes are 1.01 and 1.23 kb. In wild-type cells, a broad band centered at about 1.15 kb is observed (Fig. 9, lane 1) and this could be accounted for by the overlapping of the two expected solR transcripts. The corresponding band in mutant B appears as a smear centered at about 4.47 kb (Fig. 9, lane 2). From Fig. 4a, it is apparent the chromosomal solR promoter can generate a transcript which would contain the solR' region and since the native solR transcription terminator is not present in the insert of pO1X, the transcript would continue into the vector where it may terminate near the ori or the MLSr gene. The longer transcript is unlikely to arise from transcription of solR' from the MLSr gene promoter as this promoter is oriented in the direction opposite that of the solR' insert.


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  		FIG. 9.   Northern analysis. Total RNA was prepared from exponential-phase ATCC 824 (lane 1) and mutant B (lane 2) cells from pH >=  4.5 fermentations and probed with the solR internal fragment from pO1X.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The top-scoring fingerprint match to SolR from the PRINTS database was that of a class of transcription factors with HTH DNA-binding motifs. A putative DNA-binding motif in SolR has been identified based on the corresponding region in the CRP-FNR family of regulatory proteins among others (Fig. 2). A Chou-Fasman secondary structure prediction of SolR confirmed this to be a HTH region. This is evidence that SolR is a DNA-binding transcriptional regulator. SolR overexpression and inactivation studies presented here back up this contention while qualifying the regulatory role of SolR to be that of a repressor of butanol and acetone formation genes.

A search of protein databases using the BLASTP program revealed homology (24.7% identity and 48.1% similarity), especially in the C-terminal region, of SolR to Spo0KA (545 aa, 61.5 kDa) from B. subtilis (52), an oligopeptide permease required for sporulation and competence. Another gene involved in initiation of sporulation in B. subtilis is spo0A (response regulator, transcription repressor/activator) (27). Spo0A (267 aa, 29.7 kDa) from B. subtilis (21), a DNA-binding protein that controls the expression of genes that are involved in the transition from growth to the stationary phase, is activated by phosphorylation and has two tightly folded domains, an N-terminal phosphorylation domain and a C-terminal DNA-binding domain with specificity for the 0A box 5'-TGNCGAA-3' (25, 27, 52). A search of the 4,797-bp sequence in the revised GenBank entry (accession no. L14817 revised from the 4,800-bp sequence published previously [42]) for putative 0A boxes using the FindPatterns program (allowing one mismatch), as done earlier (68), located one such sequence in the solR-aad intergenic region (sequence 5'-TGGCGTA-3' on the noncoding strand ending at nucleotide position 1712 of the revised GenBank sequence entry), with 16 other sequences located within the coding regions of either solR or aad on either strand. A BestFit alignment of SolR and Spo0A from B. subtilis (21) (15.7% identity and 42.1% similarity) revealed that the putative HTH motif in SolR (Fig. 2) is aligned to a similar sequence, including the conserved Gly residue (Gly-165 in Spo0A [21]), present within the region in Spo0A that has been implicated in DNA binding. Putative HTH motifs believed to be responsible for binding 0A boxes have been identified in spo0A gene products from Bacillus and Clostridium sp. (8). The SolR protein shares a 20.1% identity (42.9% similarity) with a 166-aa fragment of the Spo0A protein (GenBank accession no. U09978) from C. acetobutylicum ATCC 4259 and a 18.2% identity (43.0% similarity) with a 223-aa fragment from the Spo0A protein (GenBank accession no. U09979) from C. beijerinckii (formerly C. acetobutylicum) NCIMB 8052.

After weighing all available evidence, experimental (Fig. 5, 7, and 8) and theoretical, it appears that SolR is a putative DNA-binding transcriptional repressor that negatively regulates the onset of solventogenic (primarily butanol and acetone) metabolism. Induction of this putative repressor in wild-type cells during the acidogenic (early to late exponential growth) phase and considerably lower expression during the solventogenic (beyond late exponential growth) phase would ensure the well-known induction pattern of solventogenic genes (beginning during the late exponential growth phase), which is in keeping with the proposed role of SolR. The inducer responsible for derepression of solventogenic genes could be one of several factors that are believed to trigger solventogenesis, including pH, threshold butyrate concentrations (62), and nutrient limitations.

The fermentation of mutant strain B, without any effort for optimization towards increased solvent yields, is one of the most impressive ever reported in the literature in terms of solvent production (27.0 g of total solvents per liter) and butanol tolerance (17.8 g/liter). This performance (Fig. 5) would make this genetically characterized strain quite attractive industrially (69). The results presented here indicate that earlier induction of solventogenic genes (deregulated solvent production) as opposed to overexpression of the same genes in the solventogenic phase (37, 64) is essential for generating industrially significant solvent-producing strains. So far, manipulating one gene (like solR) with a global effect appears to be the most-effective approach for strain improvement to increase solvent yields and titers.

    ACKNOWLEDGMENTS

This research was supported by NSF grants BES-9632217 and BES-9604562.

We thank Neil Welker (Northwestern University) for constructive discussions and suggestions.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical Engineering, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208-3120. Phone: (847) 491-7455. Fax: (847) 491-3728. E-mail: e-paps{at}nwu.edu.

dagger Present address: DuPont Life Sciences, Experimental Station, Wilmington, DE 19880-0328.

    REFERENCES
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Abstract
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Materials & Methods
Results
Discussion
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