APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3491-3498

Vol. 56, No. 11

0099-2240/90/113491-08$02.00/0 Copyright © 1990, American Society for Microbiology

Purification of Acetoacetate Decarboxylase from Clostridium acetobutylicum ATCC 824 and Cloning of the Acetoacetate Decarboxylase Gene in Escherichia coli DANIEL J. PETERSEN AND GEORGE N. BENNETT* Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251 Received 30 April 1990/Accepted 22 August 1990

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an -2.1-kb EcoRIlBglll fragment. A polypeptide with a molecular weight of -28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.

The acetone-butanol fermentation of Clostridium acetobutylicum has traditionally been divided into two distinct phases. In the initial acidogenic phase of growth, acetic and butyric acids are formed, with a concomitant decrease in the pH of the medium to 4 to 4.5. The accumulation of acids and other possible factors causes a metabolic shift to solvent production as the cells enter the stationary phase of growth. During this phase, acids present in the medium are reassimilated and metabolized to produce acetone, butanol, and ethanol. Acetoacetate decarboxylase (AADC; EC 4.1.1.4) plays a key role in solvent production as it catalyzes the production of acetone by the direct decarboxylation of acetoacetate. No cofactors are necessary for the reaction to occur (11). The enzyme is much more stable than many of the other enzymes of the fermentation pathway. It has a pH optimum of -5.0, well suited for the production of acetone at acidic pH values. AADC is insensitive to oxygen and can withstand acetone (10). The latter property has been used to extract the enzyme from C. acetobutylicum by acetone precipitation (11, 14). Crystalline AADC has been prepared from such acetone powders (29). Laursen and Westheimer determined the sequence of amino acids around the active-site lysine to be -Glu-Leu-Ser-Ala-Tyr-Pro-Lys*-Lys-Leu (21). Lys* is the amino acid where Schiff base formation occurs (26). Although Schiff base formation is common among decarboxylases not requiring pyridoxal phosphate as a cofactor, the active-site sequence bears no obvious relationship to published sequences of other Schiff base enzymes. Acetone formation and induction of AADC activity have been shown to occur simultaneously in continuous cultures (2, 18). This sharp induction of enzyme activity was shown to be maximal at pH 4.8 (3). Linear acids from C1 to C4 were found to induce AADC activity, whereas branched acids and linear acids C5 to C7 did not (3). Similarly, the activity of the

*

coenzyme A (CoA)-transferase which allows uptake of acids for their subsequent conversion to solvents appears to be coordinately induced just prior to the onset of solventogenesis (18, 19), although low levels of the enzyme are found in acidogenic cells (2). AADC can be considered the key enzyme in acid uptake as it effectively pulls the CoAtransferase reaction in the direction of acetoacetate formation (17). Indeed, no uptake of acids is observed by the CoA-transferase until induction of the AADC (17, 18). Despite exhaustive enzymological studies, little genetic research has been published regarding the regulation of this gene.

Recently, we have isolated a mutant deficient in both CoA-transferase and AADC activity (9; unpublished results) and a flagellar mutant that had a deficiency in AADC and CoA-transferase activity as well as in NADP-dependent butyraldehyde dehydrogenase activity (D. J. Petersen, J. W. Cary, and G. N. Bennett, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, 1-124, p. 238). Mutations affecting more than one enzyme may be regulatory in nature. That the CoA-transferase and AADC together form a pathway for the production of acetone from butyrate may dispose them to synchronous regulatory mutations that may affect both enzymes. The CoA-transferase gene of C. acetobutylicum ATCC 824 has recently been cloned (8). To better analyze the induction mechanisms and relationship of the CoAtransferase and AADC, we report here the purification of AADC and cloning of the gene encoding this enzyme. MATERIALS AND METHODS Bacterial strains, plasmids, and bacteriophage. The bacterial strains, plasmids, and phage used in this study are listed in Table 1. Growth and maintenance of bacteria and phage. C. acetobutylicum and Escherichia coli strains were routinely grown and maintained as described previously (7). The titer of the C. acetobutylicum phage library was determined and screened on Luria-Bertani (LB) medium containing 1.5%

Corresponding author. 3491

PETERSEN AND BENNETT

3492

APPL. ENVIRON. MICROBIOL.

TABLE 1. Bacterial strains and plasmids Strain, plasmid, or

phage

Strain C. acetobutylicum ATCC 824 C. pasteurianum NRRL B598 C. butyricum ATCC 19398 E. coli DH5

CSR603 XL-1 Blue

NM519

Plasmids pUC19 pJC7 pSDC2 pDP2 pDP12 pDP253 pDP253AHindIII pDP253APstI pDP253ANcoI Bacteriophage A EMBL3

Relevant

Source or reference

genotype

F- endAl hsdRJ7 (rk, mk ) supE44 thi-J recAl gyrA96 relAl F- phr-1, recAl,

uvrA6, supE44 hsdRJ7 (rk, Mk'), [F' proAb lac-19 ZAM15 TnlO (Tcr)] supF58 sbcA recBC (rkj, mk ) Apr Apr BK+ PTB+ Apr AADC+ Apr AADC+ Apr AADC+ Apr AADC+ Apr AADCApr AADCApr AADC+

16

24 6

15

28 7 This This This This This This This

study study study study study study study

15

agarose and 0.7% top agarose. Phage lysates were collected and stored in SM buffer (per liter: NaCl, 5 g; yeast extract, 5 g; MgSO4 H20, 2 g; casein hydrolysate, 10 g). DNA isolation and manipulation. Total cellular DNA from C. acetobutylicum was prepared as described previously (7). Construction of a C. acetobutylicum genomic library in the lambda cloning vector EMBL3 was accomplished as described previously (8). Rapid, small-scale plasmid DNA isolation was performed by the method of Birnboim and Doly (5). Transformations were routinely performed with competent cells prepared by the method of Hanahan (16). Phage DNA was prepared by the rapid plate lysate method described by Maniatis et al. (22). All restriction enzymes, phage T4 DNA ligase, and T4 polynucleotide kinase were obtained from either Promega Biotec or New England Biolabs and used according to the recommendations of the

suppliers. Protein purification. Purified AADC was obtained by a modification of the purification described by Fridovich (14), using cells from 8 liters of a late-stationary culture of C.

acetobutylicum ATCC 824. Step 5 (hydroxylapatite adsorption) was replaced by two steps. Step 5a was anion exchange. The suspended extracts were separated by highperformance liquid chromatography (HPLC) (LDC/Milton Roy CM 4000), using an AX-1000 column (SynChropak, 250 by 10 mm; SynChrom, Inc.). Both gradient and isocratic elution, using 0.05 M KPO4 (pH 5.9) and 0.05 M KPO4-0.5 M NaCl (pH 5.9) at a flow rate of 0.15 ml/min, were used. Fractions were tested for activity by the spectrophotometric method of Fridovich (14). Step Sb was size exclusion. Pooled

fractions were further purified by HPLC, using a GF-250 column (Zorbax, 250 by 9.4 mm; Du Pont) and an elution buffer of 0.1 M KPO4 (pH 6.5) at a flow rate of 0.5 ml/min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of active fractions confirmed the presence of only a single band in the active fractions after this step. Protein sequencing. Samples of HPLC-purified AADC were loaded onto 12.5% SDS-polyacrylamide gels and electrophoresed by the method of Laemmli (20). Free-radical scavenger (10 mM thioglycolic acid) was added to the upper buffer chamber to prevent blocking of the N terminus during electrophoresis. After electrophoresis, the gels were soaked in transfer buffer before electroblotting to polyvinylidene difluoride (PVDF), as described previously (8). The polyvinylidene difluoride membrane was rinsed briefly and stained in 1.0% Coomassie blue R-250 as described by Matsudaira (23). The AADC band was cut out with a clean razor blade and stored at -20°C. N-terminal amino acid sequencing was accomplished by sequencing directly from the polyvinylidene difluoride electroblotted membrane, using an Applied Biosystems model 477A sequenator equipped with on-line phenylthiohydantoin analysis, courtesy of Richard G. Cook, Baylor College of Medicine, Houston, Tex. Preparation of oligonucleotide probes. Synthetic oligonucleotide probes to be used in screening the phage library were derived from amino-terminal sequencing data obtained from the purified AADC. Two oligonucleotide probes were synthesized: a 4-fold degenerate 20-mer deduced from amino acid residues 1 through 7, and a 32-fold degenerate 23-mer designed with amino acid residues 13 through 20. When appropriate, the "wobble" base of each codon was biased toward A or T based on codon usage data from previously sequenced C. acetobutylicum genes (25). Oligonucleotides were synthesized and purified as described previously (8). The purified oligonucleotides were radiolabeled with [_-32P] ATP (specific activity, -4,000 Ci/mmol; ICN Pharmaceuticals, Inc.) using T4 polynucleotide kinase as described by Maniatis et al. (22). Probe purity was confirmed after labeling by electrophoresis on denaturing 20% polyacrylamide gels followed by autoradiography. DNA hybridization. Total cellular DNAs from C. acetobutylicum, E. coli, and purified phage were digested to completion with the desired restriction enzymes and separated by electrophoresis on 0.9% agarose gels. The DNA was transferred to nitrocellulose (BA85; Schleicher & Schuell, Inc.) by vacuum transfer, using a TE 80 TransVac (Hoefer Scientific Instruments). Phage plaque blots were performed by the method of Benton and Davis (4). Double-stranded DNA probes were prepared by nick translation with [tx-32P]dATP, using a nick translation reagent kit (Bethesda Research Laboratories). Blotted nitrocellulose filters were prehybridized as described previously (8). Prehybridization solution was replaced with fresh buffer with the addition of 100 ,ug of denatured, sheared, salmon sperm DNA per ml and -106 cpm of probe. Hybridization was typically conducted overnight at 37°C, and the filters were washed three times for 10 min in a solution of 2x SSPE (0.36 M NaCl, 20 mM NaPO4 [pH 7.7], 1 mM EDTA) containing 0.1% SDS. The filters were then placed on X-ray film for 24 to 72 h. Enzyme assays. Purified protein was assayed by the spectrophotometric technique of Fridovich (14). Changes in A270 were followed by using 5 mM acetoacetate in 0.1 M KPO4, pH 5.9, at 26°C. The approximate molar extinction coeffi-

VOL. 56, 1990

C. ACETOBUTYLICUM ACETOACETATE DECARBOXYLASE GENE

cient of acetoacetate at 270 nm and pH 6.0 is 55.0, while that of acetone is 28.3 (14). E. coli cells harboring plasmids containing the gene for AADC were assayed by the manometric method of Davies (11). Activity was measured with a constant-pressure differential respirometer (single-valve differential respirometer; Gilson Medical Electronics) at 30°C (18). The reaction flask contained, in a volume of 3 ml, 80 mM lithium acetoacetate, 60 mM potassium acetate (pH 5.0), and whole-cell suspension (1 to 10 mg of protein), which was prepared by suspension of washed cells in phosphate buffer. The acetoacetate and buffer were mixed in the reaction flask, while the cell suspension was placed in one of the flask side arms. After equilibration at 30°C, the reaction was initiated by quickly mixing the reactants. Micrometer readings were made every 30 s for approximately 12 min.

Antibody preparation. Antibody was prepared by Bethyl Labs, Montgomery, Tex., essentially as described previously (8), using a New Zealand white rabbit. Whole serum was collected by standard methods, and anti-AADC immunoglobulin G was prepared by 75% NH4SO4 precipitation, followed by extensive dialysis with phosphate-buffered saline (per liter: NaCl, 7.42 g; KCl, 0.2 g; KH2PO4, 0.2 g; Na2HPO4. 7H20, 2.17 g) plus 5 mM NaN3. Western blotting. Whole-cell proteins (-50 mg) were separated by SDS-PAGE by the method of Laemmli (20). Proteins were then transferred electrophoretically (12 V) for 30 min to nitrocellulose filters, using a Genie (Idea Scientific) electroblotting apparatus. Antigen-antibody binding was carried out as described previously (8). The nitrocellulose filter was blocked for 30 min in TBST (10 mM Tris hydrochloride

[pH 8.0], 150 mM NaCl, 0.05% Tween 20) plus 1% bovine nonspecific protein-binding sites. Rabbit anti-AADC antibody (1:2,000 dilution) was bound to the filter in TBST buffer for 1 h followed by three 5-min washes with TBST buffer. Goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate (1:3,000 dilution; Boehringer Mannheim) was bound in TBST for 1 h, followed by serum albumin to saturate

three 5-min washes with TBST. The presence of AADC in cell extracts was visualized upon addition of Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate color development substrates (Promega Biotech). Analysis of protein size. 35S-labeling of plasmid-encoded proteins was accomplished by using the maxicell procedure (24) (using trans-35S label; ICN) as described previously (7, 8). Molecular weight markers (14,300 to 200,000) were obtained from Amersham Corp. and Sigma Chemical Co. RESULTS Protein purification and N-terminal sequencing. The purification procedure described by Fridovich (14) failed to yield a pure preparation of AADC (29). However, the addition of the HPLC anion-exchange step resulted in a preparation containing only two major protein components (Fig. 1, lane 1). Native PAGE confirmed that the two polypeptides were of two distinct proteins. Not until the additional HPLC size exclusion of this sample was the complete purification of the AADC achieved (Fig. 1, lane 3). The AADC was electroblotted to the polyvinylidene difluoride membrane as described in Materials and Methods. N-terminal sequencing was achieved directly from the immobilized sample (23). The amino acid sequence of the first 20 amino acids is shown in Fig. 2. Methionine was found to be the first residue, as was reported previously (29). Enzyme assays on the purified protein demonstrated activities equal to or greater than those

reported previously (21, 29).

3493

AAO -44u-~

Ai

aws

4w2O "14 FIG. 1. Comparison of HPLC purification of AADC. Samples (5 p.g) from each additional HPLC purification step were denatured in l x sample buffer and fractionated by SDS-PAGE. Proteins were stained with Coomassie blue. Lanes: 1, HPLC AX-1000purified AADC; 2, molecular weight standards (molecular weight, i0', shown at right) bovine serum albumin (66,000), ovalbumin to 30

(45,000) glyceraldehyde-3-phosphate dehydrogenase (36,000),

car-

bonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), and a-lactalbumin (14,200); 3, HPLC GF-250-purified AADC. Bands corresponding to the AADC are indicated by the affow.

Cloning of the C. acetobutylicum AADC gene in E. coli. Prior to screening the EMBL3 phage library (8), the specificity of each oligonucleotide probe was determined by hybridizing the probes to dot blots of DNA from C. acetobutylicum ATCC 824, C. pasteurianum B598, C. butylicum 19398, E. coi, lambda EMBL3, and pJC7 (7). Only the chromosomal DNA from clostridia gave positive hybridization with both probes. Due to their similar degree of specificity upon hybridization, both probes were used to screen the phage genomic library simultaneously. For screening purposes, the phage library stock was diluted as to obtain 1,000 to 2,000 plaques per plate following infection of E. coli NM519 cells. Mter screening approximately 10,000 plaques with both probes, four were identified which showed homology to the probes. Following plaque purification of these putative recombinant phage, DNA was isolated from each by the plate lysate method (4). Southern hybridization of HindIII-digested DNA from each phage isolate identified two phages which contained DNA homologous to both probes. Subcloning and restriction enzyme mapping. SaiI digests of DNA isolated from one positive phage isolate revealed that this restriction site was not present within the insert DNA. Subcloning of the entire clostridial insert (--15 kb) into pUC19 formed the plasmid pSDC2, which expressed AADC activity when strains bearing this plasmid were assayed. Various restriction enzyme digestions were performed on the DNA from pSDC2, and Southern hybridization analyses

3494

PETERSEN AND BENNETT

APPL. ENVIRON. MICROBIOL.

1 2 5 3 4 9 10 6 7 8 met - leu - Iys - asp - glu - val - ile - Iys - gln - ile -

5 - ATG TTA AAA GATC GAA GTAT AT -3 11

12

13

ser - thr- pro

-

CCA

-

20 - mer

14 15 16 17 18 19 20 lou - thr - ser - pro - ala - phe - pro TTA

ACA TCA CCA GCA

-

TTT CC -3

23 - mer

FIG. 2. Sequences of mixed oligonucleotide probes specific for the amino terminus of the AADC. Numbers in the top row refer to positions of the amino acid residues derived from amino-terminal sequencing of the purified protein. Below each number is the corresponding amino acid sequence, with the residues used to design the probe in boldface print. The nucleotide sequence of each probe is shown below the respective amino acid sequence.

were performed with both oligonucleotide probev diograms of these blots revealed that the probes I to an -4.2-kb EcoRI fragment (Fig. 3). Subcloni EcoRI fragment into appropriately cleaved pUC19 pDP2 as well as pDP12 (opposite orientation) (Fig. restriction sites were found for AccI and Aval; each were found for BanIl, PstI, and BglII; three found for NcoI; and six sites were found for Hi restriction sites were found for BamHI, Clal, Ecol PvuII, Sall, or XhoI. Southern hybridization analysis of restriction fra pDP2 revealed that the region encoding the N

resided within the 0.54-kb EcoRI/HindIII fragment and, hence, the gene must extend to the right from the EcoRI site as indicated (Fig. 5) as the EcoRI/HindIII fragment is not large enough to contain the entire gene. Subsequent subcloning of the -2.1-kb EcoRIlBglII fragment of pDP2 into EcoRI/BamHI-cleaved pUC19 produced pDP253, with the lacZ promoter oriented oppositely from the AADC gene (Fig. 5). DNA homology. The origin of the 4.2-kb EcoRI insert of pDP2 was demonstrated by a Southern blot experiment with C. acetobutylicum DNA digested with restriction enzymes to compare the chromosomal fragments with those of pDP2.

IA 12 34

ii

5 67

23^456 7

_~~~~~~~~~

me,

I

_t

a lb

q

FIG. 3. Southern blot analysis of pDP2 DNA. 32P-end-labeled pDP2 was hybridized to various restriction digests of C. acetobutylicum DNA. (A) Samples were electrophoresed on a 0.9% agarose gel, stained with ethidium bromide, and transferred to nitrocellulose filter paper. Lanes: 1, HindIll digest of lambda DNA as size standards (lengths shown at left in kilobases); 2 through 6 are C. acetobutylicum DNA digested with EcoRI (2), BanII (3), NcoI (4), HindIll (5), HindIII/AccI (6); 7, E. coli DNA digested with HindlIl. (B) Autoradiogram of lanes 2 through 7 after Southern hybridization with 32P-labeled pDP2. Size estimates of the bands on the autoradiogram were made by alignment with the markers in panel A.

VOL. 56, 1990

C. ACETOBUTYLICUM ACETOACETATE DECARBOXYLASE GENE

0 -

X-

-

-

z

I L1

z co C;z Lz

I I l liu ...1

--

.

Pvu 11

Sca

=

=

w

U

UN

=

m

.s5 ;.s.s X CL = = < = co

E

3495

III El

LLJ~~~U.

I

I1

I

Pvu 11

I^f

I m n~~~~~~ I

ApR

I

kb

FIG. 4. Physical map of pDP2. Restriction endonuclease sites within the 4.2-kb EcoRI insert of C. acetobutylicum DNA derived from pSDC2 are shown. The direction of transcription of the P-lactamase gene of the pUC19 vector is illustrated by the arrow.

The results are shown in Fig. 3. The fragments exhibiting homology with the nick-translated pDP2 probe corresponded in size to those anticipated from the physical map of the 4.2-kb EcoRI insert. Specifically, hybridization signals from NcoI-digested C. acetobutylicum DNA corresponded to the internal fragments of the 4.2-kb insert of pDP2 of 0.55 kb (two fragments), and BanIl digestion gave hybridization signals at 1.0 and 0.7 kb. Hindlll digestion of C. acetobutylicum DNA produced hybridization signals at 1.6, 1.5, 0.24 (two fragments), and 0.12 kb as expected. HindIIIIAccI

C.

acetobutylicum

X

=gg S

o

Ui

double digests exhibited the expected pattern, with the 1.5-kb HindIll signal split into two bands of 0.9 and 0.6 kb. A hybridization signal corresponding to the entire 4.2-kb EcoRI fragment was also found in EcoRI digests. Analysis of enzyme activity. Table 2 summarizes the results of assays for AADC activity performed on whole cells of E. coli DH5 bearing pDP2 and derivatives of pDP2. Cells harboring pDP2 and pDP253 exhibited comparable activities, while those harboring the vector pUC19 were devoid of any AADC activity. Inversion of the 4.2-kb fragment within

DNA

0e co z ZCL

1 111 11 11

X

Z

pDP253

pDP253AHind

o

1.0

gE\m\AADC

>

2.0

Enzyme Activity

ZZ--

pDP253APst pDP253ANco FIG. 5. Physical map and enzyme expression in derivatives of pDP253. Restriction endonuclease sites within the 2.1-kb EcoRI/BglII insert shown. Asterisk denotes loss of the BglII site at the right end of the insert of pDP253 upon ligation with BamHI-cleaved vector DNA. The approximate location and direction of transcription of the structural gene are indicated. Hatched region denotes BanIIlEcoRI fragment where the N-terminal probes hybridize. Dark bars represent region of the 2.1-kb fragment retained in each subclone. AADC activity was assayed in whole cells as described in Materials and Methods. The AHind and APst deletions extend to the corresponding site within pUC19. The presence (+) or absence (-) of activity in E. coli CSR603 cells containing the various plasmids is indicated at right. are

3496

APPL. ENVIRON. MICROBIOL.

PETERSEN AND BENNETT TABLE 2. AADC activities of recombinant plasmids

Sp act

Plasmid

(U/mg of protein)" 0.00 pUC19 ....................................... 2.17 + 0.11 pDP2 ....................................... 2.18 ± 0.07 pDP12 ....................................... 2.24 ± 0.21 ........................... pDP253 ............ 0.00 pDP253APst ....................................... 3.11 ± 0.05 pDP253ANco ....................................... a Averaged over 10 min. One unit is defined as the amount of enzyme catalyzing the evolution of 14.5 ,umol of CO2 per min (29).

pUC19 (pDP12 versus pDP2) resulted in similar levels of activity. The presence of enzyme activity in constructs in which the insert is oriented in either direction relative to the lac promoter of the vector indicates that the clostridial gene is expressed from a promoter within the 4.2-kb EcoRI fragment. This promoter is also contained within the 2.1-kb EcoRIIBglII fragment of pDP253. Deletion analysis of pDP253 with PstI, HindIII, and NcoI deletions of the subclone further defined the location of the gene (Fig. 5). Both the AHindIII and APstI subclones were devoid of any AADC activity, while the ANcoI subclones exhibited high AADC activity comparable to or greater than the original 4.2-kb insert. Hence, the coding region of the gene ends between map positions 0.9 and 1.1 kb (Fig. 5). Identification of the AADC gene product. SDS-PAGE and Western blot analysis of 35S-labeled, plasmid-encoded proteins produced by the maxicell technique was used to identify the polypeptides encoded by pDP2 and its deriva-

tives. Western blot analysis of whole-cell extracts of E. coli CSR603 harboring pDP2 and its derivatives were performed with antiserum raised to the purified AADC of C. acetobutylicum (Fig. 6A). Extracts from cells harboring pDP2, pDP12, and pDP253 (lanes 3, 4, and 5) produced a band of homology which had the same mobility as the purified AADC (lane 1). This band (-28 kDa) was not detected in extracts of CSR603 harboring pUC19 alone (lane 2). Some homology was also found to two other E. coli proteins as evidenced by signals in lanes 2 to 5. The filter used in the Western blot experiment (above) was placed against X-ray film for 2 days. Analysis of the autoradiogram demonstrated that pDP2, pDP12, and pDP253 encoded two unique polypeptides of 28 and 27 kDa (Fig. 6B). The 28-kDa polypeptide had the same mobility as the purified AADC. The 27-kDa polypeptide represents an as yet unidentified protein. An additional band corresponding to the -29-kDa 3-lactamase of pUC19 was found in each lane. DISCUSSION AADC plays a key role in the conversion of acids to solvents by catalyzing the decarboxylation of acetoacetate to acetone in fermentations of C. acetobutylicum ATCC 824. The reaction is quite favorable (AG"', -26.2 kJ/mol [17]) and effectively pulls the less favorable CoA-transferase reaction (AGO', -7.1 to -9.6 kJ/mol [17]) towards the formation of acetoacetate. Thus, it is critical for the uptake of acids. Both the AADC and CoA-transferase have been shown to be highly inducible at the onset of solventogenesis (2, 18, 27). Hence, the ability to regulate this gene may allow

A

B

1 2 3 4 5

1 234 5 kDe

-69-*

_30-

AADC -_ ---

''-

*

bli A-

_

-21S9

-14.3FIG. 6. Analysis of plasmid-encoded proteins. Plasmid-encoded proteins were radiolabeled by the maxicell procedure, using E. coli CSR603 cells harboring pUC19 or pDP2 and its derivatives. Extracts (-50 ,ug) or purified AADC (-5 jig) was denatured in 1 x sample buffer and fractionated on a 12.5% SDS-polyacrylamide gel. Protein was transferred to nitrocellulose filter paper as described in Materials and Methods. (A) Western blot analysis of purified AADC (lane 1) and whole-cell extracts of E. coli CSR603 harboring pUC19 (lane 2), pDP2 (lane 3), pDP12 (lane 4), and pDP253 (lane 5). (B) Autoradiogram of lanes 1 to 5 after 2-day exposure showing 35S-labeled proteins. The positions of protein size standards (in kilodaltons) are given in the center. Bands corresponding to AADC (28 kDa), and ,B-lactamase (bla; 29 kDa) of pUC19 are indicated by arrows. Asterisks denote nonspecific binding of the AADC antibody to E. coli proteins.

VOL. 56, 1990

C. ACETOBUTYLICUM ACETOACETATE DECARBOXYLASE GENE

metabolic regulation of the fermentation yields of the organism with respect to both acetone and butanol production. Previously, the AADC was partially purified and characterized from C. acetobutylicum B527 (14, 29). We have succeeded in purifying the enzyme to homogeneity from C. acetobutylicum ATCC 824 as evidenced by SDS-PAGE

analysis.

Initial attempts to clone the AADC gene of C. acetobu-

tylicum ATCC 824 via immunological detection of plaque blots failed to yield positive signals over background levels due to cross-reactivity of the antibody with at least two other E. coli proteins. Oligonucleotides designed to the published amino acid sequence of the active site were highly degener-

ate and sufficiently nonspecific and therefore failed to generate positive signals. However, purification of the AADC

from C. acetobutylicum ATCC 824 and amino acid sequencing of over 20 amino acids from the N terminus allowed us to approach the cloning of these genes via screening with longer, more specific oligonucleotide probes. Hybridization experiments with these oligonucleotides successfully localized the gene within an -4.2-kb EcoRI fragment of C. acetobutylicum DNA. Southern hybridization mapping of C. acetobutylicum genomic DNA with radiolabeled pDP2 confirmed that the cloned DNA was of clostridial origin. Deletion mapping of pDP253 showed that the AADC gene is confined within a region spanning -1.06 kb of the 2.1-kb EcoRI/BgIII-insert fragment. The calculated DNA size necessary to encode a polypeptide of this size is -0.78 kb. Therefore, the region noted is quite sufficient to contain the entire gene and promoter sequences. Evidence that transcription originates from DNA sequences of clostridial origin includes the observation that inversion of the -4.2-kb EcoRI fragment with relation to the lac promoter of the pUC19 factor (pDP2 versus pDP12) resulted in equal levels of enzyme activity. Hence, readthrough of the lacZ gene does not appear to affect the expression of the AADC, in contrast to that of the CoAtransferase gene (8). Generally, AADC is not found in E. coli or any nonclostridial species. Although expression of the enzyme in C. acetobutylicum appears to be regulated by low pH, butyrate concentration, and other as yet undetermined factors, the addition of acids was not necessary for formation of enzyme activity in E. coli. CO gassing of acidogenic continuous cultures resulted in butyrate uptake without acetone formation presumably due to complete inactivation of the AADC by CO (18). Continued production of butanol and ethanol occurs under conditions of CO sparging. Since the enzymes involved in the synthesis of these alcohols (butanol and ethanol dehydrogenases) do not show the same induction patterns as AADC, it is not clear whether CO inhibits the AADC directly or via a regulator of the enzyme. With the gene encoding AADC removed from clostridial control mechanisms, future tests can more directly determine whether CO regulates the enzyme itself or its synthesis. Mutants with impaired or deficient AADC activity are available from several sources. A fluoropyruvate-resistant mutant was isolated in which AADC activity declined soon after induction (13). This mutant produced higher quantities of acetoin and normal amounts of butanol. Acetoin production has been found to be inversely related to acetone levels (12). It has been assumed (12, 13, 19) that acetoin competitively inhibits the AADC as a structural analog of acetoacetate, but no studies have confirmed this, and the speci-

3497

ficity of the AADC (11) may make this an unlikely assumption. Clark et al. isolated a rifampin-resistant mutant which produced no acetone and was found to be devoid of AADC activity (9; unpublished experiments). The phenomenon of degeneracy described as an irreversible, permanent loss of solvent production ability (1) has long been reported. Recent enzymatic studies of reported degenerate mutants have indicated the loss of AADC as one reason for the reported loss of acetone production ability. The genetic techniques for reintroducing genes into many bacteria are now well defined. Application of this technology to clostridia will allow complementation of mutants with the cloned AADC gene. The cloned AADC gene can also be used as a probe for monitoring the presence of the gene in degenerative and mutant strains. Hybridization studies will indicate whether chromosomal rearrangements have resulted in inactivation of the gene, or whether other control mechanisms have been altered, allowing insight into the mechanisms whereby degeneration occurs. Finally, reintroduction of a modified AADC gene may allow the possibility of controlling butanol and acetone production in vivo. ACKNOWLEDGMENTS We thank F. B. Rudolph and B. F. Cooper for the use of the HPLC unit, R. G. Cook for the sequencing of the AADC N terminus, J. W. Cary for his expertise with the phage library, and G. McGaughey for assisting with the manometer assays. This research was supported by a grant from the National Science Foundation (BCS-8912094). D. J. Petersen was supported by a National Science Foundation Predoctoral Fellowship. LITERATURE CITED 1. Adler, H. I., and W. Crow. 1987. A technique for predicting the solvent-producing ability of Clostridium acetobutylicum. Appl. Environ. Microbiol. 53:2496-2499. 2. Andersch, W. H., H. Bahl, and G. Gottschalk. 1983. Level of enzymes involved in acetate, butyrate, acetone, and butanol formation by Clostridium acetobutylicum. Eur. J. Appl. Microbiol. Biotechnol. 18:327-332. 3. Ballongue, J., J. Amine, E. Maison, H. Petitdemange, and R. Gray. 1985. Induction of acetoacetate decarboxylase in Clostridium acetobutylicum. FEMS Microbiol. Lett. 29:273-277. 4. Benton, W. D., and R. W. Davis. 1977. Screening Xgt recombinant clones by hybridization to single plaques in situ. Science

196:180-182. 5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 6. Bullock, W. D., J. M. Fernandez, and J. M. Short. 1987. XL-1 Blue: a high efficiency plasmid transforming recA Escherichia coli strain with ,-galactosidase selection. Biotechniques 5:376379. 7. Cary, J. W., D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett. 1988. Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli. J. Bacteriol. 170:4613-4618. 8. Cary, J. W., D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett. 1990. Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate: coenzyme A-transferase in Escherichia coli. Appl. Environ. Microbiol. 56:1576-1583. 9. Clark, S. W., G. N. Bennett, and F. B. Rudolph. 1989. Isolation and characterization of mutants of Clostridium acetobutylicum ATCC 824 deficient in acetoacetyl coenzyme A:acetate/butyrate:coenzyme A-transferase (EC 2.8.3.9) and other solvent pathway enzymes. Appl. Environ. Microbiol. 55:970-976. 10. Dvies, R. 1942. Studies on the acetone-butyl alcohol fermentation. 2. Intermediates in the fermentation of glucose by Cl. acetobutylicum. 3. Potassium as an essential factor in the fermentation of maize meal by Cl. acetobutylicum (BY). Bio-

3498

PETERSEN AND BENNETT

chem. J. 36:582-599. 11. Davies, R. 1943. Studies on the acetone-butanol fermentation. Biochem. J. 37:230-238. 12. Doremus, M. G., J. C. Linden, and A. R. Moreira. 1985. Agitation and pressure effects on acetone-butanol fermentation. Biotechnol. Bioeng. 27:852-860. 13. El Kanouni, A., A.-M. Junelles, R. Janati-Idrissi, H. Petitdemange, and R. Gray. 1989. Clostridium acetobutylicum mutants isolated for resistance to the pyruvate halogen analogs. Curr. Microbiol. 18:139-144. 14. Fridovich, I. 1963. Inhibition of acetoacetic decarboxylase by anions. J. Biol. Chem. 238:592-598. 15. Frischauf, A.-M., H. Lehroch, A. Poustka, and N. Murray. 1983. Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol. 170:827-842. 16. Hanahan, D. 1985. Techniques for transformation of E. coli, p. 103-135. In D. M. Glover (ed.), DNA cloning, vol. 1. A practical approach. IRL Press, Oxford. 17. Hartmanis, M. G. N., T. Klason, and S. Gatenbeck. 1984. Uptake and activation of acetate and butyrate in Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 20:66-71. 18. Husemann, M. H. W., and E. T. Papoutsakis. 1989. Comparison between in vivo and in vitro enzyme activities in continuous and batch fermentations of Clostridium acetobutylicum. Appl. Microbiol. Biotechnol. 30:585-595. 19. Jones, D. T., and D. R. Woods. 1986. Acetone-butanol fermentation revisited. Microbiol. Rev. 50:484-524. 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)

APPL. ENVIRON. MICROBIOL.

227:680-685. 21. Laursen, R. A., and F. H. Westheimer. 1966. The active site of acetoacetate decarboxylase. J. Am. Chem. Soc. 88:3426-3430. 22. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035-10038. 24. Sancar, A., R. P. Wharton, S. Seltzer, B. M. Kacinski, N. D. Clark, and W. D. Rupp. 1981. Identification of the uvrA gene product. J. Mol. Biol. 148:45-62. 25. Usdin, K. P., H. Zappe, D. J. Jones, and D. R. Woods. 1986. Cloning, expression, and purification of glutamine synthetase from Clostridium acetobutylicum. Appl. Environ. Microbiol. 52:413-419. 26. Warren, S., B. Zerner, and F. H. Westheimer. 1966. Acetoacetate decarboxylase, identification of lysine at the active site. Biochemistry 5:817-823. 27. Weisenborn, D. P., F. B. Rudolph, and E. T. Papoutsakis. 1989. Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids. AppI. Environ. Microbiol. 55:323-329. 28. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119. 29. Zerner, B., S. M. Coutts, F. Lederer, H. H. Waters, and F. H. Westheimer. 1966. Acetoacetate decarboxylase. Preparation of the enzyme. Biochemistry 5:813-816.

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of...
2MB Sizes 0 Downloads 0 Views