APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1990, p. 3760-3765

Vol. 56, No. 12

0099-2240/90/123760-06$02.00/0 Copyright © 1990, American Society for Microbiology

Cloning of a Lactate Dehydrogenase Gene from Clostridium acetobutylicum B643 and Expression in Escherichia coli PAMELA REILLY CONTAG,t MICHAEL G. WILLIAMS,4 AND PALMER ROGERS*

Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 Received 11 June 1990/Accepted 22 September 1990

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pPC58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.

During exponential growth with glucose, Clostridium acetobutylicum carries out an acidogenic fermentation, producing acetate and butyrate. The accumulation of acid products results in a switch to a decelerating growth phase and a solventogenic fermentation, in which acetone, butanol, and ethanol are formed (7). The biochemical pathways for these fermentations are known. Clostridia use the Embden-Myerhoff pathway to convert glucose to pyruvate. Pyruvate is then cleaved by pyruvate ferredoxin oxidoreductase to produce C02, acetyl coenzyme A, and reduced ferredoxin. Acetyl coenzyme A and butyryl coenzyme A serve as the branch points for the production of acids and solvents (12, 21). Under stress conditions such as iron limitation or a high pH, pyruvate is converted to lactate as a major fermentation product, probably because of the induction of lactate dehydrogenase (LDH) (1). Significant progress has been made in the analysis of enzyme activities for both the acidogenic and the solventogenic phases in C. acetobutylicum (19, 21). However, the only clostridial LDH activities to be partially purified and characterized are LDH from C. acetobutylicum (9) and two LDH isoenzymes found in C. thermohydrosulfuricum (24). These clostridial LDHs have properties very different from those of the three LDHs found in Escherichia coli (16, 23); however, they do share some characteristics in common with the LDHs of other gram-positive anaerobes, such as activation by fructose 1,6-diphosphate (FDP) (9, 10, 24). Many of the genes of C. acetobutylicum concerned with fermentative metabolism have been cloned and expressed in E. coli, and a few have been sequenced (26). In the present work, LDH from C. acetobutylicum was cloned by complementation of fermentation mutants of E. coli. This approach was used because of the lack, at present, of a good system for the identification of cloned genes in C. acetobutylicum. The cloned clostridial LDH was expressed in E. coli LDH-

deficient mutants and characterized as a nonreversible, FDP-activated enzyme. MATERIALS AND METHODS Bacterial strains and plasmids. C. acetobutylicum B643, a strain producing high levels of solvents, was obtained from L. K. Nakamura (Northern Regional Research Center, Peoria, Ill.) and preserved as spores or frozen in 10% glycerol at -70°C (8). The relevant genotypes and sources of the E. coli strains used are given in Table 1. The plasmids used for cloning and subcloning were pBR322 (New England BioLabs) and pUC19 (25). Growth and selection media. All clostridial strains were grown anaerobically at 35°C (anaerobic chamber from Forma, Marietta, Ohio) in clostridial basal medium (18) or in yeast extract medium (18). E. coli was grown anaerobically at 35°C or aerobically at 37°C in Luria-Bertani broth (LB), M9 minimal medium plus 1% glucose (15), or tetrazolium-ethanol medium (3). For the maxicell experiments, E. coli was grown in M9/MF medium, which is M9 medium plus glucose and fortified with a complete L-amino acid-vitamin mixture lacking L-methionine (17). The pH of each medium was adjusted to 7.0. For the agar base, 1.5% agar (Difco) was added. Any additions for specific strains are described in the references indicated in Table 1. Antibiotics were used at the following concentrations: ampicillin, 50 ,ug/ml; chloramphenicol, 10 jig/ml; and tetracycline, 12 ,ig/ml. Isolation of DNA from C. acetobutylicum. All restriction enzymes and cloning enzymes were obtained from IBI and used in accordance with the manufacturer's instructions. C. acetobutylicum was grown to the mid-log phase in 500 ml of clostridial basal medium with 1% glucose. The culture was centrifuged in a Sorvall GSA rotor at 10,000 rpm for 15 min. The pellets were pooled and suspended in a total of 40 ml of clostridial basal medium with 10% sucrose. Lysozyme at a final concentration of 2 mg/ml was added, and the cells were converted to protoplasts by incubation at 37°C for 1 h. The protoplasts were centrifuged in a Sorvall SS34 rotor at 4,000 rpm for 10 min and suspended in 10 ml of CBM-0.3 M

* Corresponding author. t Present address: Department of Microbiology and Immunology,

Stanford University School of Medicine, Stanford, CA 94305. t Present address: 3M Corp., St. Paul, MN 55144.

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TABLE 1. E. coli strains Strain

Relevant marker(s)a

Reference

DC272 PRC436 LCB898 FMJ39 HB101 JM109 MC4100

adhC adh+ acd+ adhC adh+ acd pfl ldh+ pfl Idh recA13 supE44 recAl lac A(lac)U169

6 6 16 16 15 25 4

a adh, Alcohol dehydrogenase; acd, aldehyde dehydrogenase; adhC, regulatory gene for aerobic respression of adh and acd; pfl, pyruvate formate lyase; Idh, lactate dehydrogenase.

sucrose. Pronase (5 mg) or proteinase K (200 ,g) and 1.2 ml of sodium dodecyl sulfate (SDS) (20%) were added to the protoplasts. The lysate was extracted once with an equal volume of phenol-saturated Tris hydrochloride (100 mM, pH 7.4). The aqueous phase was removed and placed in a clean sterile beaker, sodium acetate at a final concentration of 0.3 M was added, and the mixture was mixed well. Two volumes of absolute ethanol were layered on top of the aqueous phase. The DNA was spooled from the interface with a heat-sealed Pasteur pipette, washed in 70% ethanol, dried slightly, dissolved in sterile distilled water, and spooled again as described above. The quantity and purity of DNA were assessed spectrophotometrically. Formation of a C. acetobutylicum genomic library from size-selected DNA. Size selection of C. acetobutylicum DNA Sau3A fragments was accomplished on an NaCl gradient as described by Kaiser and Murray (13). A DNA salt gradient fraction in the size range of 5 to 7 kb or 3 to 5 kb was selected and ligated into pBR322 or PUC19 by standard methods (15, 25). Five-microliter samples of the ligation mixtures were used to transform E. coli JM109 or PRC436. Transformation into E. coli and screening for C. acetobutylicum fermentation genes. Transformations of E. coli with plasmids containing C. acetobutylicum DNA were carried out by the method of Hanahan (11). Individual clones in JM109 were grown overnight in 1 ml of LB-ampicillin medium. Clones were pooled (50 ,ul each) in groups of 30, and total plasmid DNA was isolated by an alkaline lysis procedure (2). The pooled plasmids were used to transform E. coli PRC436. Following a recovery period of 1 h at 37°C, the transformed cells were washed three times in M9 minimal medium and plated onto M9 minimal medium with 1% glucose and ampicillin (50 ,ug/ml), LB-ampicillin, or tetrazolium-ethanol-ampicillin. The M9 minimal medium-glucose-ampicillin plates were incubated anaerobically at 35°C, and the LB-ampicillin plates were incubated aerobically at 37°C. Colonies that grew anaerobically after 48 h were restreaked on minimal medium-glucose-ampicillin and LBampicillin, and the plasmid was isolated, back-transformed into the host strain, and grown as described above. The back-transformants were tested for enzyme activity and fermentation products. Plasmids from the back-transformants were isolated and subjected to restriction analysis under the enzyme reaction conditions specified by the manufacturer of the enzymes (IBI). Preparation of DNA probes and hybridization. Plasmids pPC37 and pPC58 were isolated by the alkaline lysis procedure (2) and digested with restriction enzymes EcoRI and BamHI. EcoRI-BamHI fragments were separated by agarose gel electrophoresis, eluted from the agarose by electroelution, and purified by standard methods (15). Probes 37 and 58 were prepared as 32P-DNA fragments by nick trans-

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lation by a procedure modified from that of Rigby et al. (20). Electrophoresis of DNA, transfer of DNA onto filter membranes, and hybridization with 32P-DNA probes were done as described by Maniatis et al. (15). Since Gene Screen Plus (DuPont) was used as the filter membrane, an alkaline transfer buffer was used in accordance with the manufacturer's instructions. Gas chromatography for determination of fermentation products. Acids were extracted from growth media (20-h cultures) by adding to 0.9 ml of the sample beer 0.8 g of NaCl, 0.3 ml of 9 M H2SO4, and 1.5 ml of diethyl ether. The tubes containing this mixture were capped and shaken vigorously, the ether layer (top layer) was pipetted off and placed in an Eppendorf tube, and a small amount of anhydrous CaCl2 was added to remove excess H20. Solvents were extracted from 0.9 ml of the sample beer by adding NaCl to saturation (-0.8 g), 0.3 ml of 5 M NaOH, and 1.5 ml of cyclohexanone. The tubes were capped and shaken vigorously and, if necessary, centrifugation was used to separate layers. The top layer was transferred to an Eppendorf tube, and CaCl2 was added as described above. Methyl derivatives of nonvolatile acids such as lactate were made by adding to 1 ml of the culture medium 2 ml of methanol and 0.4 ml of 50% H2SO4. This solution was mixed well and heated at 60°C for 30 min. Following this incubation, 1 ml of distilled H20 and 0.5 ml of chloroform were added, the mixture was shaken vigorously, and the bottom (chloroform) layer was transferred to an Eppendorf tube. Volatile acids, methylated acids, and solvents were measured with a Packard model 427 gas-liquid chromatograph with a flame ionization detector. For measurements of volatile acids, 2 to 10 ,ul of ether extract was injected into a glass column (1.8 m by 2 mm [inner diameter]) packed with Supelco 1-1841 (10% SP-100-1% H3PO4 on Chromosorb W) at 140°C, with He carrier gas at a 20-mllmin flow rate. Propionic acid was added as an internal standard for quantitation. For measurements of methylated acids or solvents, 2 to 10 ,ul of chloroform extract or cyclohexanone extract was injected into a glass column (1.8 m by 2 mm [inner diameter]) packed with Supelco 1-1813 (80/120 Carbopak-3% SP-1500), with a thermal program from 100 to 180°C over 8 min for solvents and 12 min for methylated acids and with He carrier gas at a 15-m/min flow rate. Solvents were quantified with propanol as an internal standard, and methylated acids were quantified with methylated lactic and succinic acids as standards. Enzyme assays. Crude extracts were prepared from overnight cultures (20 ml) by harvesting cells and suspending the pellet in 1 ml of sonication buffer (50 mM potassium phosphate) [pH 7.4], 0.01 M dithiothreitol, 20 ,ug of Leupeptin, 9.5 ml of H20). Cells were sonically disrupted at 0°C by two 20-s bursts at 1.25 mA with a Mullard Scientific Equipment Sonifier. Cell debris was removed by centrifugation at 15,000 rpm for 30 min in a Sorvall SS34 rotor. LDH activity in crude extracts was assayed for lactate oxidation to pyruvate at pH 8.5 or for pyruvate reduction to lactate at pH 7.0. The assay mixture for lactate oxidation (1 ml) contained 10 mM dithiothreitol, 50 mM sodium 2[Ncyclohexylaminolethane sulfonate (CHES) buffer (pH 8.5), 10 mM NAD, and 100 to 500 ,ug of crude extract protein. The substrate, 50 mM sodium lactate, was added last. The assay mixture for pyruvate reduction (1 ml) contained 10 mM dithiothreitol, 50 mM sodium morpholinopropane sulfonate (MOPS) buffer (pH 7.0), 10 mM NADH, and 10 to 50 ,ug of crude extract protein. Sodium pyruvate, 50 mM, was added to the assay mixture to start the reaction. FDP (1 mM) was added to some reaction mixtures to activate LDH. All

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TABLE 2. Effect of C. acetobutylicum clone pPC37 expression in E. coli on fermentation products and LDH activity Concn (mM) of the following E. coli producta: strain s.rm Ethanol Acetic Lactic Succinic

lacid

acid

acid

3.2 0.2 3.0

16 37 12

1.5 2.2 3.0

Et

DC272 PRC436(pPC37) PRC436

8.5 0.22 0.7

LDH sp act of protein)b

TABLE 3. Production of lactate and succinate and levels of LDH in an E. coli LDH-deficient mutant with clones pPC37 and pPC58 Lactate Pheotye'(mM)c

E. E. listrin' Phenotypeb ccli ostraina

(nmol/min/mg 31 67 49

a Products were determined by gas chromatography (as described in Materials and Methods) for extracts of strains grown for 20 h in minimal salts medium with 1% glucose. Strain PRC436 was grown anaerobically in LB. I Strains used in enzyme assays were grown anaerobically for 20 h in LB, washed, and suspended in buffer containing MgSO4 and 2% glucose. After 24 h of anaerobic incubation, cells were harvested, sonically disrupted, and centrifuged to clarify the lysate. LDH activity was monitored by the formation of NADH at 340 nm after the addition of sodium lactate (50 mM). The total protein of the clarified lysate was measured spectrophotometrically after the addition of BioRad protein assay reagent.

reactions were monitored with a spectrophotometer (LKB Ultrospec Plus 4054) at 340 nm to determine the rates of reduction or oxidation of NAD or NADP. Activities are reported as nanomoles per minute per milligram of protein. Protein determinations were done with the Bio-Rad miniprotein assay by following the manufacturer's instructions. Maxiceli analysis. Plasmids pPC37 and pPC58 were transformed into E. coli MC4100. E. coli MC4100(pPC37) and MC4100(pPC58) were grown in M9/MF medium and maxicells were produced by a modification of the method of Sancar et al. (22). Following UV irradiation, cycloserine (120 jig/ml) was added to reduce the background labeling of proteins with [35S]methionine. The 35S-labeled proteins of maxicell lysates were separated in 12% SDS-polyacrylamide gels at a current of 25 mA for 3 h by the method of Laemmli (14). Gels were dried onto bond paper and exposed to X-ray film or stained with Coomassie blue R-250. RESULTS Screening of C. acetobutylicum by complementation of E. coli PRC436. Since Clark and co-worker (5, 6) showed that E. coli mutants lacking aldehyde dehydrogenase or alcohol dehydrogenase were incapable of anaerobic growth on glucose, we used E. coli PRC436, which is acd (Table 1), to screen for clostridial clones that allowed E. coli PRC436 to grow anaerobically on glucose. We found that the ligation products transformed strain PRC436 inefficiently; therefore, DNA from the gene library was first transformed into E. coli JM109, pooled, and then transformed into E. coli PRC436 (see Materials and Methods). A total of 107 pools of 30 clones each were made. Two plasmids from independent pools, pPC37 and pPC58, complemented E. coli PRC436 for anaerobic growth on minimal medium-glucose-ampicillin. Fermentation products and enzyme analysis of E. coli PRC436(pPC37). Gas chromatographic analysis demonstrated that the parental strain, E. coli DC272, displayed the typical mixed acid fermentation products, while E. coli PRC436(pPC37) produced very low amounts of ethanol and twice as much lactic acid as did strain DC272 (Table 2). This observation suggested that we had cloned and expressed an LDH gene from C. acetobutylicum in E. coli, thus permitting the synthesis of additional lactic acid. The production of this additional lactic acid was apparently sufficient to permit anaerobic growth on glucose. LDH activity was assayed in crude cell extracts as described in Materials and Methods. E.

LCB898 DC272 FMJ39 FMJ39(pPC37)

FMJ39(pPC58) FMJ39(pPC582.1)

Pfl- Ldh+ AdhcPfl- Ldh-

45 35 0 25 51 68

Succinate (mM)c

LDH activity (nmol/min/mg

3 3 1 1 3 2

74.0 9.3 2.0

of protein)'

0.5 0.2 0.05

a FMJ39 was grown aerobically; all other strains were grown anaerobically in minimal medium plus 1% glucose and required supplements. b Pfl-, Pyruvate formate lyase negative; Ldh+ and Ldh-, LDH positive and negative, respectively; Adhc-, derepressed for alcohol dehydrogenase and aldehyde dehydrogenase under aerobic conditions. c Lactate and succinate were determined by gas chromatography of methylated acids. d LDH activity was determined by assay in the direction of lactate oxidation to pyruvate (see Materials and Methods).

coli PRC436(pPC37) crude extracts had greater than twofold the LDH specific activity as had DC272. However, crude extracts of strain PRC436 without pPC37 also contained high levels of LDH (Table 2). Complementation of an LDH-deficient mutant of E. coli with C. acetobutylicum clones pPC37 and pPC58. To lower the background of LDH produced by E. coli, we transformed plasmids pPC37 and pPC58 into an LDH-deficient E. coli mutant, strain FMJ39. This E. coli LDH-deficient strain does not grow anaerobically on glucose, since it is pfl, and it requires added acetate for aerobic growth on glucose (14). These strains, as well as two E. coli strains, LCB898 (pfl) and DC272 (pfl+), expressing LDH activity during anaerobic growth, were assayed for lactate production and LDH activity (Table 3). The levels- of lactate produced by strains LCB898 and DC272 were 45 and 35 mM, respectively. Strain FMJ39 (pfl ldh) produced no lactate, whereas strain FMJ39 containing pPC37, pPC58, or pPC582.1 produced at least 25 mM lactate (Table 3); plasmid pPC582.1 is an EcoRI-BamHI subclone of pPC58 into pUC19 (Fig. 1). Lactate levels were not enhanced when isopropylthio-p-D-galactoside was added to activate the lac promoter in pUC19 (data not shown). LDH activities in crude cell extracts were assayed in the direction of lactate oxidation to pyruvate. Although the LDH-deficient mutant showed a significant increase in the formation of lactate when transformed with clones pPC37 and pPC58, extracts of these transformants showed little or no increase in LDH activity (Table 3). C. acetobutylicum B643 also showed no LDH activity in crude extracts when assays were done in the direction of lactate oxidation to pyruvate. However, positive results were obtained when LDH activities were assayed in the direction of pyruvate reduction to lactate in crude extracts of C. acetobutylicum B643 and of E. coli containing pPC37 and pPC58. C. acetobutylicum B643 showed a similar LDH activity when assays were done in the direction of pyruvate reduction whether this strain was grown in yeast extract medium or in minimal medium with low iron and high phosphate concentrations. However, when activated by FDP, B643 crude extracts from minimal medium had ninefold-higher LDH activity than did unactivated crude extracts. B643 grown in yeast extract medium had LDH activity levels that increased only 2.5-fold upon activation with FDP (Table 4). It is known that C. acetobutylicum (9) and C. thermohydrosulfuricum (24) possess LDHs that are strongly activated by FDP and that the E. coli LDHs are not activated by FDP (10).

C. ACETOBUTYLICUM LACTATE DEHYDROGENASE

VOL. 56, 1990 (BamHD)

pPC58 (10kb)

Hind D

aRm BomHI

Ec

"I"

into

pUCI9

Bom HI

FIG. 1. Overlapping fragments of clones pPC37 and pPC58. Restriction maps of pPC37 and pPC58 were determined by single and double digestion of the plasmids with various restriction enzymes and ordering of the resulting fragments according to their mobility in a 0.7% agarose gel. Plasmid pPC58.2.1 is pUC19 containing the 2.1-kb EcoRI-BamHI fragment of pPC58. Probe 37 is a 4.0-kb fragment made by digestion of pPC37 with EcoRI and BamHI (see Materials and Methods). Probe 58 is a 2.1-kb fragment made by digestion of pPC58 with EcoRI and BamHI (see Materials and Methods). Parentheses around BamHI indicate that the BamHI site was not regenerated by ligation to a Sau3A site.

No LDH activity was found in crude extracts of E. coli FMJ39 (ldh) when assays were done in the direction of pyruvate reduction or lactate oxidation, while crude extracts of E. coli LCB898 (ldh+) had higher levels of LDH activity when assays were done in the direction of pyruvate reduction to lactate. However, the E. coli LDH was not activated TABLE 4. Comparison of LDH activities in C. acetobutylicum and E. coli with clones pPC37 and pPC58 LDH activity Growth medium

Straina

C. acetobu-

tylicum

(nmol/min/mg of

protein)b Plus Control

Yeast extract medium-1% glucose

16

41

Minimal medium (low iron, high P04)-1% glucose

14

131

1,200

32,000

860

13,000

0 2,400

0 1,600

B643

E. coli

FMJ39(pPC37) Minimal medium-1% glucose

FMJ39(pPC58) Minimal medium-1% glucose

FMJ39 LCB898

LB-1% glucose LB-1% glucose

Cultures were grown anaerobically at 35°C. LDH activity was assayed in the direction of pyruvate reduction to lactate (see Materials and Methods). c FDP (1 mM final concentration) was added as part of the reaction mixture, and the reaction was started with the addition of sodium pyruvate (50 mM) (see Materials and Methods). a

b

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in the presence of FDP (Table 4). In contrast, crude extracts of E. coli FMJ39 containing clones pPC37 and pPC58 had LDH activities of about 1 ,umol/min/mg at pH 7.0. These enzyme activities increased from 28- to 15-fold when FDP was added during the assay (Table 4). These data indicated that the two plasmids carried the gene for a C. acetobutylicum LDH that was expressed in E. coli. Overlapping fragments of pPC37 and pPC58. To compare and align the fragments in the LDH clones, we performed a restriction analysis of pPC37 and pPC58 with enzymes EcoRI, HindIII, and BamHI (these overlapping fragments are represented by crosshatched areas in Fig. 1). To show which portion of clone pPC37 overlapped with pPC58, we hybridized a Southern blot of HindIII-cut pPC37 to the EcoRI-BamHI fragment of pPC58 (probe 58 in Fig. 1). The probe 58 fragment of pPC58 hybridized to the 4-kb HindIII fragment of pPC37 but not to the 6.2-kb HindIII band representing about 1.8 kb of insert and 4.3 kb of pBR322 (data not shown). Maxicell analysis of clones pPC37 and pPC58. Maxicell analysis for plasmid-encoded proteins revealed that pPC37 expressed two proteins not encoded by pBR322 alone (Fig. 2). Plasmid pBR322 without an insert encodes the ampicillin resistance gene, which codes for a P-lactamase with an apparent molecular mass of about 26 kDa, and the tetracycline resistance gene, which codes for a protein of approximately 30 kDa. Clone pPC37 expressed the pBR322 P-lactamase (Apr) but not the Tetr gene product. It did, however, express two new proteins with molecular masses of 36 and 38 kDa on the SDS gel. Maxicell analysis of clone pPC58 also revealed the expression of 38- and 40-kDa proteins (data not shown). Hybridization of pPC37 to E. coli and C. acetobutylicum genomic DNA. Genomic DNA from E. coli DC272 and C. acetobutylicum B643 was cut with BglII, EcoRI, and HindIII, separated by gel electrophoresis, transferred by blotting to nitrocellulose filters, and hybridized with probe 37 (Fig. 1). Probe 37 did not hybridize to E. coli genomic DNA (Fig. 3B, lanes 1, 2, and 3). However, probe 37 did hybridize to two C. acetobutylicum BglII fragments of approximately 9.4 and 7.0 kb, one EcoRI fragment of 8.0 kb, and one HindlIl fragment of 9.4 kb (Fig. 3B, lanes 4, 5, and 6). Each genomic digest (Fig. 3) yielded the number of hybridized fragments predicted by the pPC37 restriction map (Fig. 1).

DISCUSSION Clones of C. acetobutylicum DNA that were isolated by complementation of a fermentation-deficient E. coli strain, PRC436, allowed anaerobic growth in a minimal medium with glucose. Since strain PRC436 (acd) lacked aldehyde dehydrogenase and did not produce ethanol (6), our original aim was to select clones expressing the C. acetobutylicum butyraldehyde dehydrogenase. However, strain PRC436, carrying C. acetobutylicum clone pPC37 or pPC58, produced no significant ethanol but rather produced an excess of lactic acid (Table 2). We showed that these same clones complemented E. coli FMJ39 (ldh pft) to allow anaerobic growth on glucose plus added acetate, and again significant lactic acid was produced (Table 3). These complementation data suggest that the amount of C. acetobutylicum LDH expressed from a multicopy plasmid is sufficient to allow recycling of NADH, necessary to support the anaerobic growth of E. coli FMJ39 and PRC436. Furthermore, since E. coli PRC436 is ldh+ and pfl+ but cannot produce ethanol

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B

A 1

2 3 4 5 6

23ow som

9.4-

w

6.4-

2.32.0-

FIG. with

gel.

a

3. E. coli and C. acetobutylicum genomic DNA probed fragment of pPC37. (A) Ethidium bromide-stained agarose

Lanes 1 to 3 show E.

HindIII,

coli

genome DNA cut with

Bglll,

EcoRI,

acetobutylicum genomic DNA cut with Bglll, EcoRI, and HindIII, respectively. (B) Autoradiogram of a DNA blot of the gel in panel A hybridized with 32P-labeled probe 37. Numbers are kilobases.

and

FIG. 2. Maxicell analysis of LDH clone pPC37. Plasmid-encoded proteins were labeled with [3S]methionine by the method of Sancar et al. (22). Cell extracts were electrophoresed on a 12% SDS-polyacrylamide gel. The gel was dried onto Whatman 3 MM filter paper and exposed to XAR-S film for 43 h at -100°C. All plasmids were expressed in E. coli MC4100, a host strain used for maxicell analysis. Plasmid pBR322 (lane A) expresses two proteins, a 26-kDa protein (ampicillin resistance) and a 30-kDa protein (tetracycline resistance). Clone pPC37 (lane B) expresses the 26-kDa protein, a faint 36-kDa protein, and a 38-kDa protein.

(acd), Clark and co-workers proposed that the pyruvate formate lyase competes with the cell LDH for pyruvate, preventing anaerobic growth on glucose because sufficient lactate is not produced (5, 16). Our data suggest that the cloned C. acetobutylicum LDH expressed in strain PRC436 can compete with E. coli pyruvate formate lyase, thus producing sufficient lactic acid to support anaerobic growth. Our data indicate that the C. acetobutylicum LDH is different from the E. coli LDH. LDH activities in crude extracts were determined in both directions and tested for activation by FDP in the direction of pyruvate reduction. As reported previously, the E. coli fermentative LDH is moderately reversible and is not activated by FDP (5, 10). LDH activities assayed in E. coli containing the cloned C. acetobutylicum LDH (Tables 3 and 4) indicate that this LDH is a

respectively.

Lanes 4 to 6 show C.

nonreversible enzyme that operates in the direction of pyruvate reduction to lactate and that is activated by FDP. The importance of FDP activation in differentiating various LDH enzymes was pointed out by Garvie, who also suggested that all known FDP-activated LDHs are found in gram-positive bacteria (10). Our data are consistent with results obtained from an analysis of a partially purified C. acetobutylicum LDH that is nonreversible and FDP activated (9). Turunen et al. (24) characterized two C. thermohydrosulfuricum LDH isoenzymes. One isoenzyme, LDHE, showed an almost absolute requirement for FDP. Maxicell analysis of the pPC37 and pPC58 plasmid-encoded proteins revealed a protein band in common at an approximate mass of 38 kDa in SDS-polyacrylamide gel electrophoresis. This mass is also consistent with a mass of 159 kDa for the native LDH and a mass of 36 kDa for the SDS-treated LDH of C. acetobutylicum reported previously (9). Restriction endonuclease mapping of pPC37 and pPC58, probing of Southern blots with radiolabeled DNA pieces from the two clones, and preparation of a subclone, pPC582.1, expressing LDH activity in E. coli suggest that a single LDH gene from C. acetobutylicum is carried by a 2.1-kb region of these clones. ACKNOWLEDGMENTS This study was supported by grant DE-FG02-86ER13512 from the Division of Energy Biosciences, U.S. Department of Energy, and a grant-in-aid from the Graduate School, University of Minnesota. We thank David E. Clark for supplying us with the E. coli mutant strains used in this study.

VOL. 56, 1990

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