Molecular Characterization of an Aldehyde/Alcohol ...jb.asm.org/content/176/3/871.full.pdfMolecular...

JOURNAL OF BACrERIOLOGY, Feb. 1994, p. 871-885 Vol. 176, No. 30021-9193/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Molecular Characterization of an Aldehyde/AlcoholDehydrogenase Gene from Clostridium acetobutylicum

ATCC 824-RAMESH V. NAIR,' GEORGE N. BENNETT,2 AND ELEFTHERIOS T. PAPOUTSAKIS'*

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

Received 17 June 1993/Accepted 2 December 1993

A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream ofthe previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ.Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codesfor a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bpupstream of the aad start codon. The N-terminal section ofAAD shows homology to aldehyde dehydrogenasesof bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcoholdehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AADexhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional proteinencoded by adhE from Escherichia coli. Expression ofaad from a plasmid in C. acetobutylicum showed that AAD,which appears as a -96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependentbutanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, anda small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that couldpotentially encode a 36,704-Da protein was identified upstream of aad.

The strict anaerobe Clostridium acetobutylicum is a gram-positive, spore-forming, saccharolytic bacterium capable offermenting a wide variety of sugars, oligosaccharides, andpolysaccharides to butanol, acetone, ethanol, butyrate, andacetate. Advances in the science of metabolic engineeringcoupled with today's better understanding of the regulation ofkey acidogenic and solventogenic genes may potentially lead tothe development of superior solvent-producing industrialstrains.

Butyraldehyde dehydrogenase (BYDH) catalyzes the con-version of butyryl coenzyme A (butyryl-CoA), a key interme-diate in the formation of butanol, to butyraldehyde, accompa-nied by the oxidation of NAD(P)H and the release of CoAfrom butyryl-CoA. Previously, a BYDH with a subunit molec-ular mass of 56 kDa was purified from C. acetobutylicum B643(35). However, no genes encoding this or any other BYDHhave yet been cloned from C. acetobutylicum.

Acetoacetyl-CoA:acetate/butyrate:CoA transferase (CoA-transferase) is the first enzyme in the acetone formationpathway. This enzyme catalyzes the transfer of the CoA moietyfrom acetoacetyl-CoA to either butyrate or acetate and thus isresponsible for the uptake of the two acids during the non-growth-associated solventogenic phase of the fermentation.The two genes (ctfA and ctfB) encoding the CoA-transferasehave recently been cloned and sequenced (4, 38). The end ofan open reading frame (ORF) was identified 32 bp upstream ofctfA (38). Northern (RNA) analysis revealed that the ctf geneswere contained within a 4.1-kb transcript in C. acetobutylicumDSM 792 (12). A terminator has been identified immediatelydownstream of ctJB, and the coding region of the ctfA and ctJBgenes spans only -1.4 kb. It is thus believed that at least one

* Corresponding author. Mailing address: Department of ChemicalEngineering, Northwestern University, 2145 Sheridan Rd., Evanston,IL 60208-3120. Phone: (708) 491-7455. Fax: (708) 491-3728.

other gene is encoded on the remaining -2.7 kb of transcriptupstream of ctfA. Enzymatic studies had indicated that BYDHhas an induction pattern similar to that of CoA-transferase(19), which plays a critical role in both acetone formation andacid uptake leading to butanol and ethanol formation. It thusappears likely that the gene encoding BYDH is cotranscribedwith the ctf genes.Here we report the identification, sequencing, and charac-

terization of a gene, designated aad, upstream of ctfA. Thisgene encodes a protein that demonstrates at least two differentactivities (aldehyde and alcohol dehydrogenase). Both of theseactivities are necessary for the formation of the two alcohols,butanol and ethanol, from butyryl-CoA and acetyl-CoA, re-spectively.

(Preliminary results of this study have been presentedelsewhere [32a].)

MATERIALS AND METHODS

Bacterial strains. C. acetobutylicum ATCC 824 was obtainedfrom the American Type Culture Collection (Rockville, Md.).Escherichia coli JM109 [endAL recAl gyrA96 thi hsdR17 (rK-MK+) relAl supE44 X- A(lac-proAB) (F' traD36 proABlacIqZAM15)] was obtained from Promega Corp. (Madison,Wis.) and used in all cloning steps. E. coli BL21(DE3) (48) [F-ompT (rB- mB-) (DE3)] was purchased from Novagen, Inc.(Madison, Wis.) and used as the host in T7 expression studies.E. coli ER2275 (recA mcrBC) was purchased from NewEngland Biolabs (Tozer, Mass.) and used as the methylationhost.Growth conditions and maintenance. All E. coli strains were

grown aerobically, unless otherwise stated, at 37°C in Luria-Bertani medium supplemented when necessary with ampicillin[50 ,g/ml for JM109 cultures and 200 ,ug/ml for BL21(DE3)cultures]. Both recombinant and wild-type strains were storedat - 85°C in 15% (vol/vol) glycerol (41). For [35S]methionine

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872 NAIR ET AL.

1

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TTTAACTTTTTTTTCATTTTTTATCCCCCTTATAGAATGTAATAATAACATTCTTATCTAATTTAGTTTTCTATTTTTAACTGGCAGTAATAAAGTTTTT

GCATTGCAATGGTCGGCGTTAATACGTGAACAATTGTTATTTAGCAAAAAATTATGTTCAATACAAATGACTTTGl''(AAAAC-ATl-l-TTTCTT-ATlATLACCACATT-1A

TATATTAATTTGAAATTAATGTAAACGTTTCTTGTTACAATTTTCTCTCTTAATTTTTAATTAAAAAAACTCGATAATTTTCTTTGTATGTGGTATTATT

ORFI

401

501

601

701

801

901

1001

1101

TGTGTACATAAGTATAAAAAAAGGAAAGAGTTGAATTTAG ATCAATTTATTAAATCTTTTTACATATGTCATACCTATTGCGATATGTA

AATATTTrATAATAG.TAACGCATTTTCAAATCAAATCTCTTAAC!AAAG.CGTG.rACAAGC TTTAATAAGGGTGA ArGAArGCAATCCCCTAGAAATACTATCA

AATATAAATCATAAAaTAACAA_ CC_ AAACACTTT_As_rCTTACTTTTA&

1201

1301

1401 GAAGCTATATCTTAATTCAAAATTAAGATATAGCTTCTTTTATGTAGTATTATTTCAGAAGTCTACAAATTAAGTTTATATTTAGACCCTGGGGTGTAAC+++++++++++++++++ +++++++++++++++++++

TATAGTATTTAATATTGGTACTATTAATTAGGGTTATATATACTAGAACTTATCATGGTAAACATAAATATAAACTCAATTCTATTTATGCTCCTATAAA

ATTTTATAATATAGGAAAACTGCTAAATGTAAATTATACGTTTACATTTAGCAGTTTATTTTAAACCTTCATATTTTTCTAAATATACTGATAATTCCTA+++++++++++++++++ +++++++++++++++++

AATATATATTATTACGCCAAAATATTAGATACCATTTTGTAAAAGTTGCTATTTACATTTAAATACACAGCTGTTATATTTIIGAm,TATGCTTTTTATT

0GAACTCATAAAGCATAGTAATTAGTAATTAATAATTTATATTATTAATTATCATAAAATTTATGATCCTTTAAAGTAACATTTTTATTTACTTCATA

AATTGATGTTATTTTTTTCTTGATGTATTGTAAACCTTGTTTTGTTTIZrAGTTTACAATATCTATCTCGAATICTGCTTTCAAGAATAATATCTATACT

aad

M K V T T V K E L D E K L

AAAAACAATATAGACATTATTTTTAAAATATAATAAATATTTTAGAAAGAAGTGTATATTTATGAAAGTCACAACAGTAAAGGAATTAGATGAAAAACTC

K V I K E A Q K K F S C Y S Q E M V D E I F R N A A M A A I D A R I

2101 AAGGTAATTAAAGAAGCTCAAAAAAAATTCTCTTGTTACTCGCAAGAAATGGTTGATGAAATCTTTAGAAATGCAGCAATGGCAGCAATCGACGCAAGGA

E L A K A A V L E T G M G L V E D K V I K N H F A G E Y I Y N K Y

2201 TAGAGCTAGCAAAAGCAGCTGTTTTGGAAACCGGTATGGGCTTAGTTGAAGACAAGGTTATAAAAAATCATTTTGCAGGCGAATACATCTATAACAAATA

K D E K T C G I I E R N E P Y G I T K I A E P I G V V A A I I P V

2301 TAAGGATGAAAAAACCTGCGGTATAATTGAACGAAATGAACCCTACGGAATTACAAAAATAGCAGAACCTATAGGAGTTGTAGCTGCTATAATCCCTGTA

T N P T S T T I F K S L I S L K T R N G I F F S P H P R A X K S T I

2401 ACAAACCCCACATCAACAACAATATTTAAATCCTTAATATCCCTTAAAACTAGAAATGGAATTTTCTTTTCGCCTCACCCAAGGGCAAAATCCACAA

FIG. 1. Nucleotide sequence of aad. The translation in the single-letter amino acid representation is placed above the first nucleotide of eachcodon. The transcriptional start sites of aad at nucleotide positions 1819 (S2) and 1979 (Sj) are indicated (K). Putative promoter -10 and - 35regions corresponding to these start sites are underlined, as are the nucleotide sequence of the upstream ORF1 and a portion of the downstream

ctfA. Two inverted repeat segments downstream of ORF1 are underscored (+).

labeling studies, both recombinant and wild-type strains ofBL21(DE3) were grown aerobically or anaerobically in M9minimal medium (41) supplemented with 1% (wt/vol) glucose,20 ,ug of thiamine hydrochloride per ml, and 0.01% (wt/vol) 18amino acids (minus Cys and Met). For recombinant strains, themedium was supplemented with carbenicillin (50 ,ug/ml). C.acetobutylicum was grown in clostridial growth medium (56),supplemented when necessary with 100 pug of erythromycin perml, and maintained as previously described (32).

Fermentor experiments. For enzyme assay experiments,batch fermentations of C. acetobutylicum were performed in a

BioFlo II fermentor (New Brunswick Scientific, Edison, N.J.)with a culture volume of 5 liters (clostridial growth mediumwith glucose at 80 g/liter instead of 50 g/liter). For fermenta-tions of recombinant C. acetobutylicum, the medium was

supplemented with 75 ,ug of clarithromycin (Abbott Laborato-ries, North Chicago, Ill.) per ml (31). The reactor medium(initial pH of 6.2) was inoculated with 500 ml of preculture atan optical density at 600 nm (OD6.) of -0.2. The pH droppedwith culture progression until it reached a value of 4.5, at whichpoint it was maintained through additions of 6 M NH40H or

2 M HCl. Growth was monitored through OD60 measure-

J. BACrERIOL.

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ATTTATTArACCCTA AATTAACACrAAAAACAATAAATCATaTTTACAarTTTCCACTTAGCTATrArATTCTTCCAnACC

ATATTTTTTAAGACCAaTTGAACTCCAGCCTAATGTAaGTATATC'CTACGACAACTTACCCTGGTTTT CTACTTAArArr.CAAATACCATAAGGCAATA

aAAAACTTTCAAAAAGCTATTTCCATr.r.r.rAnTACAAACTCTC TAGAACTTTACGAATTACCTATGCCAAAATACnAnATTATAAAAAATCCGAAG

AATATCT AAaAAAGC TAnACGCA CT CATATATACTTTTCGTATTTAAAAACAAAaACCAATCATATTAAACTAGCAAA

anAATATCnTnTAAAACCAATTGAaCTTAATAAAAATAATTTTCAT=TTATAAAAATnTT=TCAACTAAACCTTGCTC.AGnATr.ATTATCACCaCTTC,

TATAAAAATCTTaAAATATTTTTAaAAAAAATAAATTTTCTAACTAATr.r.Ar.AACACTTTAATCATGAAGTTTATaATAAACTTAAACATAATGAAAAGT

TTAAGGAGCTTATAr.CTAA AAATTT TTT AATCGATr. TTAACCCAAAATTTTTAGTATAAAA


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aad GENE FROM C. ACETOBUTYLICUM ATCC 824 873

L A AK T I LD A A VK SG A P E N I1GW ID E P SI ELIT Q Y

2501 TCACGTAAATCTAGACGTAGGGTCCGAATTAAGTGAAAGACTATGATATAT

L M QK A DIIT L A G G P S L V K S A YS S G K P A I G V G P G

2601 TTTAATGCAAAAAGCAGATATAACCCTTGCAACTGGTGGTCCCTCACTAGTTAAATCTGCTTATTCTTCCGGAACCAGCAATAGGTGTTGGTCCGGGT

NITP V II D ES A I K M AV S SI I L SKIT Y D N G V I C ASKE

2701 ACCCATAATGTATTCCTTAATGATATCATTTACAACTTAATGGTTTTCTT

Q S V I V L K S I Y N K V K D F QOK R G AY I IK K N L D K V

2801 AACAATCTGTAATAGTCTTAAAATCCATATATAACAAGGTAAAAGATGAGTTCCAAGAAAGAGGAGCTTATATAAT TAAGAACGAATTGGATAAGT

R V I F K D G S V N P K I V G Q S AY T I A A M A G I1KV P K

2901 CCGTGAAGTGATTTTTAAAGATGGATCCGTAAACCCTAAAATAGTCGGACAGTCAGCTTATACTATAGCAGCTATGGCTGGCATAAAGTACCTAAACC

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

3001 ACAAGAATATTAATAGGAGAAGTTACCTCCTTAGGTGAAGAAGAACCTTTTGCCCACGAAAAACTATCTCCTGTTTTGGCTATGTATGAGGCTGACAATT

DOD A L K K A V IL I N L G G L G S G I Y A DOK I K A R D K I

3101 TTGATGATGCTTTAAAAAAAGCAGTAACTCTAATAAACTTAGGAGGCCTCGGCCATACCTCAGGAATATATGCAGATGAAATAAGCACGAGATA~AAA

D R F S S A M K T V RIT F V N I P ITS Q G AS G D L Y N F R IP P

3201 AGATAGATTTAGTAGTGCCATGAAAACCGTAAGAACCTTTGTAAATATCCCAACCTCACAAGGTGCAAGTGGAGATCTATATAATTTTAGAATACCACCT

SF1T L G C G F G GMN S V S EN V G P K L L N I1K VA EKR R E

3301 TCTTTCACGCTTGGCTGCGGATTTTGGGGAGGAAATTCTGTTTCCGAGAATGTTGGTCCAAAACATCTTTTGAATATTAAAACCGTAGCTGAAGGAGAG

N M L W F R V P K V Y F K F G C LOQ F A L K D L K D L K K K R A

3401 AACTCTGTTGGTCCTAGAATTATCGTTTCATGTTAAATAAGTTAGAAAGG

F I V D P Y N L N Y V S I IK ILK L D ID F K V F N K

V G RE A D L K T I K K EAT M S SF M P D ITII AL G G P EKM

S S A K L M W V L Y P V K F L A IK F M DI R K R I Y

3701 TGAGCTCTGCAAAGCTAATGTGGGTACTATATGAACATCCAGAAGTAAAATTTGAAGATCTTGCAATAAAATTTATGGACATAAGAAAGAGAATATATAC

F P K L G K K A M L V A I TT S A GS G S KEV P F A LV IT D N N

3801

3901

4001

T G N K Y M L A D YE M T P N M A I V D A E LM M K M P K G L T A YACTGGAAATAAGTACATGTTAGCAGATTATGAAATGACACCAAATATGGCAATTGTAGATGCAGAACTTATGATGAAAATGCCAAAGGGATTAACCGCTT

S G I DA L V N S I K AY T S V Y A S E Y T N G L A L EA I R L IATTCAGGTATAGATGCACTAGTAAATAGTATAGAAGCATACACATCCGTATATGCTTCAGAATACACAAACGGACTAGCACTAGAGGCAATACGATTAAT

F K Y L P AY K N G RITN K A RE K KMA A S T M A G M A S A

N A F L G L C S M A I1K L S SKE N NI PS G I AN A L L IKEE V I

4201 AATGCATTTCTAGGTCTATGTCATTCCATGGCAATAAAATTAAGTTCAGAACACAATATTCCTAGTGGCATTGCCAATGCATTACTAATAGAAGAAGTAA

K F N A V D N P V K Q A P C P Q Y K Y P NIl F RY A R I A D YI4301 TAATACCGTAATCGAACACCTGCAATTATTCACCAATAAAGTGATCGTAA

K L GG NTID E K VD L L I NK INK L K KA LN I PITSI K D

4401

4501

A G V L EE N F Y SS L D R I SE L A L D D Q C T G A N P R F P LITGCAGGTGTTTTGGAGGAAAACTTCTATTCCTCCCTTGATAGAATATCTGAACTTGCACTAGATGATCAATGCACAGGCGCTAATCCTAGATTTCCTCTTA

SKEI K M Y I N F V L K N N L K P S Y F N Y F L *

4601 CAAGTGAGATAAAAGAAATGTATATAAATTTTGTTTTAAAAAACAACCTTAAACCTTCATATTTCAACTACTTTITATAATTTTAATAAAGAATTTAAAA

ctffA

4701

FIG. 1-Continued.

ments with a Beckman DU-65 spectrophotometer (Beckman

Instruments, Fullerton, Calif.).DNA isolation, transformation, and manipulation. Isolation

of C. acetobutylicum chromosomal DNA was performed as

described previously (32). Plasmid isolation from E. coli was

done by the method of Birnboim and Doly (2), with additional

steps of the procedure of Wu and Welker (59) when the DNA

was to be sequenced. Previously published methods were used

for electrotransformation of coli (11) and C. acetobutylicum

(30, 32), using a Bio-Rad Gene Pulser (Bio-Rad, Hercules,

Calif.). Restriction endonucleases and T4 DNA ligase were

purchased from New England Biolabs and used according to

the manufacturers' specifications.

Southern blots. Appropriately digested DNA was trans-

ferred from agarose gels to nitrocellulose filters (Schleicher &

Schuell, Inc., Keene, N.H.) as previously described (47). DNA

probes were labeled with [cx-32p]dATP (Du Pont Co., NEN

Research Products, Wilmington, Del.), using the a nick trans-

lation system (Bethesda Research Laboratories [BRL], Life

Technologies, Inc., Gaithersburg, Md.). Unincorporated radio-

nucleotides were removed by using Select-D, G-25 spin col-

umns (5 Prime->3 Prime, Inc., Boulder, Colo.).DNA sequencing. Both DNA strands were sequenced by the

dideoxy-chain termination method (42), using synthetic oligo-nucleotide (20-mer) primers. Double-stranded plasmid DNA

was prepared for use as a template, and sequencing reactions

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874 NAIR ET AL. J. BACrERIOL.

x Z, -94 ~~~~z C.)Zp~ ~~l iIe8 Zu3 ZZ fi 88

Lii F Ai1iN 1I I aadadc

0 1.0 2.0 3.0 4.0 5.0 6.0 fiagmentFIG. 2. Transcriptional organization ofaad and the acetone formation genes. ctfA and cB encode CoA-transferase, and adc encodes acetoacetate

decarboxylase. The restriction map of the 6.7-kb SalI-XbaI fragment of C. acetobutylicum DNA in pHXS5 containing the genes is shown. Theorientation of transcription of aad, ctfA, and ctfB is indicated by arrowed boxes. Intergenic spans are 663 bp between ORFi and aad, 32 bp betweenaad and ctfA, 27 bp between ctfA and ctfB, and 65 bp between ctfB and adc. Thin baselines indicate regions where the DNA has been sequenced. Thickbaselines indicate regions which have not been sequenced, and consequently, restriction sites within these regions were physically mapped.

were performed by using the Sequenase version 2.0 kit (U.S. studies was isolated from cells that had been producingBiochemical Corp., Cleveland, Ohio) as specified by the man- butanol for 5 h, at which point the butanol concentration wasufacturer. 30% of the final culture concentration.RNA isolation. Total RNA was isolated from C. acetobuty- Primer extension. Primer extension reactions were per-

licum as previously described (52). RNA for primer extension formed as previously described (12), using Moloney murine

aad 1 K T K E LD E K L K V I E K K F S C Y S M E NFRID A R I E L A K A LadhE 1 iA NVA ELN A L V E R V KK R E Y A S F T Q K| F A AL AD A R I P L A K M A

aad 56 TMGLV E D K V I KN H F A|G|E Y I Y N|KY K D E K T C G I I E R N E PYG I T K|IA E P I G|V V A AadhE 56 JS M G|IV E D K V I KN H F A|S|E Y I Y N A Y K D E K T C G|V L S E D D T F T I T IA E P I GII I C G

aad 111 I V T N P T S T T I F KS L I S L K T R N G F F S P H P R A K K STI L K T I D V K S E NadhE 111 V T T N P T S T A I F KS L I S L K T R N A IIF S P H P R A K|D ATN K D I V AII A A| D

aad 166 I G W I D E I TQ Y Q K A T L A T G G PIS L S A Y S S G K P A IG V GIP|G N T P V|IadhE 166 LII G W I D QPV SN A H H P N L I|L A T G G P|G M KAA Y S S G K P A I G V GG N T P VIV

aad 219 rI S H M S I I L K D N G V I C A S E Q S V L K I N K K D E Q E R A I I KadhE 221 TD I KIRIA V L M F D N G V I C A S E Q S V V VV D V D A R E R A T H G L L Q

aad 274 K N D K R E F D S V[P K|I V G Q1SAiYTI|A M|;I K K TRK I L I G E V T S L GW E[adhE 276 G K K A Q D L N A L A A I V G Q PA Y K EL FS E NUK I L IG E V T V V D S

aad 329 P F A H E K L S P VL A MY EAD NFDD L KK V TI N LGL G H T S GIYAD EI K A R D K I DRIadhE 331 |P F A H E K L S P|TL A MY RLK DJED AV E| AE KV A M| G|IIG H T SIC LWT JQD N Q P A R V S Y

aad 384 S S A V T F VRIRTT |A S|G D L Y N FR IP FT L G C G|F|W G G N SIVISE N V G P K HLK LadhE 386 G Q K|M K T A I L I T A G I!G D L Y N FIK L APL T L G C GISIW G G N SIISE N V G P K H LIN

aad 439 I[K T V A E[RE N M L WF RVfH K VI K FfCIQ F K D L K D L K K F|I VT D|S D P Y L NadhE 441 K K T V A K A E N M L W|H K L K S I R R S P I D E V I T D G HK I VT D|R F LFJN GS

aad 494 VfSfI K IfE H L D ID F K NKfG R E A D K T I KiA TIE M S MIT| IIA L G G|T P E1SadhE 496 AIQWT S VUK A A G V E T E F EE A D P TS I VRL GAA L ANL K VL II A L G GiG SPP D

aad 549 S L L Y E H P EV K D I K|F M D I R K R I Y|T P K L K M L V I S A S G S E VadhE 551 A I M Y E H P EIT HFEE L R F M D I R K R I YK F P K M V K M I V TT S TG S E V|

aad 604 T P F AILV NNN1 N ML A D Y|E M1N M A I V DA1EM KFMK7 G TT YSfIWL V N S I1adhE 606 TPF AVV T DD A QKYPL A D Y A LT PDM A I V D ANLVMDM PKSCF GGLDAV T H A M

aad 659 T S Y T N L E I R I F K| L E AK NR TE K| K M A HS M M iSadhE 661 V L F S D Q LQL K L K E| A SH ES K P V R R V H S AI I dF

aad 714 |N A F L G L C H S M A I S E H N S IA N A L L I|E E K F| N V A P C P K Y NadhE 716 |N A F L G V C H S M A HKG 0 F H H L A N A L L IC N R Y N T T A F SQ D R Q

aad 769 T I F Y A R A DY I K G G N TD- - E E V D L I N K I H E K A N IT K D A G V L E NadhE 771 A R R R Y A E AH L GS A PGAR T AAAI E K L A W L E TLIAEJ GI KI REE A G QL A D

aad 822 Y S S L R I L L D D Q C T G A N P R F T I E M Y I N F V L K N N L K P S Y F N Y F LadhE 826 L A N V K L D F D D Q C T G A N P R Y I L Q I L L D T Y Y G R D Y V E G E T A A K K E A A P

FIG. 3. Amino acid alignment of aad and adhE gene products. The two proteins share a very high identity of 56% (75% similarity). Boxedpositions indicate strict conservation. Abbreviations: aad, C. acetobutylicum AAD (this study); adhE, E. coli ADH/ACDH/PFL deactivase (13, 21).


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TABLE 1. ALDHs with high identities to AAD

Organism and protein Identity Size Reference(% (aa)

Mus musculus cytosolic ALDH 23 500 40(AHD-2)

Vibrio cholerae ALDH 23 506 37Aspergillus nidulans ALDH 22 497 39Escherichia coli ALDH 22 495 17E. coli succinate semialdehyde 22 482 34

dehydrogenaseE. coli lactaldehyde 21 478 18

dehydrogenaseBos taurus mitochondrial 20 520 15ALDH

E. coli betaine ALDH 20 491 3Pisum sativum ALDH-related 20 508 16

turgor-responsive proteinPseudomonas oleovorans ALDH 17 483 23

leukemia virus reverse transcriptase (U.S. Biochemical Corp.).Two different end-labeled oligonucleotides, PE-1 (5'-TT'1'ACTGTTGTGACTITTCAT-3') and PE-2 (5'-TTACCTTGAGT'1TTI1'CATCT-3'), complementary to the N-terminal re-gion of aad were used in separate primer extension reactions.To map the exact transcriptional start sites, sequencing reac-tions were performed on the corresponding DNA by using thesame primers that were used for the primer extension reac-tions.PCR. DNA fragments were amplified by PCR in a reaction

mixture which included 300 ,uM each dATP and dTTP, 200,M each dGTP and dCTP, 4 mM MgSO4, 0.01 ,g of eachprimer per RI, 100 ,ug of nonacetylated bovine serum albuminper ml, template DNA, and 0.05 U of Vent DNA polymerase(New England Biolabs) per RI1. A total of 15 cycles wereexecuted in a DNA Thermal Cycler (Perkin-Elmer Cetus,Norwalk, Conn.), with each cycle consisting of annealing at40°C for 1 min, extension at 72°C for 3.5 min, and denaturationat 94°C for 1 min.

Construction of plasmid pCADEX1. aad, together with itstwo putative promoters, was amplified by PCR using plasmidpHXS5 DNA (which contains aad; see Results) as a template.The upstream primer, 5'-AAATATACTGAGAAHCCTAAATATATA-3' was generated by substituting a G for a T atnucleotide position 1692 (Fig. 1) to provide an internal EcoRIsite (underlined). The downstream primer, 5'-AATCTAATTATTCITAGGTTCAT1'TAA-3', resulted from a substitu-tion of a C for a T at nucleotide position 4723 (Fig. 1) on thecomplementary strand to provide an internal XbaI site (under-lined). The amplified product was sequentially digested withEcoRI and XbaI and ligated into the similarly digested vectorpT7/T3a-18 to yield plasmid pCADEX1 (see Fig. 7). Theprecise orientation of aad with respect to the T7 promoter wasverified by sequencing the region upstream of aad in theconstruct pCADEX1 by using primer PE-1.

Construction of plasmids pCAAD and pCCL. AccI-digestedpCADEX1 was ligated to CiaI-digested pIMl3 (28) to yieldthe 8.1-kb plasmid pCAAD (see Fig. 8a). The Bacillus subtilisplasmid pIMl3 provides MLS' and a gram-positive origin ofreplication (28). Plasmid pCAAD was digested sequentiallywith BamHI (position 2923; Fig. 1) and BglII (position 4434;Fig. 1) and religated to generate the control plasmid pCCL(see Fig. 8b), which contains a truncated aad (58% of thestructural gene deleted).T7 expression. For T7 expression studies, aad was inserted

TABLE 2. ADHs with high identities to AAD

Organism and protein Identity (%) Size (aa) Reference

Clostridium acetobutylicum 42 388 62P262 ADH1

Zymomonas mobilis ADH2 34 382 8Bacillus methanolicus methanol 33 382 10

dehydrogenaseSaccharomyces cerevisiae ADH4 32 382 57Escherichia coli 1,2-propanediol 30 383 5

oxidoreductaseClostridium acetobutylicum 25 390 52ATCC 824 BDH I

C. acetobutylicum ATCC 824 22 391 52BDH II

into the vector pT7/T3a-18 (BRL). For assay experiments, 500ml of E. coli BL21(DE3) cultures were grown to an OD600 of-0.7. The T7 RNA polymerase under the control of theisopropylthiogalactopyranoside (IPTG)-inducible lacUV5 pro-moter was then induced for 2 h with 0.4 mM IPTG (49) at 37°Cunder either aerobic or anaerobic (Forma Scientific AnaerobicSystem, Forma Scientific, Inc., Marietta, Ohio) conditions withanother addition of 200 ,ug of ampicillin per ml. For therifampin studies, rifampin (200 pg/ml; Sigma Chemical Co., St.Louis, Mo.) was added to cultures 30 min after the start of theinduction. The cells were harvested by centrifugation at 5,000x g for 5 min at 4°C. For [35S]Met (Du Pont) labelingexperiments, BL21(DE3) cells were grown to an OD60 of 0.4and then subjected to IPTG induction for 20 min and a further30-min incubation subsequent to rifampin addition.Enzyme assays. Cells from 500 ml of C. acetobutylicum

culture were harvested, and the pellets were stored at - 850Cuntil used (always within 3 weeks). Cells were suspended in 15mM potassium phosphate buffer (pH 7.0) containing 1 mMdithiothreitol and 0.1 mM ZnSO4 (l g of frozen cells per 5 mlof cell lysis buffer). The cell suspension was sonicated on icewith an ultrasonic cell disruptor (model W-225R; Heat Sys-tems-Ultrasonics, Farmingdale, N.Y.) at level 10 for 18 minwith a pulse setting of 90%. The cell debris was removed bycentrifugation at 35,000 x g for 20 min at 40C. Crude extractswere prepared from E. coli in a similar manner, with thefollowing modifications: the cell lysis buffer contained 1 mg oflysozyme per ml, cells were incubated for 1 h on ice in cell lysisbuffer prior to sonication, and cells were sonicated at level 5 for5 min at a pulse setting of 90%. Butyraldehyde dehydrogenase(BYDH) and acetaldehyde dehydrogenase (ACDH) assayswere performed in the reverse physiological direction aspreviously described (19). One unit is defined as the amount ofenzyme necessary to reduce 1 ,mol of NAD(P) per min.Butanol dehydrogenase (BDH) assays were performed in thephysiological direction as previously described (54), with bu-tyraldehyde at a final assay concentration of 25 mM. Anidentical procedure was adopted for ethanol dehydrogenase(EDH) assays, with acetaldehyde at a final assay concentrationof 50 mM. One unit is defined as the amount of enzymenecessary to oxidize 1 ,umol of NAD(P)H per min.

[35S]methionine labeling. Five milliliters of E. coli cellsamples was withdrawn prior to induction, at the end of theinduction period, and after incubation with rifampin addition.Five milliliters of C. acetobutylicum cultures was withdrawn atthe early exponential (OD60 of about 0.6) and late exponen-tial (onset of solventogenesis) stages of growth. These sampleswere then transferred to prewarmed tubes containing [35S]Met(final concentrations of 50 ,Ci/ml for E. coli cultures and 100

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aad 1 M KV TT V KEL----------DE K -L KV IKERKK F SC YSEMV D - --E - I FRN AadhE 1 MAVTNVAEL----------N A L -VERV KKIAIQR E YASF TQE QVD- --K- - -I FRAA1-aid 38 IPD GQAEDA----------RKA - IDAA E RIAIQPE WEEALPAIPRAERA- -S-s- - WLRKEIvibch 49 -RVARS S sQ ---------- D VE -L-ALDAjH NALE SW ST T SAV- --E-----RS Naspni 23 IN NE FV KGV---------- EG K TFV INP SN E KV IT SV HEATE KDVD- - -VAVAAaid 55 KSV DI D RAM ---------- SAA - RGV FE RGD W SL S- -SP AKR K- - -A- - -VLNK Ls-aid 38 -KL G SV P KMGA--------DE T -RA A IDAfN RAL PAWRAL TA K- - -E RAT IL RN Wbovin 7i VAE GDEKADV---------- D RA -VEKA A RAIAIFQLG SP WR RMDAS- - -E R GR LL NR L

aad 39 AMAAI DA---------------R ------------------------ IE- K AadhE 39 ALAAA DA---------------R ------------------------ IP KMi-aid 76 5- AG I RE.---------------R.------------------------AS--EIS Avibch 83 IL LRIAD.---------------R.------------------------I E SNLET Laspni 65 ARAA F EG--------------- P WRQ VTP SER G IL IN KLADLME RD IDT--LAAI

s-aid EH.---------------Q.------------------------D --LAbovin ii2 ADL IERD---------------R------------------------T Y--LAALmouse 64 VK AA RQA F QIG SPW RTMDA SE R G --------------E-------KL

aad 53 A--------VQETfM GLVE DjV-IEKNHFAG-EY-------- I--YNK Y KDE - --

adhE 53 A- . ----VA ESIGIM G IV E D V IKNH FAS--EY.--------I-- YNA Y KDE -- -

i-aid 89 L--------I V EEGJGEKI Q QLAE VEVA FTA-DY-------- ID YMAEWAR R- --

vibch 99 A- .----IV E -- - SWDElNG 7P-IRETL AA-DL PL TID H FR Y- - FAACIRS -- -

aspni 103 E--------LD N GKAFTMA V DLAN SI GC-LR -------- Y-- YAGWADK- --

aid 105 E--------TLDT G KP IR H S- --L RDD IPG--AA-------- R- -A IRWYAEAI Ds-aid 94 M. LEQGKPLAE Ali]G-EISYAA SF IE W .--------F - -AEEGKRI - --

bovin 126 E--------TD NGK P YII SY L-VDLDMVL- -KC--------LR Y YA GWADK -- -

mouse 9ADLMERDRLLATMEAL NGGDV -FANAYLS- - D LGG CI KAL KY- - CAGWADK -- -

aad 84 KT C GI IE RNEP -Y G ITKI A - FIGVVA AI IVTN PT ST T I FKSLI SLKTRNGIF FadhE 84 KT CGV L SEDDT- F GT IT IA-jIGIIC G I VPT TNPT STAIF KS L I SLKTRNAII Fi-aid 122 Y EG EII Q SD RP GE N ILL FK - RALGVTTG I LP WN F P FFL IAR KMAPALLTGNTIfIvibch 135 QE GAA SE L DSR- TL T Y HL P-[EPI GVVGQI I PWN F PL LMAA W KLA PALLAAAGTVIVIL

mouse 140 IH G QTI P SDGD- IF T YT R R - IG V,C GQLIIP WNFP]MLM F I WEIGPASCGTV jV

aad 137 S PH P RA KEKS TI LAA K T I L D AA V K S GA P E N I1IGW I DE P S I E L- T - --Q Y - L - M Q9EAadhE 137 SPH P RAKD ATNEKAIAID IV L QWAIAAGA P KDL IG W IDQ P SV EILI- S- - -NA-L-MHH Pi-aid 176 KP S E F TP N N0I A F A K I V D ElI G L PR G VF N L V L G R G ET V G Q EL - AG N P K V - A-M V SM

aspni 188 KTAQ Q TPIL SRL YARKELIEEfEPI PIA GV IINIVI SG FIGIRT AIGIAAI - S-- - SH -MDID j~V

bovin 212 KVA E Q TPL TIAIL YVIAIN L I KE[jG FjP jGVVNV IVGFPGGT AGAA I -A -- - SH -EDV D

JV

sad 186 DI - -0ELAT-G-GP SLVKSY S SG -- - -EKPA IGVGPGNTP------VOI D E SAHIEKadhE 186 DIN L ILA T-G-GPG MVEKAIAIY S SG--- -EKPAIGVGAGNTP ------V VIDETADIKi-aid 228 TG--SVA -G-EEKI MA TAIAIK N IT ---EK--EVCLELGG A P .------AV MDDAL E

vibch 237 AFl- -TG ST-EIGNH ILEKCIAIADN L ---I P S-TIE LG GK S PNIY F PDI F SH EDQYL Daspni 238

AF--TGSTLV KSN--

- - -NZV FDDADI Daid 241 AF- -T G ST RT-GGEQL LDADUG D SN ---MKR1V1VW L EA GGEKSAN ----- IVFADCPDL Qs-aid 230 SF--TGST-E -IKKVSLELGG A ------F V F D DADL Dbovin 262 AF--TGST-E L I M SDADM Dmouse 243 AU- - T G ST-Q-VG KL IKE AAG K SNLK R - -V:L L G G KSP------CIVFAD I

aad 227 MAVjS SI I LSEKT YDNGVICA SEQ SV IVlLKSI Y NK -- - -V KD EFQ E RG---------adhE 229 R A V A S V L M SEKT F D N G V I C A S EQ S V V V V D SV Y D A - - - - V R E R F A T H G.-----i-aid 268 LAVJK A IVDSRV IRSGQV CN CA E iVYIV KGIY D Q -- - -FVNRL GE AM---------vibch 285 KC IEG AL LA -fFINIQ GEV CTCP SIR IILIVHEIHEYE K -- - -F IAEKI IE RV.-----aspni 281 NRI SW A N FGIFIFINIHGCCC A G SIRIILQE GI YDEK-- - -F VA RF KEERA---------aid 285 QAA SAT A A GIFIYINIGQGVQVCIIAGTIRILLLLEEEIADDELLA L L KQQAQN WQ PG H PLDPA Ts-aid 272 KIAVIEG A L A SEKFIRINNAGQTVCVcANIRRILYHGDGVR -- - -FAE KL QQ AV---------bovin 305 WIA VIEQ0A H FALIFIFOGQCC CCAG SRjTFVQEDyA E- -- - FVER SVA RA---------mouse 286 IA V E FAH HG V Y H QG QCCV AA SRIFE ES------D E FVEKR S .-----

FIG. 4. Amino acid alignment of nine ALDHs. Boxed positions indicate conservation in at least six of the nine proteins. Abbreviations: aad,C. acetobutylicum AAD (this study); adhE, E. coli ADH/ACDH/PFL deactivase (13, 21); l-ald, E. coli lactaldehyde dehydrogenase (18); vibch,Vibrio cholerae ALDH (37); aspni, Aspergillus nidulans ALDH (39); ald, E. coli ALDH (17); s-ald, E. coli succinate semialdehyde dehydrogenase(34); bovin, Bos taurus mitochondrial ALDH (15); mouse, Mus musculus cytosolic ALDH (40).


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aad 269 - - A Y I I K K N E L D - - - - - - - K V R E V - - - - - - - - - - - - - - - - - I F K D G S V N P K----adhE 271 - - G Y L L Q K E L K - - - - - - - A V Q D V - - - - - - - - - - - - - - - - - I L K N G A L N A A----i-ald 310 - - Q A V G F G N P A E - - - - - - - R - - - - - - - - - - - - - - - - - - - - - - - N D I A M G P L----vibch 326 - - A L I K Q G N P L D - - - - - - - T E T QI G A Q V S K E Q Y D K I L G Y I Q I G K D E G A E L Iaspni 323 - - Q K N K V N P F E Q D T F Q G P Q V S QL - - - - - - - - - - - - - - - - - Q F D R I M E Y I N----ald 340 T M G T L I D C A H A D - - - - - - - S V H S F - - - - - - - - - - - - - - - - - I R E G E S K G Q L-s-ald 314 - - S K L H I D G L D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N G V T I G P L

bovin 347 - - K S R V V N P F D S R T E Q G P Q V D E T - - - - - - - - - - - - - - - - - Q F K K V L G Y I K

mouse 324 - - V E R A K K Y V L G - - - - - - - N P L T P - - - - - - - - - - - - - - - - - G I N Q G P Q I D K E Q H D

aad 294 - - - - I V G QS A Y T I A A M A G I K V P K T T R I L I G E V T S L G - - - - - - - - - - - - - - - E E E P

adhE 296 - - - - I V G QP A Y K I A E L A G F S V P E N T K I L I G E V T V V D - - - - - - - - - - - - - - - E S E P

1-ald 329 - - - - I N A A A L E R V E Q K V A R A V E E G A R V A F G G K A V E G K G Y Y Y P P T L L L D V RQ E M S Ivibch 368 - - - - F G G H P N N Q E N Y L S G G Y Y I K P T L F F - - - - - G H N - - - - - - - - - - - - - - - Q M H I

aspni 355 - - - - H G K K A G A T V A T G G D R H G N E G Y F I Q P T V F T D V T - - - - - - - - - - - - - - - S D M K

ald 367 - - - - L L D G R N A G L A A A I G - - - - P T I F V D V D P N A S L S - - - - - - - - - - - - - - - R E E I

s-ald 332 - - - - I D E K A V A K V E E H I A D A L E K G A R V V C G G K - - - - - - - - - - - - - - - - - - - - - - -

bovin 379 - - - - S G K E E G L K L L C G G G A A A D R G Y F I Q P T V F G D L Q - - - - - - - - - - - - - - - D G M T

mouse 353 K I L D L I E S G K K E G A K L E C G G G R W G N K G F F V Q P T V F S N V T - - - - - - - - - - - - D E M R

aad 330 F H K - L S P L A M Y E A D N F D D A L K K V T L I i.L G G L H T S G I Y A D E I K A R D K I D R FadhE 332 F H E K - L S P L A M Y R A K D F E D A V E K A E K L V A M G G HH T S C L Y D Q D N Q P A R V S Y Fi-ald 380 M H E E T - F G P V L P V V A F D T L E D - - - - A I S M A D S D LT S S I Y T Q N L N V A M K A I K -

vibch 399 F Q E E I - F G P V I A I - - - T KF K D E I - EWL H L A N D T V Y G L G A G V W T R D I - - - N I A H R Maspni 391 II Q E I F G P V V T I Q K F K D V A E A I K - - - - I G N S T D Y G L A A A V H T K N V - - - N T A I R V

ald 399 F G - - - - - - VL V V T R F T S E EQ A L - - - - L A N D S FY G L G A A V WLAR D L - - - S R A H R M

s-ald 360 - H R - - - - - - - - - G G N F F QP T I L V D V P A N A K V S K EAATF G P L A P L F R F K D E A D V I

bovin 415 I A K E E I F G M QI L K F K S M E E V V G R - - - - N N S | A A A V F K D L - - - D K A N Y Lmouse 396 I A K E E I F G QQI M K F K S V D D V I K R - - - -N N T T GLA A G L F K D L - - - D KAI T V

aad 384 S S A M K T V R T F V NI P T S Q G A S G D L Y N F R I P P S F T L G C G F W G G N S V S E N V G P K H L L NadhE 386 G Q K M K T A R I L I N T P A S Q G G I G D L Y N F K L A P S L T L G C G S W G G N S I S E N V G P K H L I N

i-ald 429 - - G L K F GE T Y I N R E N F E A M Q G - - - - - - - - - - - - F H A G W R K S G I G G - - - - - - - - - -

vibch 446 A K N I K A G R V W V N C Y H A Y P A H A A F G G Y K K S - - - - - - - - - - G I G R E T H K L T L S H Y Q N

aspni 439 S N A L K A G T V W I N - - - - - - - - - - - - N Y N M - I S Y Q A P F G G F K Q S G L G R E L G S Y ALENald 441 S R R LK A GS V F V N - - - - N Y N D G D M - - - - - - - - - - - - T V P F G G Y K Q S G N G R D K S L H As-ald 405 A A N D T E F G L A Y F Y A R D L S R V - - - F R V G E A L E Y G I V G I N T G I I S N E V A P --F G Gbovin 463 SQ A L QA T V W V C Y D V F G A QS P F G G Y K L S G S - - - - - - - - - G R E L G E - Y G L QA Y T E

mouse 444 S S A L QA V V W V C Y I M L S A QC P F G G F K M S G - - - - - - - - - N G R E L G E H - G L Y E Y T E

aad 439 - - - I T A E R R E N M L W F R V P H K V Y F K F G C L Q F A L K D L K D L K K K R A F I V T D S D P Y N

adhE 441 - - - K T A K R A E N M L W H K L P K S I Y F R R G S L P I A L D E V I T D G H K R A L I V T D R F L F Ni-ald 460 - - - - - - - A D G K H G L H E Y L Q T Q V V Y L Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

vibch 491 - - - I N ---- - - L I S H E I H P L G L F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

aspni 481 Y T Q I T H Y R L G D A L F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

ald 480 - - - L E K F T E L K T I W I S L E A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

s-ald 455 - - - I K A S G L G R E G S - K Y G I E D Y L E I K Y M C I G L - - - - - - - - - - - - - - - - - - - - - - -

bovin 508 - - - VKTIT - - - - - - - - V R V P QK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

mouse 489 - - - L T A - - - - - - - - M K I S QK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

FIG. 4-Continued.

,uCi/ml for C. acetobutylicum cultures) and mixed. Labeling byincubation at 37°C for 10 min was followed by a chase ofunlabeled Met (300 ,ug/ml), and the tubes were then chilled onice. The cells were washed thrice with 1.5 ml of cold buffer (50mM Tris-HCl [pH 8.01-2 mM EDTA for E. coli samples and 25mM Tris [pH 5.5] for C. acetobutylicum samples), and cellpellets were stored at - 85°C until used.

SDS-polyacrylamide gel electrophoresis (PAGE). Cells from[35S]Met experiments were suspended in sample buffer (41)and placed in a 100°C water bath for 5 min. Cell debris wasspun out twice, and an aliquot of the resulting supernatant wassubjected to liquid scintillation counting (Model 1900CA Tri-Carb liquid scintillation analyzer; Packard Instrument Co.,Meriden, Conn.). Samples were loaded at 500,000 cpm perlane in a sodium dodecyl sulfate (SDS)-8% polyacrylamide gel(25) alongside 14C-labeled high-molecular-weight protein stan-dards (BRL). Upon electrophoresis, gels were dried on What-man 3MM paper, and imaging plates (exposed for at least 24h) were scanned in a model 400S Phosphorlmager (Molecular

Dynamics, Sunnyvale, Calif.). C. acetobutylicum samples frombioreactor runs (early exponential, late exponential, and sta-tionary stages of growth) were suspended in sample buffer,boiled, and loaded as described above. High-range (BRL) andmid-range (Promega) protein molecular weight markers wereloaded alongside. Gels were stained with 0.25% (wt/vot)Coomassie brilliant blue R-250 (Sigma).

Analysis of fermentation products. The concentrations ofbutanol, acetone, ethanol, butyrate, and acetate were deter-mined by gas chromatography as described previously (29).Computer programs. Sequence comparisons and homology

searches of the GenBank (release 75.0, February 1993) andSwissProt (release 24.0, January 1993) data bases were done byusing the Wisconsin Genetics Computer Group (9) sequenceanalysis software package (version 7.2). The programs usedincluded FastA, TFastA, BestFit, StemLoop, Fold, Termina-tor, Composition, PeptideStructure, and PlotStructure. Phos-phorlmager scans were performed by using ImageQuant ver-sion 3.15 (Molecular Dynamics).

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406 L Y N F R I P P S F T L G C G F W G G N S V S E N V G P K H L L N I K T V A E R R E N M L W F - - R V H K V408 L Y N F K L A P S L T L G C G S W G G N S I S E N V G P K H L I N K K T V A K R A E N M L W H - - K L P K S I

1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M L S F D Y S I P T K V6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y - - S I P T R I1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M M R F - - T LPL R D I1 M T N F F I P P A S V I G R G - - - - - - - - - - - - - - - - - A V K E V G T R - - - - - - - - - - - - - - -

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

5 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R M - - I L N E T A1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M S S V T G F - - Y IEP I S

45946113131124171314

Y F K F G C L Q F A L K D L0D L K K T S D P Y N - L N Y V - D S I I K I LE H L D I D F K V F

Y F R R G S L P I A L D E V I T D G H RL IV TR F L F N - NfEjY A - D Q I T S V L K A AIV E T E V FF F G K G K I D V I G E E I K Y - G S R VL IV Y G G G S I K - R N G I Y D R A T A I L K E N N I A F Y E LF F G K D K I N V L G R E L K K Y G S KV L I V Y G G G S I K R - N G I Y - D K A V S I L E K N S I K F Y E LY Y G KjS L E Q L K N L K G K K L G G G S M K R F G F V D K V L G Y K E AG I E V K L I

- - - - - - - - - L K Q I G A K K A L I V T DA F L H S - T G L S - E E V A K N I R E A G L D V A I F

GS L E K A I K D L N G S G F IKN A L I V S D A F M N K - S G V V - K Q V A D LQK A Q G I N S A V Y

W FG R G A V G A L T D E VK R R G Y QK A L I V T D K T L V Q - C G V V - A K V T D K M D A A G L A W A I YF E TAGDLY IUI YIK ALI T PG I A A- I ULS- G RV QK M E ER L N V AIY

512 N K V G R E A D L K T I K K A T E E M S S F M P D T I I A L514 F E V E A D T L S I V R K A E L A N S F K P D V I I A L66 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

66 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 I61 E G VE P D P S V E T V F K G A E L M R Q F EPGF W I I AM64 P K A Q P D P A D T Q V H E G V D V F K Q E N C D A L V S I

66 D G 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 L66 D G V P N P T I T V V K E G L G V F Q N S GALEN Y LIE I

67 D K T Q P N PN I A N VT A GL K V L K E E N S E I V V S I

PE M S SAKG G G S P MfDA A K

G G G S A I D CS KG G G S A I D C A KG G G S P I D A A KG G G S S H D T A KG G G S P H D C A KG G G S P Q D T KGGGSAAHDNAK

- L M W V L Y E H P E - - V K- I M W V M Y E H P E - - T H- V I - - - - - A A G - - V Y- V I A A A C E Y D G - - - -

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

- A I A L L A T N G G E I G D

F E D L A I K F M D I R K R I Y T F P K L G K K A M L S G S E

- F E E L A L R F M D I R K R I Y K F P K M G V K A K - M - - IAV T TTS T G S E- Y D G - - - - - - D T W D M V K D P S K I T K V L P - I - - A SIL T LST G S E- - - - - - - - - - N P W D I V L D G S K I K R V L P - I - - A SI L A T G S E- F D D I - - - - - - - - K D P F T V P E L R N K A K - F - -LAI P STSG T A SE- N D Y Q G V N - - S V E K P V V P - - - - - - - - - - V - - VL A I T TT A G T G| ED Y E G I D - - - - - - - - - - - - - - K S K K P A L P L - - M I N T T A G T A SE- - - - - - - - F A D V R S L E G L S P T N K P S V P - I - - L AI P T T A G T A. E- Y E G V - - - - - - - - - - - - - - - N Q S K K A A - L P L F A IN T T A G T A| E

V TP F A L V TDN N TV P F A T D D A TM D Q I A V I S N M E TMD TWAVI NNMD TV fA F S V I T D Y K TT T S L A V I T D S A RM T R F C I T D E V RV T I N Y I T D E E K

M R F T I SNEE K

Y M L AY E MTY P L A Y A L T PL G V G H D DM R PLI AAH P DMAPY P L A D F N IT P

M P V I D E K I T PM A I V D R H V T PF V C V D P H P

MA IIDN NV P

N M I VD MWI VK FS V LKF S I LD V V VT VWI VM V S V NQ V F IA V V N

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AELMMKMANLVMDMP T Y T F T VP T Y T Y T VS E L A E T MP E L M V K KP L L M V G MAD MMD GMP S T M F G L

KGLT AY Sf I

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K N Q T AA G T A

T N QT A A G T AP K L T A H T GMA G L TE T G MK G L T A A T G MPALTAATfMLP A LiA A T IG V

PA L T A A T GL

DALIVNS IDAT H A MD IM S H T FD I M S H I FD A L T H A ID A L S H A ID A L T H A FD A L T H A ID A L T HC I

E A YE A YE YELJYE A YE A YE A YE YE A Y

T SV Y A SEYVS V L A S E F SF SG V E G A Y VFWNTKTAY LV A T L H S P F TV A K G A T P V TSVAKGATPVITIS0T A A T P1 ITII T R G A W A L TV0T A SN PI

aad 670 N G - L L E AI- R L]F KY[P E[Y K N G R T NE K-A R K9A H A S T M M S A N A F GL - -

adhE 672 [G -QWLQ A L - K L L K E YWJP A S Y H E G S K N P V - A R ARV H S A A T I AGI[ A N A F L GV - -

bdhA 213 Q D - G I R E A I L R T C I K Y G K I M E K T D D Y E - - A R N L M W A S S L A - - - I N G L L SL G K

bdhB 213 Q D R M E A L L - R T C I K Y G G I L E K P D D Y E - - A R A N L M W A S S L A - - - I N G L L T Y G K

adhl 211 P - L A M Q I - E M I N E H - - - F K S Y E G D K E - A R Q H Y A Q C L M A F S N A L L I - -

mdh 211 D A - F A I Q A M - K L I N E Y L P K RV A N G E D I E - - A R E A M A Y A Q Y M A G V A F N N G G L G L - -

zymeso 213 D A -CtAL K A A - S M I A K N L K T A|C D N G K - D M P - A R E AMAYAQ F L A G M A F N N A S L G y _ _fucO 215 D A - L HI K A I - E I I - - -_ A G A L R G S V A G D K D A E E M A L G Q Y V A G M F S N V G L G L - -

yeast 214 UA - C'L K G I - D LI N E SIT1V A Y K D G K D K K-AT D M'C Y ['E Y L A G M N A S L G y - -

aad 720 - - - - C I K S S EH N I I A L I E E I K FA V D N P V K Q A P C P Q Y K Y P

adhE 722 - - - - - C|H|S|M A HK L S Q F H IH GL ANALL I C NUI R Y A - - N D N P T K Q T A F S Q Y D R PD R K W S C H P M H E L A Y D IH G V G I L T N W M E Y I L - - N D D T L H K F V S - - - - Y G

bdhB 261 D T N W S V H LMWHELSA YWD I TH G V GLAIWTIPIN W M E Y I L - - N N D T V - - Y K F V E Y G

adhl 258 - - - - - C H S M A H K TGA V F H IPH GAC ANYI Y L PIYVI K FNS - - - - - - - - - - - - - - - - - K

mdh 260 - - - - - V HSIHQV GG VfK LQH G I C NSV N M|P|H|V|C A F|N|L - - I A K T E - - - - - - - - - - -

zymnmo 262 - - - - - VH AMA H QLG G Y|YIN L PH G|V CN AV LLP H V L A Y N A S V V A G R L K D V G - - - - - - -

fucO 262 - - - - - V H GMA H|P|L G|A FIYIN T|P H G|V A|N A|I|L|L|P|H|V|M R Y|N|A - - - - - - - - - - - - - - - D F Tyeast 263 - - - - - VA LAHQLGG FH LP H GV CNAVLLPHVQ E ANM - - - - - - - - - - - - - - - Q C P

FIG. 5. Amino acid alignment of nine ADHs. Boxed positions indicate conservation in at least six of the nine proteins. Abbreviations: aad, C.acetobutylicum AAD (this study); adhE, E. coli ADH/ACDH/PFL deactivase (13, 21); bdhA, C. acetobutylicum BDH I (52); bdhB, C.acetobutylicum BDH 11 (52); adhl, C. acetobutylicum ADH1 (62); mdh, Bacillus methanolicus methanol dehydrogenase (10); zymmo, Zymomonasmobilis ADH2 (8); fucO, E. coli 1,2-propanediol oxidoreductase (5); yeast, Saccharomyces cerevisiae ADH4 (57).

aadadhEbdhAbdhBadhlmdhzymmofucOyeast



aad 564adhE 566bdhA 113bdhB 116adhl 113mdh 116zymmo 119fucO 116yeast 121

aad

adhE

bdhA

bdhB

adhlmdh

zymmofucO

yeast

615 G N617 G Q158 N E158 N E156 E I156 K V158 H V160 R R159 K I

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768 N T I F - Y A R I D Y I -

770 Q A R R - Y A E I D H L -

310 I N V W G - I D K N KD N Y -

310 V N V W G - I D K E K N H Y -

291 T S L E - Y A K I XQ I -

297 - - - - - F A H I AE L L G305 - - - - - - - - - V M G L -

297 G E K Y D I A R V M G - V -

298 K A K K R- L G E Ira] - - - -

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

- - E E K V D L LQ]N K I H E lK K A LNR T A A K I E K L L A W L E T L K A EL G I P- - - - - - - --.KN T R E Y F N S L G I P- - Q K T R D Y F V N - - - - - - - V L G P- - E E L V D S L IN L V K EQN K K M QI P- - A A A A E R A I V A L E R Y N K N F G I P- - K E G A E A T Q A V R D LA A S I G I P- - E E A R N A A V E A V F A L N R D V G I P- - Q E D P E E TOK A L H VLN R T MIP

818 EQN F Y S S L D R I S E L L D G A F P L T S I K E M Y I N F V L K N N L K P S Y F N Y F L822 E A D F L A N V D K L S E D A F D G A Y P L I S E L K Q I L L D T Y Y G R D Y V E G - - - - - -

352 K D K - - - - L E L M A 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 K S Y - - - - -

353 E K L D IM A K ES V K L T G G TI G P V N A S E V L Q I F K KS V-- - - - - -

341 EQ E F K N K V D L I S E R A I G ACT G S N P R Q L N K D M K K I F E C V Y Y G T E V D - - - - - - - -

345 E D - - - -I E L LA KN A F E D V C T QS N P R V A T V Q D I A Q - - - -- I I K N A L - - - - - - - - -

347 K E D - - - - V P L LA DH A L K D A C L T N P R Q G D Q K0DV E EL F L S- - - - - - - - - - - - - - - -

347 K E D - - - - I P A L A Q A A L D D V C G G N P R E A T L E D I V E L Y - - - - - - - - - - - - - - - - - -

344 T DF - - - D I L A E H A M HDA LTNP- V F T---KEQV V A I I K K A - - - - - - - - - -

FIG. 5-Continued.

Nucleotide sequence accession number. The 4,800-bp se-quence (Fig. 1) that includes aad and the upstream ORF1 hasbeen submitted to GenBank (1) and has been assigned acces-sion number L14817.

RESULTS

Plasmid pHXS5. Previously, a bacteriophage lambdaEMBL3 library of C. acetobutylicum DNA was screened byusing both ctfA and cfB subunit probes to identify a positivephage isolate, Hi (4). A -6.7-kb SalI-XbaI DNA fragment(Fig. 2) from this phage isolate, which was later found tocontain intact ctfA and ctfB genes and a -5.0-kb regionupstream of ctfA, was cloned into the similarly digested pUC19vector to yield plasmid pHXS5. The Sall end of this fragmentcorresponds to that of the 2.0-kb SalI-BglII DNA fragment(containing both ctf genes) previously cloned on plasmidpCoAT9 (4).

Southern blotting. NcoI-digested C. acetobutylicum chromo-somal DNA was probed with nick-translated pHXS5. Theresulting Southern blot (not shown) showed that up to 3.8 kbof DNA upstream of ctfA was intact. Figure 2 shows thelocations of the NcoI sites.

Sequence analysis. Both strands of a 4,712-bp segment of C.acetobutylicum DNA upstream of ctfA were sequenced (Fig. 1)from pHXS5. Two large complete ORFs were identified onthis segment (Fig. 2). The 2,619-bp ORF immediately up-stream of ctfA was designated aad. On the basis of homologysearches of protein and DNA data bases, this ORF was foundlater to encode an aldehyde/alcohol dehydrogenase (AAD).This AAD is a 873-amino-acid (aa) protein with a calculatedmolecular mass of 96,517 Da. A putative ribosome binding site(RBS) (44) (5'-AAGAAG-3') was found 9 bp upstream of theaad start codon. The sequence and spacing of the RBS aresimilar to those of other C. acetobutylicum genes (36).The function of the second ORF (ORF1) upstream of aad is

unknown. If this were a gene, the encoded 319-aa proteinwould have a molecular mass of 36,704 Da. A putative RBS(5'-GGAAAGAG-3'), similar in sequence and spacing tothose of other C. acetobutylicum genes (36), was found 11 bpupstream of the ORFi start codon. Two inverted repeatsegments (Fig. 1) were identified in the region of DNAbetween ORF1 and aad (AG = -20.0 kcal [1 kcal = 4.184

kJ]/mol [63], positions 1399 to 1439; AG = -19.4 kcal/mol,positions 1617 to 1657).Amino acid comparison. Data base searches revealed that

AAD shares a high amino acid homology (56% identity and75% similarity) with the trifunctional 96,008-Da protein en-coded by adhE from E. coli K-12 (13, 21). Identity wasobserved over the entire spans of these proteins except for thelast 22 aa at the C-terminal end of AAD (Fig. 3).AAD also showed homology to several aldehyde dehydro-

genases (ALDHs) (Table 1) and alcohol dehydrogenases(ADHs) (Table 2). The 10 ALDHs listed in Table 1 (sixbacterial, two mammalian, one fungal, and one plant) have a17 to 23% identity (41 to 46% similarity) to AAD. Homologyof these 10 ALDHs (average size = 496 aa) is restricted towithin -460 aa residues at the N-terminal end of AAD. Theamino acid alignment of seven of these proteins which showthe best identities to AAD and the adhE gene product(ADH/ACDH) is shown in Fig. 4. There are 10 amino acidpositions that are conserved in all nine proteins and anadditional 16 positions that are conserved in eight of nineproteins.The seven ADHs, which include three clostridial ADHs (52,

62), that show homology to AAD are listed in Table 2 (sixbacterial and one yeast). Here the identities to AAD are higherthan those of the ALDHs, with the range being 22 to 42% (46to 65% similarity). Homology of the seven ADHs in Table 2(average size = 385 aa) is restricted to within -420 aa residuesof the C-terminal section of AAD. The amino acid alignmentof these seven proteins with AAD and ADH/ACDH is shownin Fig. 5. There are 19 amino acid positions that are strictlyconserved and an additional 20 positions that are conserved ineight of nine proteins.

Transcriptional start sites. It was shown previously that thectfA and ctfB genes were contained on a 4.1-kb transcript in C.acetobutylicum DSM 792 (12). These genes have also beenfound on a similar-size transcript in strain ATCC 824 (51).These findings indicate that aad, ctfA, and ctfB are expressed asan operon, since the corresponding size of the transcript basedon sequence analysis agrees with the Northern blot results.A primer extension experiment was performed to identify

the precise 5' end of the aad-ctfoperon. The results are shownin Fig. 6. Two bands were observed that corresponded topositions 83 bp (Sj; intense band) and 243 bp (S2; faint band)

aadadhEbdhAbdhBadhlmdhzyinmofucOyeast

aadadhEbdhAbdhBadhlmdhzymnmo

fucOyeast

T S I K D AK S I R E AS K LR E VS R L R D VT T LK E YS G Y A E MA N LT E LP H L R D VR N L K D L

GGGGGGGGG

V LV QI GI EI HV KA KV RV K

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GATC PE

24-S2

FIG. 7. Plasmid pCADEX1. The 3.0-kb insert (shaded region)containing aad and its two natural promoters (PI and P2 upstream oftranscriptional start sites SI and S2, respectively) was amplified byPCR, using single-base-substituted primers to generate EcoRI andXbaI ends, and cloned into the T7 expression vector pT7/T3a-18. TheT7 promoter is designated PT7.

sMM.

1 2 3 4 5FIG. 6. Primer extension analysis. Primer extension products made

by using primer PE-1 complementary to aad are shown in lane 5. Thearrows indicate bands corresponding to transcriptional start sites SIand S2 (83 and 243 bp, respectively, upstream of the aad start codon).RNA used in this experiment was obtained from C. acetobutylicumcells which had been producing butanol for 5 h. Regions of plasmidpHXS5 were sequenced by using the same primer, and the resultingDNA sequences are shown in lanes 1 to 4.

upstream of the aad start codon. To rule out the possibility ofnonspecific hybridization of the PE-1 primer with differentmRNA, the experiment was repeated (not shown) with a

second primer (PE-2) complementary to a different N-terminalregion of aad. Both experiments revealed identical dual startsites with the same relative band intensities. The - 10 and - 35regions of the apparent promoters, along with those of other C.acetobutylicum solventogenic genes, are shown in Table 3. Thedistal, apparently weaker, aad-ctf promoter was similar to thatpreviously identified as a consensus clostridial promoter (61).

However, the other aad-ctf promoter differed from those ofother solventogenic genes, especially in the - 10 region.As expected, no terminator structures could be located in

the 32-bp intergenic region between aad and ctfA or the 27-bpintergenic region between ctfA and ctfB. The presence of abidirectional terminator has been identified downstream ofctfB (38).ALDH and ADH activities of AAD in E. coli and C. acetobu-

tylicum. Cell crude extracts were assayed for BYDH, ACDH,BDH, and EDH activities. Preliminary attempts to detectALDH and ADH enzyme activities in JM1O9(pCADEX1)crude extracts were unsuccessful, suggesting either that thenatural promoters could not function in E. coli or that theprotein itself was not functional. To overcome this potentialdifficulty, pCADEX1 (Fig. 7), whereby aad is transcribed fromthe T7 promoter, was used to transform E. coli BL21(DE3).Under aerobic growth conditions, low activities were detectedfor NAD-specific BYDH, NAD- and NADP-specific ACDH,and NADPH-specific BDH (results not shown). Previously, anALDH from Clostridium beijerinckii NRRL B592 was found tobe sensitive to oxygen (60). Moreover, adhE-lacZ fusionstudies have indicated a 10- to 20-fold induction of ADH/ACDH under anaerobic conditions (26). Hence, the assayswere repeated with cells grown anaerobically. Rifampin wasalso added to these cultures to selectively inhibit the host RNApolymerase (50) and thereby reduce any background activity ofthe host. Under these conditions, more pronounced activitieswere detected for NAD-specific BYDH and NADH-specificBDH, with both vector and host controls yielding no detectable

TABLE 3. Mapped promoter regions of solventogenic C. acetobutylicum genes

SequenceGene Reference

- 35 region - 10 region -1

aad-cf TTATATTT TTGACC TATGCTTTTTATTGAAC TATAAT AAAAGCAT(S2) a This studyGTTTTGTT TTGCAG TTTACAATATCTATCT CCAAAT CTGC (S1) a This study

adc TGTAAAAA TTTACT TAAAAAAACAATATGTGT TATAAT GTAAAT 12Consensus clostridial sequence -T------ TtaAcA ----T- AAtATga TAtAAT ---T--- 61bdhA TTAGATGC TTGTAT TAAAATAATAAAATAG TAAAAT ATAAGTA 52bdhB AAATATTA TTGTAA TAATTTTAAGTAGGTT TAAAAT ATATATA 52

a SI and S2 are the transcriptional start sites referred to in Fig. 6.

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aad GENE FROM C. ACETOBU7YLICUM ATCC 824 881

(a)

E(

(b)

XbaI Cla l/Acc

EcoR

FIG. 8. (a) Plasmid pCAAD. AccI-digested pCADEX1 was ligatedto ClaI-digested B. subtilis plasmid pIM13 (28) (which confers MLSrand a gram-positive origin of replication). (b) Plasmid pCCL. pCAADwas sequentially digested with BamHI and BglII and religated (BamHIat position 2923 [Fig. 1] and BglII at position 4434 [Fig. 1]) to yield thiscontrol plasmid carrying a truncated aad (58% of structural genedeleted).

activities (results not shown). Under no conditions couldNADH- or NADPH-specific EDH activity be detected fromplasmid pCADEX1.When assays were performed with crude extracts of C.

acetobutylicum samples from fermentor experiments at pH 4.5of wild-type and recombinant strains carrying plasmid pCAAD(Fig. 8a) or pCCL (Fig. 8b), activities were detected forNAD-specific BYDH and ACDH and for NADH-specificBDH and EDH (Table 4). While BDH and ACDH activitiesfrom pCAAD were higher than those of the wild-type strain(up to 2.5- and 70-fold, respectively) and ATCC 824(pCCL)control strain (up to 2- and 60-fold, respectively), the pCCLcontrol also reflects some degree of enhancement of theseactivities (up to 2- and 45-fold higher, respectively). EDHactivities were lower, but the same trend can be observed.However, BYDH specific activity indicates a more selectiveamplification in the strain carrying the intact aad on pCAAD.Although the control 824(pCCL) fermentation shows higherfinal butanol and acetone concentrations, the ethanol-to-acetone and butanol-to-acetone ratios (Table 5) indicate thatthe recombinant strain carrying aad (on pCAAD) producesalcohol with improved selectivity compared with either of thecontrols. In addition, the 824(pCAAD) fermentation shows 2-

TABLE 4. ALDH and ADH specific activities in C. acetobutylicumfrom fermentor experiments at pH 4.5

Sp actb (mU/mg of protein)

C. acetobutylicum Growth NADHATCC 824 phasea NAD specific specific

BYDH ACDH BDH EDH

Wild type A <1 <1 120 <1B <1 3 161 <1C <1 <1 372 <1

Harboring pCCL A < 1 1 268 4B < 1 138 329 1C <1 16 166 <1

Harboring pCAAD A <1 63 252 5B 69 224 426 3C < 1 60 418 2

a A, early exponential growth phase; B, late exponential growth phase; C,stationary phase.

b NADP-specific BYDH and ACDH activities and NADPH-specific BDH andEDH activities were very low and hence are not reported. Errors in activitynumbers are typically of the order of 20% (53).

and 2.7-fold-higher ethanol concentrations compared with thecontrol fermentations 824(pCCL) and 824, respectively.

Detection ofAAD. Amplification of aad in C. acetobutylicumATCC 824(pCAAD) revealed a strong induction of a -96-kDaprotein during the onset of solventogenesis (late exponentialstage) in Coomassie blue-stained denaturing gels (Fig. 9, lane7) and in gels (not shown) with [35S]Met-labeled proteins. Instrain 824, this band is seen faintly at the onset of solvento-genesis (lane 3). Strain 824(pCCL), like strain 824, has only asingle intact copy (chromosomal) of aad. However, plasmid(pCCL)-imposed metabolic stress (see Discussion) seems tolead to elevated expression of chromosomal aad (lane 12).Gels (not shown) run with [35S]Met-labeled proteins from T7expression studies in E. coli did not reveal overexpression of a-96-kDa protein which would be expected from the molecularweight of the translated aad. Coomassie blue-stained gels oftotal protein also confirmed this finding. However, an intenseband corresponding to -42 kDa was observed (gel not shown)in the rifampin-treated sample of cells carrying pCADEX1.Several fainter bands corresponding to higher-molecular-weight proteins were observed for both aerobically and anaer-obically grown E. coli cells (results not shown).

DISCUSSION

C. acetobutylicum AAD, which demonstrates both aldehydeand alcohol dehydrogenase functions, shows high identity tothe trifunctional adhE from E. coli. Under anaerobic condi-tions, the adhE gene product has ACDH and ADH activities,which are responsible for ethanol formation (13), and pyru-vate-formate-lyase (PFL) deactivase activity (21). PFL deacti-vase inactivates PFL, which is required in the nonoxidativecleavage of pyruvate to form acetyl-CoA and formate (6, 22).The anaerobically induced PFL deactivase exists as an iron-dependent homopolymeric rod-shaped protein (-40 x 96kDa) (21). Amino acid alignment of AAD and ADH/ACDH(Fig. 3) revealed that identity is established at the veryN-terminal end and the loss in identity is in the C-terminalregion (22 aa residues of AAD and 37 aa residues of ADH/ACDH). E. coli ADH/ACDH is 19 aa residues longer thanAAD. The presence of PFL deactivase activity has not beenverified for AAD, since no evidence exists for the presence ofPFL in C. acetobutylicum. PFL activity has been detected in

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TABLE 5. Final product levels in C. acetobutylicum fermentor experiments at pH 4.5

C acetobutylicum Concn (mM) RatioATCC 824 Butanol Acetone Ethanol Butyrate Acetate Butanol/acetone Ethanol/acetone

Wild type 74 66 15 36 53 1.12 0.23Harboring pCCL 138 95 21 6 12 1.45 0.22Harboring pCAAD 118 72 41 5 31 1.64 0.57

Clostridium butyricum (a species substantially different from C.acetobutylicum). In contrast to the PFL of E. coli, the C.butyricum PFL functions mainly to furnish formate for ana-bolic Cl-unit formation (58).The location of the 957-bp ORF1 upstream of the aad-ctf

operon suggested that it could perhaps encode a regulatoryprotein. However, data base searches did not reveal homologyto DNA-binding proteins.As previously observed (18), the N-terminal ends of ALDHs

appear to be poorly conserved; however, ALDH domains(N-terminal regions) of AAD and ADH/ACDH show homol-ogy to several ALDHs (Fig. 4). Strictly conserved residues arefound in 10 positions, half of which are Gly residues. The twostrictly conserved Pro residues are present as part of a highlyconserved 15-aa peptide (starting at position 102 [Fig. 4] inAAD). The dodecapeptide that includes the last 12 of these 15residues has previously been reported to be conserved amongALDHs (18). Proteins which demonstrate only ALDH func-tion have the highly hydrophobic residues Phe and Trp be-

824 824 824(pCAAD) (pCCL)

kDa A B C A B C A B CI1 2 3 4 5 6 7 8 9 10 11 12 13

200.0 -

tf~ ~ ~~r"ir0ti;0:000xt 's eXyi X 0 kDa

97.4- RM -0~~~~~~~-iA97.497.4- _ AAD

68.0 - 66.2

- 55.0

43.0- 42.7- 40.0

29.0 4;=- | | - 0 [-31.0

FIG. 9. SDS-PAGE. Cell samples from C. acetobutylicum con-trolled-pH (4.5) batch fermentations of both wild-type and recombi-nant strains were taken at the early exponential (A), late exponential(B), and stationary (C) stages of growth, and proteins were electro-phoresed in SDS-8% polyacrylamide gels and Coomassie blue stained.Lanes 2 to 4 correspond to the wild-type strain, lanes 5 to 8 correspondto the recombinant strain carrying intact aad on plasmid pCAAD, andlanes 9 to 12 correspond to the recombinant control strain carrying atruncated aad on plasmid pCCL. Lanes 5 and 9 represent earlyacidogenic stages (prior to stage A) of the corresponding recombinantstrains. The -96-kDa band representing AAD is indicated. Theprominent -39-kDa-band protein observed in lanes 2 to 12 is probablythe butyrate kinase (36). High-range (lane 1) and medium-range (lane13) protein molecular weight markers are shown alongside.

tween the Pro residues. AAD and ADH/ACDH have hydro-philic (Thr) to moderately hydrophobic (Val) residues in thisregion of unknown function. A decapeptide of sequenceVTLELGGKSP (starting at position 261 in the protein desig-nated aspni in Fig. 4) is highly conserved among ALDHs (55)but not in AAD or ADH/ACDH. The heptapeptide EExFGPV(starting at position 382 in the protein designated l-ald in Fig.4; x is any amino acid) is also conserved among several ALDHs(18), but the corresponding sequence in AAD and ADH/ACDH is a poor match. These comparisons clearly indicatethat the N-terminal regions of AAD and ADH/ACDH, whilerelated to ALDHs, have diverged in some ways from thisgroup.The ADH domains (C-terminal regions) ofAAD and ADH/

ACDH show homology to several ADHs (Fig. 5). Of the 19positions that are strictly conserved, there are four Gly andthree Pro residues. This overrepresentation in conserved Glyresidues has been suggested as typical of ADHs with largelyconserved conformation (20). Cys and His residues have beenimplicated as metal-binding ligands in zinc-containing long-chain ADHs (20). A comparison of AAD and ADH/ACDH(Fig. 3) reveals seven His (four in the N-terminal part) andfour Cys (two in the N-terminal part) residues that areconserved throughout both proteins.The coenzyme specificity of dehydrogenases has been fairly

well documented (20, 24, 43). A highly conserved glycine-richregion with a GxGxxG pattern within a Pot3 Rossmann foldwas found to be common to the NADH-binding domain ofmany ADHs (20, 43). A GxGxxGG pattern is observed in thecentral regions (Fig. 3) of AAD (starting at residue 418) andADH/ACDH (starting at residue 420). With the exception ofglyceraldehyde-3-phosphate dehydrogenase (43), a coenzyme-binding consensus sequence among ALDHs seems to containan additional amino acid between the second and third glycines(18). Such a sequence is observed in several ALDHs (Fig. 4;aligned to initiating Gly-168 in AAD) but not in AAD andADH/ACDH. However, a similar sequence is present in AADstarting at position 362 in a region that is not very wellconserved in the initiating glycine. A GxGxxA sequence withina Pctp fold (with another alanine almost always present afurther four residues downstream) is conserved among someNADP(H)-binding proteins (43). However, some significantdifferences among NADPH-binding domains (24, 43) preventsidentification of such a region on the basis of sequencecomparisons alone. A GxGxxVxxxA sequence exists in AADstarting at position 598 (Fig. 5) in a region highly conservedamong ADHs. The ADH portion of AAD has a 42% identity(65% similarity) with the NADPH-dependent ADHI from theindustrial strain C. acetobutylicum P262 (62).

It appears that in strain ATCC 824, ALDH (both BYDHand ACDH) functions are present as part of a multifunctional-96-kDa (monomer size) protein (Fig. 9 and Table 4) which isthe product of aad. Earlier, a CoA-linked BYDH (monomersize of 56 kDa and native size of 115 kDa) was purified from C.acetobutylicum B643 (35), and a CoA-acylating ALDH (mono-

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mer size of 55 kDa and native size of 100 kDa) was purifiedfrom C. beijerinckii NRRL B592 (60). It is not known whetherproteins similar to these proteins exist in strain ATCC 824.However, different genetic organizations and coenzyme spec-ificities of similar enzymes in different strains of clostridia isnot an unusual phenomenon. Of the three CoA-linked ALDHsisolated from Clostridium kluyveri, an NAD-specific enzymefrom the soluble fraction (45) has a molecular mass of 290kDa, an enzyme (NAD and NADP specific) from the particu-late fraction of strain DSM 555 has a molecular mass of 194kDa composed of 55- and 42-kDa subunits (27), and anNADP-specific succinate semialdehyde dehydrogenase fromC. kluyveri DSM 555 has a monomer size of 55 kDa and nativesize of 115 kDa (46).

Table 5 shows that AAD confers a superior ethanol forma-tion capability compared with both the wild-type strain and the824(pCCL) plasmid control strain. This is possibly due toelevated activities (Table 4) of the NAD-specific ACDH and toa lesser extent the NADH-specific EDH. Since no attempt wasmade to enhance acetone pathway genes, it is meaningful tocompare ethanol-to-acetone and butanol-to-acetone ratios ob-tained in wild-type and recombinant strain fermentations(Table 5). The ethanol-to-acetone ratio for the recombinantstrain carrying intact aad (on pCAAD) is more than doublethat for either control (Table 5). The butanol-to-acetone ratioalso reflects an enhancement (Table 5) although not quite aspronounced. As observed earlier (31), host-plasmid interac-tions also lead to elevated final solvent levels and decreasedfinal acid levels (Table 5) due to a possible induction of stressproteins upon plasmid-imposed metabolic stress. We havefound this to be a general effect with several other plasmidconstructs, as we shall report shortly (52a). We also note theappearance of a strong band at -65 to 66 kDa, which parallelsin intensity the AAD band and solvent production in all threefermentations (Fig. 9; Table 5). This band could representDnaK, a 65.6-kDa C. acetobutylicum stress protein (33). Thiswould be consistent with the proposed connection between thestress protein induction and solventogenesis (33).The high BDH activities observed in C. acetobutylicum

(Table 4), which are likely in large part due to BDH I andBDH II isozymes (52), coupled with the use of the samesubstrate (butyraldehyde) in both BYDH and BDH assays,leads to a masking of BYDH activities due to an uptake byBDH of the NAD(P)H generated in the BYDH reaction. Thiscould explain the low BYDH activities detected (Table 4).However, detectable ACDH activities are higher, possiblybecause of low detectable levels of EDH (Table 4), whichwould minimize such a masking effect on the ACDH activities.

Expression of aad in E. coli from its natural promoters wasundetectable even though one of the natural promoters (Table3) shows conservation to the E. coli o70 RNA polymerasepromoter in 11 of 12 positions in the -10 and -35 regions.Also, the T7 expression system described in this report did notlead to the expected high ALDH and ADH activities of AADin E. coli. Since no overexpressed -96-kDa-band protein wasdetected in any of the E. coli BL21(DE3) gels, the questionremains as to whether AAD exists in E. coli as a 96.5-kDaprotein or whether it undergoes posttranslational processinginto an ALDH (average size of -55 kDa [Table 1]) and anADH (average size of -42 kDa [Table 2]). The idea that the42-kDa band observed in E. coli protein gels is perhaps atruncated AAD could be explained by the presence of aconsensus E. coli RBS starting at position 3395 (Fig. 1)upstream of a Met start site at position 3406 (Fig. 1), which isprecisely where the ADH homology region is initiated (Fig. 5).However, this does not explain the observed ALDH activities

in E. coli. These ambiguous results in E. coli prompted the C.acetobutylicum studies described in this report. Internal trans-lation initiation at position 3406 could also occur in C.acetobutylicum, and the prominent bands of -43 to 44 kDaappearing in lanes 3, 6 to 8, and 10 to 12 of Fig. 9 couldrepresent such a translated product from the chromosomal aad[for 824 and 824(pCCL)] and the chromosomal and plasmidaad [for 824(pCAAD)].The aad-ctf operon seems to play a very important role in

solvent formation. CoA-transferase and AAD are induced andderepressed simultaneously at the onset of solventogenesis.Activity data suggest that AAD could provide both ALDH andADH functions in butanol and ethanol formation pathways.Specific mutations in ALDH and ADH regions ofAAD wouldbe necessary to verify that these are mutually exclusive func-tions of the same protein. Coregulation of butanol and acetoneformation genes implies that a mutation in a key regulatoryregion in this common locus could lead to loss of severalsolventogenic enzyme functions. The existence of a commonregulatory factor for acetone and butanol formation has beenproposed (14). Chemical mutagenesis of ATCC 824 has re-sulted in a mutant, M5, which lacks BYDH (NAD and NADPspecific) and CoA-transferase activities but expresses bothNADH- and NADPH-specific BDH activities (7). The pres-ence of the BDH isozymes (BDH I and BDH II) in ATCC 824(52) in a different region on the chromosome could perhapsexplain the BDH activities detected in mutant M5.

ACKNOWLEDGMENTS

We thank K. Walter and L. Mermelstein for advice and technicalassistance, J. Cary for isolation of the clone that was sequenced, and N.Welker and M. Heaton for helpful suggestions.

This work was supported by grants BCS-9210108 and BCS-9209627from the National Science Foundation.

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