Vol. 174, No. 22JOURUAUL OF BACTERIOLOGY, Nov. 1992, p. 7149-71580021-9193/92V227149-10$02.00/0Copyright X) 1992, American Society for Microbiology

Molecular Characterization of Two Clostridium acetobutylicumATCC 824 Butanol Dehydrogenase Isozyme Genes

KARL A. WALTER,1 GEORGE N. BENNETTI,2 AND ELEFTHERIOS T. PAPOUTSAKISl*Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208,1 and

Department ofBiochemistry and Cell Biology, Rice University, Houston, Texas 772512

Received 10 July 1992/Accepted 11 September 1992

A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D.Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834, 1991) was sequenced. Twocomplete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF(ORF1). The translation products of bdka4 and bdhB corresponded to the N-terminal sequences of the purifiedBDH I and BDH H proteins, respectively. The two isozymes had a high amino acid identity (73%) and showedhomology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhBdid not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bpupstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for thesegenes were almost identical. bMUA and bdhB were found to be induced or derepressed immediately prior tosignificant butanol production in controlled pH 5.0 batch fermentations.

The gram-positive, obligately anaerobic bacterium Clos-tridium acetobutylicum is one of the few organisms known toproduce 1-butanol as a major fermentation product. Interestin the use of butanol as a fuel extender and chemicalfeedstock has prompted several investigations into the mo-lecular means by which C acetobutylicum produces buta-nol.The butanol dehydrogenase (BDH) is involved in the final

step of the butanol formation pathway in C. acetobutylicum.In this step, butyraldehyde is converted to butanol, with thecofactor NAD(P)H being oxidized in the process. Evidencesuggests that there are two distinct types of BDHs in C.acetobutylicum, one which is NADH dependent and onethat is NADPH dependent (11, 25). The relative physiolog-ical importance of these two dehydrogenases is unclear atpresent.

Previously, Youngleson et al. (42, 43) cloned and se-quenced the adhl gene from C. acetobutylicum P262. Thisgene codes for a 43-kDa NADPH-dependent alcohol dehy-drogenase (ADH). No activity was observed with NADH asa cofactor. The enzyme utilized ethanol and butanol nearlyequally well and was thus classified as an ADH.More recently, two BDH isozymes (BDH I, BDH II) were

purified and cloned from C. acetobutylicum ATCC 824 (25,35, 36). These isozymes were primarily NADH dependent,although some NADPH-dependent activity was observed.Both enzymes were dimers with a subunit molecular size of-42 kDa. The BDH II enzyme was found to have 46-foldgreater activity with butyraldehyde than with acetaldehyde,whereas the BDH I enzyme had only 2-fold greater activity.

In this study, the nucleotide sequence and putative aminoacid sequence of the bdhA and bdhB genes are presented.These two genes code for proteins showing homology to anewly described class of ADHs. Although the bdhA andbdhB genes were found to exist in tandem on the chromo-some, RNA studies revealed that they do not form anoperon. In addition, both genes were shown to be induced or

* Corresponding author.

derepressed near the onset of butanol formation in batchculture.

MATERIALS AND METHODS

Bacterial strains. C. acetobutylicum ATCC 824 was ob-tained from the American Type Culture Collection (Rock-ville, Md.). Escherichia coli ER2275 [trp3l hisl tonA2rpsL104 supE44 xyl-7 mtl-2 metBi e14+ A(1ac)U169 endAlrecAl R(zgb-210::TnlO) Tets A(mcr-hsd-mrr)114::1510/F'proAB lacIqZAM15 zz::min-TnlO (Kmr)] was obtained fromNew England Biolabs (Beverly, Mass.) and was used for allcloning steps.Growth conditions and maintenance. E. coli was grown

aerobically at 37C in Luria-Bertani (LB) medium supple-mented when necessary with ampicillin (50 ,ug/ml), kanamy-cin (25 ,g/ml), and chloramphenicol (250 ,ug/ml). E. coli wasstored in LB medium containing 10% glycerol at -85°C. C.acetobutylicum was grown in clostridial growth medium (37)and was maintained as previously described (19). Batchfermentations for RNA experiments were performed in aBiostat M bioreactor (B. Braun Instruments, Burlingame,Calif.) with a culture volume of 1.5 liters, and fermentationsfor assay experiments were performed in a BioFlo II biore-actor (New Brunswick Scientific, Edison, N.J.) with aculture volume of 5 liters. The reactor was inoculated with1:10 (vol/vol) preculture at an optical density at 600 nm ofapproximately 0.2. The pH was maintained automaticallythrough the addition of 8 M NH40H or 1 M HCI.DNA isolation and manipulation. Isolation of C. acetobu-

tylicum chromosomal DNA was performed as previouslydescribed (19). Plasmid isolation from E. coli was done bythe method of Birnbaum and Doly (3), with additional stepsof the procedure ofWu and Welker (40) when the DNA wasto be sequenced. Cosmid transfection was performed by themethod of DiLella and Woo (9). Restriction enzymes werepurchased from New England Biolabs and used under therecommended conditions. DNA fragments were purifiedfrom agarose gels by using a Geneclean II kit (Bio 101, SanDiego, Calif.).Cosmid library preparation. C. acetobutylicum ATCC 824

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chromosomal DNA was partially digested with Sau3A andsize fractionated on a 1.0% agarose gel. DNA fragments of30 to 45 kb were purified from the gel and ligated with theBamHI-digested cosmid pWE15 (Pharmacia, Piscataway,N.J.). NotI and EcoRI cassettes surrounding the BamHI siteon pWE15 allowed for easy removal of C. acetobutylicumDNA from cosmid clones.Colony hybridizations. E. coli cosmid library transfor-

mants were gridded onto nitrocellulose filters (Schleicher &Schuell, Inc., Keene, N.H.) resting on LB plates containingampicillin and incubated at 37°C. When growth appeared onthe nitrocellulose, the filters were transferred onto LB platescontaining chloramphenicol and incubated for an additional24 h. The gridded colonies were then lysed, and the liberatedDNA was fixed by using the method described by Sambrooket al. (30). The nitrocellulose filter was incubated at 42°C for4 h in a prehybridization solution consisting of 50% formam-ide, Sx Denhardt's solution, Sx SSPE (lx SSPE is 0.18 MNaCl, 10 mM NaPO4, and 1 mM EDTA [pH 7.7]), 0.1%sodium dodecyl sulfate (SDS), and 100 ,ug of denaturedsalmon sperm DNA per ml. Single-stranded radiolabeledprobe was then added to the prehybridization solution, andthe hybridization was allowed to continue at 42°C for 14 h.After the hybridization was finished, the following washeswere performed: five washes for 10 min at room temperaturein 2x SSC-0.1% SDS, one wash for 1 h at 68°C in lxSSC-0.1% SDS, one wash for 4 h at 68°C in lx SSC-0.1%SDS, and one wash for 1 h at 68°C in 0.2x SSC-0.1% SDS.The filter was air dried and then exposed to X-ray film.DNA sequencing. Sequencing of both strands of DNA was

done by the dideoxy chain termination method of Sanger etal. (31) by using synthetic oligonucleotide (20-mer) primers.Double-stranded plasmid DNA was prepared for use as a

template, and sequencing reactions were performed by usingthe Sequenase version 2.0 kit (U.S. Biochemical Corp.,Cleveland, Ohio) according to the specified conditions.

Isolation of total RNA. Total RNA was prepared by amodification of the procedure of Gerischer and Durre (12).Indicated stock solutions were treated with diethyl pyrocar-bonate (DEPC) as described previously (9). Cells from 10 mlof growing culture were harvested by centrifugation at 4°C,and the pellets were stored at -85°C. Cell pellets were

suspended in 3 ml of ice-cold AE buffer (20 mM sodiumacetate [pH 5.5], 1 mM EDTA). To this suspension, 6 ml ofacid phenol (AMRESCO, Solon, Ohio) and 150 ,ul of 20%SDS, both preheated to 60°C, were added. The suspensionwas shaken by hand for 6 min at 60°C and then placed on icefor 5 min. After centrifugation (4°C), the upper phase wasrecovered and adjusted to 0.25 M sodium acetate with a 2 Msodium acetate (0.1% DEPC-treated) stock solution. Threemore acid phenol extractions were performed, and the RNAwas precipitated by the addition of 2.5 volumes of 100%ethanol. RNA was centrifuged, dried, and then resuspendedin 40 ,ul of 62.5 mM Tris-HCl (pH 7.5)-12.5 mM MnCl2.After the addition of 5 ,u1 of RNAguard (Pharmacia; 29,000U/ml), this mixture was treated with 5 ,ul of DNase I (U.S.Biochemical Corp.; 2,126 U/ml) and incubated at 37°C for 45min. After extraction with an equal volume of phenol-chloroform (AMRESCO), the RNA was precipitated with1/10 volume of 3 M sodium acetate (0.1% DEPC treated) and2.5 volumes of 100% ethanol. The integrity of the RNA was

checked by standard gel electrophoresis, and the concentra-tion was determined from A260 measurements (1 A260 unit =40 ,ug of RNA per ml).

Northern blot analysis. Total RNA was separated on 1%(wt/vol) formaldehyde agarose gels (30). Transfer of the

RNA to nitrocellulose (Schleicher & Schuell), prehybridiza-tion, hybridization with 32P-end-labeled oligonucleotides,and wash steps were performed as described by Sambrook etal. (30). Hybridizations were performed at 33°C for 20 h. Sizedetermination was done with an RNA ladder (GIBCO/Bethesda Research Laboratories, Gaithersburg, Md.).Lanes containing the standards were sliced from the gel,stained for 15 min in 1 ,ug of acridine orange per ml,destained in several changes of water (0.1% DEPC treated)for 3 to 16 h, and then photographed next to a ruler underUV light.Primer extension analysis. Primer extension reactions were

performed as recently described by Gerischer and Durre(12). Moloney murine leukemia virus reverse transcriptasewas obtained from U.S. Biochemical Corp. The cDNA wasexamined on 8% (wt/vol) polyacrylamide sequencing gels.To map the exact transcriptional start sites, sequencingreactions were performed on the corresponding DNA byusing the same primers that were used for the primerextension reactions.BDH assay. Cells from 500 ml of C. acetobutylicum culture

were harvested, and the pellets were stored at -85°C untilneeded. Cells were suspended in 15 mM potassium phos-phate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.1mM ZnSO4 (1 g of frozen cells per 5 ml of lysis buffer). Thecell suspension was sonicated (Heat Systems, Farmingdale,N.Y.) for 18 min at a power setting of 38 W and a pulsesetting of 90%. The lysed cell suspensions were clarified bycentrifugation at 35,000 x g for 20 min. Assays wereperformed in the physiological direction as previously de-scribed (36). One unit (U) is defined as 1 ,umol of NADHoxidized per min.

Analysis of fermentation products. The concentrations ofbutanol, ethanol, acetone, acetate, and butyrate were deter-mined by gas chromatography as previously described (18).A Varian (Walnut Creek, Calif.) model 6000 gas chromato-graph was used to analyze acidified samples. Sec-butanol(0.1% [vol/vol]) was used as an internal standard. Peak areaswere determined by using a Hewlett-Packard model 3394Aintegrator (Avondale, Pa.).Computer programs. Sequence comparisons were done

with the Wisconsin Genetics Computer Group sequenceanalysis software package, version 6.0 (8). The programsused included FastA, StemLoop, PeptideStructure, Plot-Structure, BestFit, Terminator, and Composition.

Nucleotide sequence accession numbers. The bdhA andbdhB sequences have been submitted to GenBank (2) andhave been assigned the accession numbers M96945 andM96946, respectively.

RESULTS

Recloning the C. acetobutylicum BDH genes. It was desir-able to reclone the BDH genes on a larger segment of DNAthan they were originally cloned for studies localizing othercloned fermentative genes to this region. Hence, a pWE15cosmid library of C. acetobutylicum 824 genomic DNA wasscreened by colony hybridization by using as a probe a0.9-kbAvaII-PvuII fragment from the plasmid pBDH51 (25),on which the genes were originally cloned. Preliminarysequencing of this AvaII-PvuII fragment had revealed aDNA segment which, when translated, matched the N-ter-minal amino acid sequence of the purified BDH I (35).Several positive colonies were identified, and two wereselected for further characterization. Cosmid DNA wasisolated from these two colonies. The cosmid pCP14 con-

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tained an insert of 33 kb, and the cosmid pCP23 contained aninsert of 36 kb. The presence of four EcoRI bands ofidentical size in these two clones indicated that they likelycontained a 16.7-kb segment of common DNA. Southernanalysis of EcoRI-digested pCP14 and pCP23 by using theprobe mentioned above localized the BDH I gene to a 6-kbEcoRI fragment present in both cosmids. The probe alsohybridized to an identical-size fragment of EcoRI-digestedC acetobutylicum 824 chromosomal DNA.The 6-kb EcoRI fragment from pCP14 was subcloned into

the EcoRI site of pUC18 to yield the plasmid pECO14. Thisplasmid was used for all subsequent sequencing experi-ments.

Nucleotide sequence of the bdzA and bdhB genes. Bothstrands of a 3,893-bp segment of C. acetobutylicum DNAcontained on the plasmid pECO14 were sequenced and areshown in Fig. 1. Two complete open reading frames (ORFs)were contained on this segment as well as part of a thirdORF. Comparison of the predicted amino acid sequences ofthe two complete ORFs with those of the N-terminal por-tions of the purified BDH I and BDH II (28, 35) revealed thatthe first complete ORF coded for BDH I and the secondcoded for BDH II. These genes were named bdhA and bdhB,respectively.bdhA and bdhB are 1,170 and 1,173 bp in length, respec-

tively. Putative ribosome-binding sites were located 8 bpupstream of the bdhA start codon (5'-AGGAGG-3', posi-tions 710 to 715) and 7 bp upstream of the bdhB start codon(5'-AGGAGG-3', positions 2157 to 2162). The spacing andsequence of these ribosome-binding sites matched well thosefound for other C. acetobutylicum genes (24). The bdhAgene is terminated by two consecutive stop codons (UAA,UAG), and the bdhB gene is terminated by a single (UAA)stop codon.Amino acid analysis. The bdhA and bdhB genes code for

proteins containing 389 and 390 amino acid residues, respec-tively. The calculated molecular masses of the BDH I andBDH II proteins were 43,039 and 43,227 Da, respectively.These molecular masses agreed well with those previouslydetermined by SDS-polyacrylamide gel electrophoresis (42kDa) for the purified proteins (35, 36). There was 72.9%identity (85.8% similarity) between the amino acid se-quences of BDH I and BDH II. This homology was foundover the entire length of the two proteins. The BDH proteinsshowed homology to several other proteins, which are listedin Table 1. These proteins have 24 to 29% identity (48 to 54%similarity) with the two BDH proteins. The alignment ofthese proteins is shown in Fig. 2. There are 32 amino acidpositions which are strictly conserved in all seven proteins,with an additional 19 positions that are conserved in six ofthe seven proteins.

Transcriptional start and stop sites. Northern (RNA) blotswere performed by using total mRNA isolated from C.acetobutylicum cells which had been producing butanol for 2to 3 h. To prevent any possibility of cross-hybridization,probes were made to corresponding regions of bdhA andbdhB, where little homology existed at the DNA level. Alack of probe cross-hybridization was also demonstrated inprimer extension experiments, where no extension productscorresponding to the other transcript were observed foreither probe. When probing with an end-labeled oligonucle-otide complementary to bdhA (5'-ATCAAAACTTAGCATACTTCT-3'), a single 1.3-kb band was identified on aNorthern blot (not shown). Use of an oligonucleotide (5'-TTCGAAATCAACCACT`ETAA-3') complementary to thebdhB gene yielded a single band of -1.35 kb. From these

data, it was evident that bdhA and bdhB were not expressedas an operon.Primer extension experiments were conducted to identify

the transcriptional start sites of both genes. The results ofthese experiments are shown in Fig. 3. A major band in lane5 of Fig. 3 indicates that transcription of bdhA starts at a site37 bp upstream of the start codon. Analysis of lane 6 of Fig.3 indicates that the transcriptional start site for bdhB lies 58bp upstream of its start codon. Two smaller bands of lesserintensity can also be seen in this lane. These bands likely donot represent alternate promoters since there are no corre-sponding -10 and -35 regions similar to consensus clostrid-ial promoters; instead, these bands likely represent eitherspecific degradation products or regions where the reversetranscriptase has difficulties reading through secondarystructure. An inverted repeat region has been identifiedwhich corresponds to the location of these minor bands. Noother transcriptional start sites were found within 250 bp ofthe start codon of either gene. Promoter regions for bdhAand bdhB along with others mapped from C. acetobutylicumare shown in Table 2.

After the sizes of the two transcripts and their transcrip-tional start sites were determined, it was possible to identifylikely terminators for the two transcripts. An inverted repeatwhich would be capable of forming a stem-loop structurewith a stem of 14 nucleotides in length was located down-stream ofbdhA. There were several U residues at the base ofthe stem and immediately following it, making it likely thatthe terminator is rho independent (29). A large invertedrepeat resembling a rho-independent terminator was alsofound downstream ofbdhB. This repeat would correspond toa stem-loop with a stem length of 20 nucleotides.mRNA analysis of the bdhA4 and bdhB genes. A controlled

pH 5.0 batch fermentation was performed to determine therelative levels of both the bdhA4 and bdhB transcripts duringa C. acetobutylicum fermentation. The butanol formationprofile for this fermentation is shown in Fig. 4. Cells begin toproduce butanol as they enter the stationary phase ofgrowth. Cell samples were taken at six time points (Fig. 4,points a to f), and total RNA was purified from each sample.Primer extension reactions were performed on these samplesby using primers complementary to the bdhA and bdhBtranscripts. The same concentration of total RNA was usedfor each primer extension reaction. The primer extensionproducts were examined on an 8% polyacrylamide sequenc-ing gel and are shown in Fig. 5. Low levels of bdhAtranscript are present at time point a when trace amounts ofbutanol are first detected in the culture. A dramatic increasein bdhA transcript from point b to c is observed, followed bya gradual decrease at points d and e. No bdhA transcript isdetectable at point f, at which time butanol formation hasceased. No bdhB transcript is found at point a. However, apattern similar to that of the bdhA transcript is observed forthe remaining time points. There appear to be higher levelsof bdhA4 transcript than bdhB transcript for all the timepoints where transcripts were present.The activity proffle of the BDH was measured in a

separate pH 5.0 batch fermentation under fermentationconditions identical to those used for the RNA transcriptstudies. The resulting activity, butanol, and biomass concen-tration profiles for this fermentation are shown in Fig. 6.BDH activity is clearly present prior to any accumulation ofbutanol in the culture. The largest activity was observedsomewhere between the onset of butanol formation and 3 hafterwards. This corresponds to the time period during

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ORF 11

101 CTCTAGGACCTCTTATTACAGTTTCAACAGTTGGTTCTGTTATAGCTCTTTCAGGGGGTTTTCCAATACTTATAATAATTGCTTTACTTTCACCATCTAG

201

301 TCAAATA GTTTAAATGGGGATGGTTAZTTGTTCTTCTTCTATAAGTTTTTTTATAACAGAGGATTCTATTACATCAGATTGGATAAGATTATTTATGT

401 AGACAATCATTGCAGAAAAATTTCTATTATTAGCTATTTTAAATTCTCTAATCGTTAAATCTGAGCAATTTGTAAATAAGGTTTCTATAGTATGTTTATT

501 TGTTTTAAGGCTAGTTGAAACCGTCTTCGTCGTTATTTTTAGATGCTTCTTCTTTATTAAAAATTTTATTAAACAACGAAAAATTCACCCCCTCTATTTA

601 TTTATATAATAGTAGTTTGCATGAAATTTCGTTGTTTATTCATATTAGATGCIIGITAAAATAATAAAATAG AMAATTAAGTAGACAAACTATAAA+ +++ ++++ ++++ +++ ,+

bdhAM L S F D Y S I P T K V F F G K G K I D V I G E E I

701 TCTATTACTAGGAGGTAAGAAGTATGCTAAGTTTTGATTATTCAATACCAACTAAAGTTTTTTTTGGAAAAGGAAAAATAGACGTAATTGGAGAAGAAAT

K K Y G S R V L I V Y G G G S I K R N G I Y D R A T A I L K E N N801 TAGGAAATATGGCTCAAGAGTGCTTATAGTTTATGGCGGAGGAAGTATAAAAAGGAACGGTATATATGATAGAGCAACAGCTATATTAAAAGAAAACAAT

I A F Y E L S G V E P N P R I T T V K K G I E I C R E N N V D L V L901 ATAGCTTTCTATGAACTTTCAGGAGTAGAGCCAAATCCTAGGATAACAACAGTAAAAAAAGGCATAGAAATATGTAGAGAAAATAATGTGGATTTAGTAT

A I G G G S A I D C S K V I A A G V Y Y D G D T W D M V K D P S K1001 TAGCAATAGGGGGAGGAAGTGCAATAGACTGTTCTAAGGTAATTGCAGCTGGAGTTTATTATGATGGCGATACATGGGACATGGTTAAAGATCCATCTAA

I T K V L P I A S I L T L S A T G S E M D Q I A V I S N M E T N E1101 AATAACTAAAGTTCTTCCAATTGCAAGTATACTTACTCTTTCAGCAACAGGGTCTGAAATGGATCAAATTGCAGTAATTTCAAATATGGAGACTAATGAA

K L G V G H O D M R P K F S V L D P T Y T F T V P K N Q T A A G T A1201 AAGCTTGGAGTAGGACATGATGATATGAGACCTAAATTTTCAGTGTTAGATCCTACATATACTTTTACAGTACCTAAAAATCAAACAGCAGCGGGAACAG

D I M S H T F E S Y F S G V E G A Y V Q D G I R E A I L R T I K1301 CTGACATTATGAGTCACACCTTTGAATCTTACTTTAGTGGTGTTGAAGGTGCTTATGTGCAGGACGGTATACGAGAAGCAATCTTAAGAACATGTATAAA

Y G K I A M E K T D D Y E A R A N L M W A S S L A I N G L L S L G1401 GTATGGAAAAATAGCAATGGAGAAGACTGATGATTACGAGGCTAGAGCTAATTTGATGTGGGCTTCAAGTTTAGCTATAAATGGTCTATTATCACTTGGT

K D R K W S C H P M E H E L S A Y Y D I T H G V G L A I L T P N W M1501 AAGGATAGAAAATGGAGTTGTCATCCTATGGAACACGAGTTAAGTGCATATTATGATATAACACATGGTGTAGGACTTGCAATTTTAACACCTAATTGGA

E Y I L N D D T L H K F V S Y G I N V W G I D K N K D N Y E I A R1601 TGGAATATATTCTAAATGACGATACACTTCATAAATTTGTTTCTTATGGAATAAATGTTTGGGGAATAGACAAGAACAAAGATAACTATGAAATAGCACG

E A I K N T R E Y F N S L G I P S K L R E V G I G K D K L E L M A1701 AGAGGCTATTAAAAATACGAGAGAATACTTTAATTCATTGGGTATTCCTTCAAAGCTTAGAGAAGTTGGAATAGGAAAAGATAAACTAGAACTAATGGCA

K Q A V R N S G G T I G S L R P I N A E D V L E I F K L S Y *

1801 AAGCAAGCTGTTAGAAATTCTGGAGGAACAATAGGAAGTTTAAGACCAATAAATGCAGAGGATGTTCTTGAGATATTTAAAAAATCTTATTAATAGAAAC

1901 TGTAGAGGTATTTTTATAATTTAAAAGATGTTAAAGAGTGAGGAGTAATTTTGTTCTAACGCCTCACTCTTTTCATTTTATGATTAAATGTATGCTGATT

2001 TACGCTAACTTAAATCCTAAATAATAACCTAATGTTAATATTTTGTAACAAATGGATAAAAGCGTAAAATATTATTGTAATAATTTTAAGTAGGTTZI&bdhB

* V V D F E Y S I P T R2101 MAZATATATAATGTAGAAGCATTCCTACATTATATTATTTAAATAATAATCTAAACAGGAGGGGTTAAAGTGGTTGATTTCGAATATTCAATACCAACTA

+++++++++++ + + +++++++++++

I F F G K D K I N V L G R E L K K Y G S K V L I V Y G G G S I K R2201 GAATTTTTTTCGGTAAAGATAAGATAAATGTACTTGGAAGAGAGCTTAAAAAATATGGTTCTAAAGTGCTTATAGTTTATGGTGGAGGAAGTATAAAGAG

N G I Y D K A V S I L E K N S I K F Y E L A G V E P N P R V T T V2301 AAATGGAATATATGATAAAGCTGTAAGTATACTTGAAAAAAACAGTATTAAATTTTATGAACTTGCAGGAGTAGAGCCAAATCCAAGAGTAACTACAGTT

E K G V K I C R E N G V E V V L A I G G G S A I D C A K V I A A A C2401 GAAAAAGGAGTTAAAATATGTAGAGAAAATGGAGTTGAAGTAGTACTAGCTATAGGTGGAGGAAGTGCAATAGATTGCGCAAAGGTTATAGCAGCAGCAT

which the greatest levels of bdhA and bdhB transcripts were ously cloned genes, a Southern blot was performed. A 33-kbpresent in the fermentation. NotI fragment from the cosmid pCP14, on which bdhA and

Southern analysis. To identify whether bdhA and bdhB bdhB we-re contained, was nick translated with [t-3bP]dATPwere located on the chromosome close to any other previ- and used as a probe. DNA fragments containing the thiolase

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E Y D G N P W D I V L D G S K I K R V L P I A S I L T I A A T G S2501 GTGAATATGATGGAAATCCATGGGATATTGTGTTAGATGGCTCAAAAATAAAAAGGGTGCTTCCTATAGCTAGTATATTAACCATTGCTGCAACAGGATC

E M D T W A V I N N M D T N E K L I A A H P D M A P K F S I L D P2601 AGAAATGGATACGTGGGCAGTAATAAATAATATGGATACAAACGAAAAACTAATTGCGGCACATCCAGATATGGCTCCTAAGTTTTCTATATTAGATCCA

T Y T Y T V P T N Q T A A G T A D I M S H I F E V Y F S N T K T A Y2701 ACGTATACGTATACCGTACCTACCAATCAAACAGCAGCAGGAACAGCTGATATTATGAGTCATATATTTGAGGTGTATTTTAGTAATACAAAAACAGCAT

L Q D R M A E A L L R T C I K Y G G I A L A K P D D Y E A R A N L2801 ATTTGCAGGATAGAATGGCAGAAGCGTTATTAAGAACTTGTATTAAATATGGAGGAATAGCTCTTGAGAAGCCGGATGATTATGAGGCAAGAGCCAATCT

M W A S S L A I N G L L T Y G K D T N W S V H L M E H E L S A Y Y2901 AATGTGGGCTTCAAGTCTTGCGATAAATGGACTTTTAACATATGGTAAAGACACTAATTGGAGTGTACACTTAATGGAACATGAATTAAGTGCTTATTAC

D I T H G V G L A I L T P N W M E Y I L N N D T V Y K F V E Y G V N3001 GACATAACACACGGCGTAGGGCTTGCAATTTTAACACCTAATTGGATGGAGTATATTTTAAATAATGATACAGTGTACAAGTTTGTTGAATATGGTGTAA

V W G I D K E K N H Y D I A H Q A I Q K T R D Y F V N V L G L P S3101 ATGTTTGGGGAATAGACAAAGAAAAAAATCACTATGACATAGCACATCAAGCAATACAAAAAACAAGAGATTACTTTGTAAATGTACTAGGTTTACCATC

R L R D V G I E E E K L D I M A K E S V K L T G G T I G N L R P V3201 TAGACTGAGAGATGTTGGAATTGAAGAAGAAAAATTGGACATAATGGCAAAGGAATCAGTAAAGCTTACAGGAGGAACCATAGGAAACCTAAGACCAGTA

N A S E V L Q I F K K S V *3301 AACGCCTCCGAAGTCCTACAAATATTCAAAAAATCTGTGTAAACCTACCGGGGTTTGGGCGTCAGATTATATTCATGAACTCCAAGAAAGCAGTATGCTA

3401 GCAAAGAAATAAAACTCAAAGCAGAGAGAAAATTTAGACATTCAACTATAAATAAAAAATACCCCCCAAAGCATTAATATCTTGGGGAGTATTTTTTATT

3501 TTGAAGTATTCTGTTCAGCTAAATATTCTTCTAAGGTAATACCTCTGTTCATAATTTCTTGTGAGGAGGAAGACCGATATATCTTACATGCCATGGCTCA

3601 AAATTATACTTTGTTATGTTTTCTTTATCCTTAGGATATCTTATTATGAAACCATATTTACCACAATTTTGTTGAAGCCATTTATAAGAATTTGTATTCA

3701 TAAATCCATCATCTAAAGAAGAGTATTCGGTTGATAGTAAGTCCATTGCCAATCCAGTTTGATGCTCACTTGTACCAGGTTCAGCTACATATTTATCAGC

3801 TTCGGCTTTTCCGTCTCGTGCTCATTTTTCATTATATAATTTTTGCTGATACGAATAAGGTCTATAACCTGAAACAGCTAGAAGTGTAAGACCFIG. 1. Nucleotide sequence and translation of the bdhA and bdhB genes from C. acetobutylicum. Promoter -10 and -35 regions are

underlined, as well as the portion of ORF1 that has been sequenced. The transcriptional start sites for bdhA and bdhB are denoted (*).Putative terminator regions (=) and other inverted repeat segments (+) have been underscored. Standard one-letter amino acid abbreviationsare listed above the first nucleotide of each codon.

(pTecoll, EcoRI [27]), the phosphotransbutyrylase and bu-tyrate kinase (pJC7, BamHI-PstI [4]), the ADH (pCADH1A2, BglII [42]), and a 14-kb segment of DNA containingthe acetoacetate decarboxylase and coenzyme A-transferasegenes (pSDC2, SalI [26]) from C. acetobutylicum wereprobed. Southern blot (not shown) analysis revealed onlyhybridization to the control (pCP14, EcoRI-digested) lane.Thus, none of these previously cloned genes were found onthe 33-kb segment of DNA containing the BDH genes.

DISCUSSION

Previously, a DNA fragment containing the coding regionfor both BDH genes was cloned (25). In this study, a

TABLE 1. Proteins with high identities to BDH I and BDH II

Identity (%) Size Refer-Organism and protein ,w

BDH I BDH II (aa), ence

Clostridium acetobutylicum ADH 29 28 388 42Saccharomyces cerevisiae ADH4 29 26 382 38Zymomonas mobilis ADH2 28 28 382 7Eschenichia coli 1,2-propanediol 27 26 383 6

oxidoreductaseBacillus methanolicus methanol 25 24 382 33

dehydrogenasea aa, amino acids.

3,893-bp segment of DNA containing these BDH genes wassequenced. The BDH proteins were found to show homol-ogy to a newly described class of ADHs (34). This class ofADHs is distinct from the long-chain, zinc-dependent ADHstypically found in eucaryotes (41) and also distinct from theshort-chain, non-zinc-dependent ADHs found in Drosophilaspp. (32). While the BDHs showed identities of 24 to 29%with this class of proteins (Table 1), an average identity ofover 42% was found between all the proteins of this class(not including the BDHs). Thus, while the BDH proteinsshow identity to this class of ADHs, they are likely asubclass. These differences might derive from the fact thatthe BDH proteins are involved in formation of butanol ratherthan ethanol, methanol, or 1,2-propanediol.The BDH proteins also showed identities of 23 and 24% to

a previously described (13) E. coli ADH coded by the adhEgene. This enzyme is substantially larger, at 891 amino acidresidues, than all of the ADHs listed in Table 1 and has beenshown to exhibit both alcohol and coenzyme A-linked ace-taldehyde dehydrogenase activities. It has been suggested(13) that this enzyme perhaps represents the evolutionaryfusion of the ADH and acetaldehyde dehydrogenase en-zymes. Although it is clear that a portion of this enzyme issimilar to the ADHs listed in Table 1, the differencesmentioned above merit distinction of this enzyme from thisclass of ADHs.The BDH polypeptides were slightly larger than the ADHs

listed in Table 1. When the ADH proteins are aligned with

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V

M L S F D Y S I P T K V F F G K

V V D F E Y S I P T R I F F G K

M M R F T L P R D I Y Y G K

M A S S T F Y I P F V N E M G E

M M A N R M I L N E T A W F G R

M S S V T G F Y I P P I S F F G E

MTNFFIPPASVIGR

G K I D V I G E E I K K Y

D K I N V L G R E L K K YG S L E Q L K N L

G S L E K A I K D L N G SG A V G A L T V E V K R R

G A L E E T A D Y I K N K

G A V K E V G T R L K Q I

G S R V L I V Y G G G S I K R N G I Y

G S K V L I V Y G G G S I K R N G I Y

K G K K A M L V L G G G S M K R F G F V

G F K N A L I V S D A F M N K S G V V

G Y Q K A L I V T D K T L V 0 C G V V

D Y K K A L I V T D P G I A A I G L S

G A K K A L I V T D A F L H S T GL S

bdhA D R A T A I L K E N N I A F Y E L S G V E N P R I T T V K K G I E I C R E N N V D L V L A I G G G

bdhB D K A V S I L E K N S I K F Y E L A G V E P N P R V T T V E K G V K I C R E N G V E V V L A I G G G

adh D K V L G Y L K E A G I E V K L I E G V E P D P S V E T V F K G A E L M R Q F E P D W I I A M G G G

zym K Q V A D L L K A Q G I N S A V Y D G V M P N P T V T A V L E G L K I L K D N N S D F V I S L G G G

fucO A K V T D K M D A A G L A W A I Y D G V V P N P T I T V V K E G L G V F Q N S G A D Y L I A I G G G

yeast G R V Q K M L E E R G L N V A I Y D K T Q P N P N I A N V T A G L K V L K E E N S E I V V S I G G G

mdh E E V A K N I R E A G L D V A I F P K A Q D A D T Q H EGV D V F K Q E N C D A L V S I

bdhA A I C S V I A A G V Y Y D G D T W D M V K D P S K I T K V L P I A S I L T L S A

bdhB S A I D C A K V I A A A C E Y D G N P W D I V L D G S K I K R V L P I A S I L T I A A

adh S P I D A A K A M W I F Y E H P E K T F D D I K D P F T V P E L R N K A K F L A I P S T S G

zym S P H D C A K A I A L V A T N G G E V K D Y E G I D K S K K P A L P L M S I N T T A G

fucO S P Q D T C K A I G I I S N N P E F A D V R S L E G L S P T N K P S V P I L A I P T T A G

yeast S A H D N A K A I A L L A T N G G E I G D Y E G V N Q S K K A A L P L F A I N T T A G

mdh SSHDTA KAIGLVAANG GR INDYQGVNSVEKPVVPVVAITTT AG

bdhA T G S E M D Q I A V I S N M E T N E K L G V G H D D M R P K F S V L D P T Y T F T V P K N Q T A A G

bdhB T G S E M D T W A V I N N M D T N E K L I A A H P D M A P K F S I L D P T Y T Y T V P T N Q T A A G

adh T A T E V T A F S V I T D Y K T E I K Y P L A D F N I T P D V A V V D S E L A E T M P P K L T A H T

zym T A S E M T R F C I I T D Y K T E I K Y P L A D F N I T P D V A V V D S E L A E T M P P K L T A H T

fucO T A A E V T I N Y V I T D E E K R R K F V C V D P H D I P Q V A F I D A D M M D G M P P A L K A A T

yeast T A S E M T R F T I I S N E E K K I K M A I I D N N V T P A V A V N D P S T M F G L P P A L T A A T

mdh TGSETTSLAVITDSARKVKJMPVIDEKITPTVAIVDPELMVKKPAGLTIAT

bdhA T A D I M S HT FESfF S G V E G A Y V Q D G I R E A I L R T C I K Y G K I A M

bdhB T A D I M SIH|I FIEIVIYIF S N T K T A Y L Q D R M A E A L L R T C I K Y G G I A L

adh G M D A L TIHIA I EIAIY V A T L H S P F T D P L A M Q A I E M I N E H L F K

zym G M D A L TIHIA FIEIAIY|S S T A A T P I T D A C A L K A A S M I A K N L K T A C

fucO G V D A L TIHIA I EIGIY I T R G A W A L T D A L H I K A I E I I A G A L R

yeast G L D A LT|HG CIUELAY V S T A S N P I T D A C A L K G I D L I N E S L V A A Y K D G K D K K

mdh G M DA L SHA IUEAYV A K G A T P V T D A F A I Q A M K L I NE Y LP K AV

VFV

bdhA E K T D D Y E AR A N L M W A S S L Al I N G L L S L G K D R K W S C HP M ER E L S A Y Y

bdhB E K P D D Y EIAIR A N L M W A S SLLA I N G L L T Y G K D T N W S VH L M EI HIE L S A Y Y

adh S Y E G D K E|A|R E Q M H Y A Q C LIA|G M A F S N A L L G I CIHS M AIHIK T G A V F

zym D N G K D M P|A|R E A M A Y A Q FLLA G M A F N N A S L G YVLHIA MAMH|Q L G G Y Y

fucO G S V A G D K D|A|G E E M A L G Q Y VUA G M G F S N V G L G L V[H G M AUHP L G A F Y

yeast A R T D M C Y|A E Y L A G M|A F N N A S L G Y V|H A L A H|Q L G G F Y

mdh A N G E D IEAR E A M AYGAQ YMVG v A F N N G GL G LVLUS I S QV G G V Y

bdhA D I T HGV G L A I L TIPN W M E Y I L N D D T L H K F V S Y G I N V W G I D K N K D N Y E I A R

bdhB D I TIH GIV G L A I L T|P|N W M E Y I L N N D T V Y K F V E Y G V N V W G I D K E K N H Y D I A H

adh H I PIH GIC A N A I YLLP Y V I K F N S K T S L E R Y A K I A K Q I S L A G N T N E E L V D

zym N L PiH GIV C N A V L LIPIH V L A Y N A S V V A G R L K D V G V A M G L D I A N L G D K E G A

fucO N T P H G V A N A I L L P H V M R Y N A D F T G E K Y R D I A R V M G V K V E G M S L E E A R

yeast H L PLH GGV C N A V L L|P H V Q E A N M Q C P K A K K R L G E I A L H C G A S Q E D P E

mdh K L QHG I C N S V N M H V C A F N L I A K T E R F A H I A E L L G E N V S G L S T A A A A E

V V-bdhA E A I K N Y R E Y F N S L G I P S K L R E V G I G K D K L E L M A K Q A V R N S G G T I

bdhB Q A I Q K T R D Y F V N V L G L P S R L R D V G I E E E K L D I M A K E S V K L T G G T I

adh S L I N L V K E L N K K M Q I P T T L K E Y G I H E E F K N K V D L I S E R A I G D A C T G

zym E A T I Q A V R D L A A S I G I P A N L T E L G A K K E D V P L L A D H A L K D A C A L

fucO N A A V E A V F A L N R D V G I P P H L R D V G V R K E D I P A L A Q A A L D D V C T G

yeast E T I K A L H V L N R T M N I P R N L K D L G V K T E D F D I L A E H A M H D A C H L T

mdh R A I V A L E R Y N K N F G I S G Y A E MGV K E E D I E L L A K N A F E D V C T Q

bdhA G S L R P I N A E D V L E I F K K S Y

bdhB G N L R P V N A S E V L Q I F K K S V

adh S N P R Q L N K D E M K K I F E C V Y

zym T N P R Q G D Q K E V E E L F L S A F

fucO G N P R E A T L E D I V E L Y H T A

yeast N P V Q F T K E Q V V A I I K K A Y

mdh S N P R V A T V Q D I A Q I I K N A L

FIG. 2. Amino acid alignment of seven ADHs. Boxed positions indicate strict conservation. The symbol V denotes a position that is

conserved in six of the seven proteins. Abbreviations: bdhA, C. acetobutylicum BDH I (this study); bdhB, C. acetobutylicum BDH II (this

study); adh, C. acetobutylicum ADH (42); zym, Z. mobilis ADH2 (7); fucO, E. coli 1,2-propanediol oxidoreductase (6); yeast, S. cerevisiae

ADH4 (38); mdh, Bacillus methanolicus MDH (33).

7154

bdhAbdhBadhzymfucOyeastmdh

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BUTANOL DEHYDROGENASE ISOZYME GENES 7155

PEA PEjI G A T C

5 6

Eu.

l:* "liii .#**-;S

10>-e S

FIG. 3. Primer extension analysis. Primer extension productsmade by using primers complementary to the bdhA and bdhB genesare shown in lanes 5 and 6, respectively. RNA used in theseexperiments was obtained from C. acetobutylicum cells which hadbeen producing butanol for 2 h. Regions of the plasmid pECO14were sequenced by using the same primers, and the resulting DNAsequences are shown in lanes 1 to 4 and 7 to 10.

the BDHs, the additional amino acid residues found in theBDHs are primarily found in interior positions rather than atN-terminal or C-terminal positions. Six of the strictly con-served residues in this class of ADHs are prolines, suggest-ing possible similarities in the three-dimensional structuresof these proteins. Interestingly, four His residues are strictlyconserved, with three of them in a 15-amino-acid stretch.Both His and Cys residues have been implicated as metal-binding ligands in long-chain, zinc-dependent ADHs (16).While there are no Cys residues strictly conserved amongthis class of ADHs, three Cys residues (Cys-84, -103, -266)are conserved in the BDHs.

E

c00

Ca0

10

10

10

200

150

.Ea

100 o0

c:50

im

O

0 10 20 30 40 50

time, hFIG. 4. Butanol proffle (E) and optical density profile (-) for a

C. acetobutylicum controlled pH 5.0 batch fermentation. Cell sam-ples were taken at points a to f for RNA analysis.

An NADH-binding region has been identified in long-chain, zinc-dependent ADHs which contains a conservedglycine-rich region of GxGxxG or GxxGxxG within a PapRossmann fold (16). No such regions were found in either ofthe BDHs, and only one such region (6) has yet beenidentified in any of the enzymes in this new ADH class,despite the fact that all of the proteins with the exception ofthe C. acetobutylicum ADH have been shown to be able toutilize NADH as a cofactor (6, 7, 10, 33, 35, 43).

All but one of the -35 and -10 region nucleotides areconserved between the bdh promoters (Table 2). In addition,there is extended identity immediately downstream of boththe -35 and the -10 regions. These extended -35 and -10regions may be required for recognition of the promoterregions or may simply be coincidental. The -35 and -10regions of these two promoters are spaced slightly closer (16bp) together than those found for all other promoters iden-tified in C acetobutylicum (17 to 19 bp).The only other solvent formation enzyme whose promoter

has been mapped is the acetoacetate decarboxylase (adc).Like the bdh genes, this gene is also induced near the onsetof solvent formation. However, the -10 and -35 regions ofthe adc promoter more closely resemble those of the glnA

TABLE 2. Mapped promoter regions of C. acetobutylicum genes

SequenceGene Reference

-35 region -10 region -1

bdhA TTAGATGC TTGTAT TAAAATATAAAATAG IAAA ATAAGTA This studyI II 11111 III II 111111 111 II

bdhB AAATATTA TT(NAA TAATTTTAAGTACGTT TAAIAT ATATATA This studyadc TGTAAAAA TACT TAAAAAAACAATATGTGT TAMATA GTAAAT 12gln4 AAAATCAC fTT TCTTAAAAAAAAGGGAAG TATA&I TTAGTTA 15

CAAAGGCG TTCAI AGAAGTTTATACTTGTC TATTGT GOCGOC 15TAATTTTT TTGCTA TCTCAACATTGCTTAATGC TATAM AAGT 15

dnaK TGGAAAAGfGACA AAGATAATGTCAGGTGA TATT ATAAACA 21GACTAATT TTATGA AAATAAGAAAAGTTGAC AAA,C AATGT 21TATAGGAA GACCGACAAGGATACCT ITCA AAAGT 21

groESL ATTGATG TTGCTA ATATATTCAGGATTATT TATTAT AATAATTG 20

bdhB 10 1bdhA

G A T C

_-w :SSi*d,,==5 .... ..

_-w_....=s_ .:....u

e ;_=_ _

.. __0 ww _i* E .,.,, ...........

=;33-_ ............._* a ;; . .

.......... _..... =_ : '* ........$S:..'-:.

.. _S_ ...._ _

: ::-:.s* ::. : : ,.

* :

_ ''' :.:.... :

_ w_ ...:':..__:.., *... . L__

_ _ ,'.,.s_ ..

*;:_

.... . .. ., 71

:*Q;w o.

......

1 2 34

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7156 WALTER ET AL.

-bdh/ ------- I I------- bdhl. -------I

i ) c cl e I a 1) c cl e f

FIG. 5. Time course primer extension analysis. Primer extensionreactions were performed on RNA purified from cells at varioustime points (Fig. 4, points a to 0 in a controlled pH 5.0 batchfermentation. The same amount of total RNA was used for allprimer extension reactions. Primer extension products correspond-ing to the bdhA and bdhB transcripts are located in lanes a to f (left)and a to f (right), respectively. The sizes of the bdhA and bdhBextension products are identical to those obtained in Fig. 3.

promoters (primarily constitutive under normal conditions)than they resemble those of the bdh promoters. Thus, it isnot likely that a single sigma factor is directly responsible forthe induction of all of the solventogenic genes in thisorganism.

In the pH 5.0 batch fermentation shown in Fig. 6, NADH-dependent BDH activity increases dramatically 2 to 3 h priorto the appearance of butanol in the culture. This observationis consistent with what others have found (14, 35). Levels ofbdhA and bdhB mRNA monitored by primer extensionanalysis (Fig. 5) indicate clearly that both the bdh genes areinduced or derepressed immediately prior to accumulation ofsignificant amounts of butanol in the culture. The appear-ance of bdh mRNA and BDH activity at approximately thesame time suggests that the BDHs are, at least initially,regulated at the transcriptional level. It is difficult to findmajor differences in the expression patterns of the bdhA andbdhB genes under the conditions employed in these fermen-tations. Qualitatively, there appear to be larger levels of

E

80

0

10

10

10

10

E

c0

0

-a0

m

-0.05

-0.04

-0.03 k_

-0.02 X

m

c

z

-0.00

0 10 20 30 40 50

time, h

FIG. 6. NADH-dependent BDH activity (A), butanol concentra-tion (El), and optical density (-) profiles of a C. acetobutylicumcontrolled pH 5.0 batch fermentation.

bdhA than bdhB mRNA at the time of induction or derepres-sion. In addition, at the earliest time point when trace levelsof butanol were initially detected in the culture (Fig. 4, pointa), only the bdhA transcript was detected.Sequence analysis revealed the location of segments of

inverted repeats in the promoter regions of both the bdhAand bdhB genes (Fig. 1). The inverted repeats in the bdhApromoter segment overlap both the -10 and -35 regions.These inverted repeats are fairly short, have two mis-matches, and are separated by a large number of nucleotides(18 bp); taken together, these factors would tend to rule outthe possibility that the inverted repeats represent a site for aDNA-binding protein. The location of these repeats, how-ever, would be amenable to binding of a repressor that wouldinterfere with binding of the RNA polymerase (5). The bdhBinverted repeats more closely resemble typical sites ofDNA-binding proteins (1). The inverted repeat segment ofthe bdhB starts 2 bp downstream of the -10 region of itspromoter and overlaps the transcriptional start site. Theserepeats are located in a position where repressor bindingsites are commonly found in other organisms (5). There is nosimilarity between the inverted repeat regions of bdhA andbdhB. Searches of the GenBank (2) data base using thesequence of either inverted repeat region revealed no signif-icant identities with other sequences. Clearly, further exper-iments are necessary to discern the role of these regions, ifany, in the transcription of these genes.

Previously, both genes in the butyrate formation pathway(phosphotransbutyrylase and butyrate kinase) were found toexist together on the chromosome, as were both genes in theacetone formation pathway (coenzyme A-transferase andacetoacetate decarboxylase) (4, 12, 26). Considering thisgenetic organization, it seemed possible that the gene codingfor the other butanol formation pathway enzyme, the bu-tyraldehyde dehydrogenase, might exist proximal to the bdhgenes. However, thus far only a single potential openreading frame (ORF1) has been identified in this region. Thelast -330 bp of ORF1 have been sequenced. Data basesearches using the translation product of ORF1 did notreveal any high homologies to any previously sequencedproteins; however, almost all of the proteins that showedhomology to the ORFi translation product were membrane-associated proteins.The relative role of these two BDH isozymes in C.

acetobutylicum is as yet unknown. Under the culture con-ditions employed in these experiments, their expressionpatterns are similar. In animal and plant cells, multipleADHs often form intergenic heterodimers (41). This possi-bility cannot be ruled out for the BDH isozymes; however,plasmids containing only the bdhA gene were capable ofgenerating BDH activity in E. coli. In Zymomonas mobilis,two ADHs, one containing zinc and one containing iron,have been identified (39). The relative levels of these twoADHs are dependent upon the availability of iron or zinc(17), the presence of alcohol (22), and the growth phase (23).Interestingly, two of the five ADHs that showed homologyto the BDHs have been demonstrated to be iron activated (6,7). Although previous experiments have indicated that bothBDHs likely utilize zinc (35, 36), further experiments may benecessary.The generation of null mutants lacking one of the two

BDH activities would facilitate identification of their relativeroles. However, until successful methods for performingchromosomal integrations are developed in C. acetobutyli-cum, studies utilizing different medium conditions (pH,

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metal ions, carbon sources, etc.) may help elucidate theroles of these two BDH isozymes.

ACKNOWLEDGMENTS

We thank L. Mermelstein, N. Welker, D. Petersen, R. Welch, andL. Wu for their advice and technical assistance, J. Cary forpreparation of the cosmid library, and M. Heaton for instruction onthe use of the sequence comparison programs.

This research was supported by a National Science Foundationgrant (BCS-8912209).

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2. Bilofsky, H. S., and C. Burks. 1988. The GenBank (R) geneticsequence data bank. Nucleic Acids Res. 16:1861-1864.

3. Birnbaum, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

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6. Conway, T., and L. 0. Ingram. 1989. Similarity of Escherichiacoli propanediol oxidoreductase (fucO product) and an unusualalcohol dehydrogenase from Zymomonas mobilis and Saccha-romyces cerevisiae. J. Bacteriol. 171:3754-3759.

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8. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-sive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.

9. DiLella, A. G., and S. L. Woo. 1987. Cloning large segments ofgenomicDNA using cosmid vectors, p. 199-212. In S. L. Bergerand A. R. Kimmel (ed.), Guide to molecular cloning techniques.Academic Press, Inc., New York.

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12. Gerischer, U., and P. Dfirre. 1992. mRNA analysis of the adcgene region of Clostridium acetobutylicum during the shift tosolventogenesis. J. Bacteriol. 174:426-433.

13. Goodlove, P. E., P. R. Cunningham, J. Parker, and D. P. Clark.1989. Cloning and sequence analysis of the fermentative alco-hol-dehydrogenase-encoding gene of Eschenichia coli. Gene85:209-214.

14. Hfusemann, M. W., and E. T. Papoutsakds. 1989. Comparisonbetween in vivo and in vitro enzyme activities in continuous andbatch fermentations of Clostridium acetobutylicum. Appl. Mi-crobiol. Biotechnol. 30:585-595.

15. Janssen, P. J., D. T. Jones, and D. R. Woods. 1990. Studies onClostridium acetobutylicum ginA promoters and antisenseRNA. Mol. Microbiol. 4:1575-1583.

16. JdrnvalH, H., B. Persson, and J. Jeffery. 1987. Characteristics ofalcohol/polyol dehydrogenases. Eur. J. Biochem. 167:195-201.

17. Mackenzie, K. F., C. K Eddy, and L. 0. Ingram. 1989.Modulation of alcohol dehydrogenase isoenzyme levels in Zy-momonas mobiis by iron and zinc. J. Bacteriol. 171:1063-1067.

18. McLaughlin, J. K, C. L. Meyer, and E. T. Papoutsakds. 1984.Gas chromatography and gateway sensors for on-line stateestimation of complex fermentations (butanol/acetone fermen-tation). Biotechnol. Bioeng. 27:1246-1257.

19. Mermelstein, L. D., N. E. Welker, G. N. Bennett, and E. T.Papoutsalds. 1992. Expression of cloned homologous fermenta-tive genes in Clostridium acetobutylicum ATCC 824. Bio/Technology 10:190-195.

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