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Applied and Environmental Microbiology, May 2003, p. 2831-2841, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2831-2841.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Expression of a Cloned Cyclopropane Fatty Acid Synthase Gene Reduces Solvent Formation in Clostridium acetobutylicum ATCC 824

Yinsuo Zhao, Lucia A. Hindorff, Amy Chuang, Melanie Monroe-Augustus, Michael Lyristis, Mary L. Harrison, Frederick B. Rudolph, and George N. Bennett*

Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005

Received 17 December 2002/ Accepted 26 February 2003


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ABSTRACT
 
The cyclopropane fatty acid synthase gene (cfa) of Clostridium acetobutylicum ATCC 824 was cloned and overexpressed under the control of the clostridial ptb promoter. The function of the cfa gene was confirmed by complementation of an Escherichia coli cfa-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing cfa were introduced into C. acetobutylicum hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the cfa gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the cfa-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of cfa results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a marR homologous gene preceding the cfa gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.


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INTRODUCTION
 
Clostridium acetobutylicum is a gram-positive, spore-forming, obligate anaerobe that is able to ferment various sugars to form the widely used solvents acetone and butanol. The commercial use of acetone-butanol fermentation ceased in most countries in the early 1960s due to the inability to compete economically with petrochemical sources, but recent developments in the molecular genetics of solventogenic clostridia have revived interest in this fermentation process (27, 33, 39). An important limiting factor in the economics of batch fermentation processes is the low concentration of products, which affects recovery costs. The main fermentative product, butanol, is quite toxic to cells. Other products, including acetate, butyrate, ethanol, and acetone, are also growth inhibiting but to a lesser extent.

The effects of ethanol have been reviewed (26); the toxicity of alcohols is correlated with chain length, with the longer-chain alcohols being toxic at a lower concentration (28). Alcohol inhibition has been suggested to be the result of damage to the semipermeable property of the cell membrane (38) and the direct inhibition of metabolism (24). An increase in fatty acid chain length seems to be generally related to ethanol tolerance (26), and the very alcohol tolerant Lactobacillus heterohiochii contains C20 to C30 chains (49). The unsaturated fatty acid acyl chains of bacterial membrane phospholipids have a major influence on membrane properties. In cis -> trans isomerization, catalyzed by cis-trans isomerase, the double bond is reconfigured. The cis-unsaturated chain contains a bend which increases membrane fluidity, whereas the trans-isomer is more adapted for increased rigidity and is associated with increased ethanol tolerance in Escherichia coli (26). For Pseudomonas strains with a high resistance to organic solvents such as toluene and xylene, several adaptive mechanisms are involved, including solvent efflux pumps and cis-trans isomerization of unsaturated fatty acids (41). In C. acetobutylicum, efflux pumps might exist, based on homology searches of the recently published clostridial genome (37), although no demonstration of their function has been reported. But no cis-trans isomerization appears as a solvent resistance mechanism in C. acetobutylicum because no cis-trans isomerase homolog exists and trans-fatty acids have not been reported.

In C. acetobutylicum, butanol specifically inhibits membrane-related functions, such as active nutrient transport, the membrane-bound ATPase activity, and glucose uptake (6). Butanol has also been found to affect the composition and decrease the fluidity of the cell membrane (4, 28, 50). In response to the presence of butanol, C. acetobutylicum increases both the proportion of saturated fatty acids and the mean acyl chain length of fatty acids in its membrane (28). Butanol in the medium increases the proportion of cyclopropane fatty acids (CFAs) at the expense of unsaturated chains (50).

CFAs occur in many bacterial cell membranes and are synthesized from unsaturated fatty acid acyl chains of membrane phospholipids by CFA synthase, which adds a methylene carbon bridge from S-adenosyl-L-methionine (SAM) across the double bond (19). CFA composition increases in response to osmotic stress (20, 35) and high temperature (13). An E. coli cfa mutant survives poorly in 20% ethanol or when exposed to acidic conditions (pH 3) (9). Therefore, cyclization of fatty acid acyl chains is generally regarded as a means to reduce membrane fluidity and prevent the penetration of undesirable molecules in order to adapt the cells for adverse conditions (19).

It was initially considered that overexpression of CFA synthase and the concomitant change in lipid composition might increase solvent tolerance and allow higher solvent production in C. acetobutylicum. However, the results presented here suggest a more complex interaction between CFA content and cell physiology related to solvent production in batch culture.


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MATERIALS AND METHODS
 
Bacteria and plasmids.
All bacterial strains and plasmids used in this study are listed in Table 1.


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  		TABLE 1. Bacterial strains and plasmids

Growth conditions.
E. coli was grown aerobically at 37°C in Luria-Bertani (LB) medium, C. acetobutylicum was grown anaerobically at 37°C in clostridium growth medium (CGM) (23). For E. coli recombinant strains, the medium was appropriately supplemented with ampicillin (100 µg/ml), chloramphenicol (35 µg/ml), kanamycin (35 µg/ml), and erythromycin (300 µg/ml). For C. acetobutylicum recombinant strains, erythromycin (40 µg/ml) and thiamphenicol (25 µg/ml) were used. For long-term storage of C. acetobutylicum, strains were cultivated and stored in ampoules after lyophilization.

Static flask fermentation and product analysis.
Cultures of C. acetobutylicum were inoculated with 1% overnight culture in 100 ml of CGM medium with appropriate antibiotics in 150-ml static flasks and grown at 37°C in an anaerobic chamber. A 10-ml sample was taken from each flask during growth. The cells were collected by centrifugation at 10,000 x g for 10 min at 4°C, and the pellet was then frozen at -20°C for fatty acid analysis or ß-galactosidase enzyme assay. The supernatant fluids were stored at -20°C for analysis of the concentrations of butanol, acetone, ethanol, butyrate, and acetate by gas chromatography with a Hewlett-Packard 5890 as described previously (16). The variability of values for individual components was less than 10%.

Fatty acid analysis.
E. coli strains were grown overnight in LB medium. C. acetobutylicum cultures were grown in static flasks or a bioreactor, and samples were removed at the various growth stages. Cells were harvested and stored at -20°C. Fatty acid methyl esters were prepared from intact cells rather than the cell membrane because no statistical difference was observed in early reports with C. acetobutylicum ATCC 824 (4). Fatty acid methyl esters of whole cells were prepared and analyzed by gas chromatography (32) at Microbial ID, Inc. (Newark, Del.), a professional company for fatty acid analysis. The composition of individual fatty acids was calculated as a percentage of total fatty acids identified. The variability of values for fatty acid composition reported in each figure was less than 5%.

Growth rate of E. coli in ethanol and propanol.
An inoculum of 1% overnight cultures was added to fresh LB medium containing 5% ethanol or 2.5% propanol with and without antibiotics. A spectrophotometric reading was immediately taken at 600 nm and termed the 0-min reading. Subsequent readings were taken at intervals for 6 h, and the doubling time was calculated.

Butyric acid and butanol shock.
Butyric acid or butanol was added to early-exponential-phase cultures in CGM containing appropriate antibiotics. The optical density at 600 nm (OD600) was monitored over time.

{alpha}-Amylase activity test.
{alpha}-Amylase activity was detected by the method described previously (22, 43). The cells were diluted and plated on 2YTGMA medium, and the plates with colonies (less than 30 on each plate) were overlaid with iodine solution (5% solid iodine, 10% potassium iodine). The colonies with {alpha}-amylase activity showed starch-hydrolyzing ability, i.e., the colonies were surrounded by a clear halo against a blue background, indicating the presence of the {alpha}-amylase encoded by a gene on the pSOL1 plasmid, demonstrating that the cells had not degenerated (22, 43). Colonies of a degenerated strain, M5 (10), did not show haloes.

DNA isolation, manipulation, and transformation.
C. acetobutylicum ATCC 824 chromosomal DNA was prepared with the Puregene genomic DNA purification kit (Gentra System, Minneapolis, Minn.). E. coli plasmid DNA was prepared with the Qiaprep miniprep and midiprep kits (Qiagen, Valencia, Calif.) following the manufacturer's instructions. Individual DNA fragments were isolated by agarose gel electrophoresis and extracted from the gel with the QIAquick gel extraction kit (Qiagen, Valencia, Calif.). Restriction endonucleases, T4 ligase, and T4 polymerase were purchased from New England Biolabs (Beverly, Mass.) and used according to the manufacturer's specifications. Transformation of E. coli was conducted by electroporation. In preparation for transformation into clostridial cells, plasmid DNA was methylated in E. coli DH10B containing the plasmid pDHKM. Approximately 15 µg of methylated plasmid DNA was used to electrotransform C. acetobutylicum as described previously (31).

Cloning of cfa gene from C. acetobutylicum.
The cfa gene was cloned by PCR amplification from C. acetobutylicum ATCC 824 chromosomal DNA. The forward primer (FP1) was the oligonucleotide 5'-ATA GGA TCC AAC AAA AAA ATA GGA GTA, and the reverse primer (LN7) was TCT GAA TTC CTC CTA CCG TAT AGG. BamHI and EcoRI sites were introduced in the primers (italic) to aid subsequent manipulation. The PCR-amplified fragment without the restriction enzyme sites corresponded to positions 1728 to 3673 in the C. acetobutylicum genome sequence (37) with GenBank accession no. AE007603. The fragment also included the coding region and a 27-bp noncoding region, containing a putative ribosome-binding site, GGAGTA, but no -35 or -10 elements, of the marR-like gene which preceded the cfa gene.

The resulting 1.95-kb fragment was ligated to the corresponding cloning sites of the shuttle plasmid pSOS84 (52) after digestion with BamHI and EcoRI to form pSOS84-cfa. This construct placed the cfa gene under control of the C. acetobutylicum ATCC 824 phosphotransbutyrylase (ptb) promoter, which contains -35 and -10 elements but no ribosome-binding site, and acts as a strong and constitutive promoter in both E. coli and C. acetobutylicum. The sequence of the cloned PCR amplification product was confirmed by DNA sequencing with an ABI 377 sequencer at Lone Star Labs (Houston, Tex.). A few nucleotide variations were observed which were different from the whole genome sequence but did not change the amino acids encoded.

Construction of pDHKM.
When the plasmids were transformed into C. acetobutylicum, usually they were first methylated by growth in E. coli(pAN1). The plasmid pAN1 expresses the Bacillus subtilis phage {phi}3TI methyltransferase, which protects the plasmid DNA from restriction by the clostridial endonuclease Cac824I (30). However, the pAN1 plasmid (encoding chloramphenicol resistance) can be lost when used in the methylation of chloramphenicol-resistant plasmids. Therefore, a new plasmid for methylation was constructed. A pAN1-derived plasmid, pBA (56), was constructed by cloning the 2.6-kb ClaI fragment containing the {phi}3TI methyltransferase into the ClaI site of pBR322. Plasmid pBA was digested with EcoRI and BamHI, and the 3.4-kb fragment containing the {phi}3TI methyltransferase was recovered and inserted into the corresponding sites of pDHK29 to yield pDHKM. pDHK29 encodes kanamycin resistance and contains the replicon of a high-copy-number mutant of RSF1030 that is able to coreside with ColE1-, pMB1-, p15A-, and pSC101-derived plasmids (40).

Construction of shuttle vector pSC12.
The pSC12 vector was obtained by replacing the erythromycin resistance determinant in pSA12 with one for chloramphenicol (or thiamphenicol) resistance. The pSA12 vector (3.7 kb) was constructed by special assembly (53) of the 1.6-kb PCR-amplified fragment containing the E. coli ColE1 ori, multiple cloning site, and lacZ{alpha} from pBluescript SK(+) (Stratagene) with primers PBLFD (5'-AGC CGG CuC CCT CCC GTA TCG TAG TTA TCT-3') and PBLRP (5'-AAG TAC TuC GTA ACC ACC ACA CCC GCC GCGC-3'), and the 2.1-kb PCR-amplified fragment containing the erythromycin resistance determinant and the pIM13 (34) ori from pSOS84 (52) was constructed with the primers PSOS84FP (5'-AAG TAC TuC GCC ATT CCA ACC AAT AGT T-3') and PSOS84RP ('-AGC CGG CuC CTC TTC GCT ATT ACG CCA GCT G-3'). (The deoxyuracils in the oligonucleotides are shown in lowercase.)

The chloramphenicol (or thiamphenicol) resistance gene (catP), originally from Clostridium perfringens plasmid pIP401 (44), was PCR amplified from the template plasmid pIMPTH (15) with the primers catP-N (5'-TTT AGT ACT CGG CAA GTG TTC AAG AAG TTA-3'), containing a ScaI site (italic), and catP-C (5'-TGA ATG CAT GGT CTT TGT ACT AAC CTG TGG-3'), containing an NsiI site (italic). The PCR product was first subcloned into pCRII-TOPO (Invitrogen), and then the 0.9-kb fragment isolated from ScaI and NsiI digestion was ligated to the corresponding sites of pSA12 to yield pSC12 (3.5 kb).

Construction of pJcfa.
The 2.1-kb fragment obtained from digestion with SalI and EcoRI and containing marR-cfa and the ptb promoter from pSOS84-cfa was recovered and inserted into the corresponding sites of pJIR750 (5) to yield pJcfa.

Construction of pPTBcfa.
The 2.1-kb SalI-EcoRI fragment containing marR-cfa and the ptb promoter from pSOS84-cfa was recovered and inserted into the corresponding sites of pBluescript SK(+) from Stratagene (La Jolla, Calif.) to yield pSK-cfa. The plasmid pSK-cfa was digested with SalI and PstI, and the 2.1-kb fragment was isolated and inserted into pGEM-11Zf(+) (Promega, Madison, Wis.) digested with SalI and NsiI to form p11Zf-cfa. Plasmid p11Zf-cfa was digested with BamHI, filled in with T4 polymerase, further digested with SwaI, and then self-ligated to result in p11Zf-PTBcfa. Plasmid p11Zf-PTBcfa was digested with SalI and EcoRI, and the 1.7-kb fragment was recovered and inserted into the corresponding sites of pSC12 to yield pPTBcfa.

Construction of pPTBMR.
The original construct, pSOS84-cfa, was digested with PstI and AflIII, and the 0.7-kb fragment was recovered and inserted into the corresponding sites of shuttle vector pSC12 to yield pPTBMR.

Construction of pJPTBcfa.
Plasmid pPTBcfa was digested with SalI and BamHI, and the 1.7-kb fragment was recovered and inserted into the corresponding sites of pJIR750 (5) to yield pJPTBcfa.


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RESULTS
 
Fatty acid composition of C. acetobutylicum strains.
The fatty acid composition of cells from various cultures, including batch fermentation and flask fermentation, was determined and confirmed the general trends reported earlier (4, 28). The fatty acids of C. acetobutylicum ATCC 824 identified here included 13:1, 14:0, 15:1, 16:0, 16:1, 17cyc (C17-cyclopropane), 18:0, 18:1, and 19cyc (C19-cyclopropane). The fatty acids 13:1, 15:1, and 18:0 accounted for less than 10% of the total fatty acids identified; 16:0 accounted for 36 to 55%, 14:0 for about 9 to 16%, 17cyc for about 1 to 7%, and 19cyc for about 3 to 20%. With the change in growth phase during the fermentation, the composition of fatty acids varied (Fig. 1). The unsaturated fatty acids (13:1, 15:1, 16:1, 17:1, and 18:1) decreased, whereas saturated fatty acid and cyclopropane fatty acid composition (14:0, 16:0, 18:0, 17cyc, and 19cyc) increased, consistent with the previous report (28). The fatty acid profile of a high-solvent-production mutant strain, SolRH (36), was also analyzed, but no difference was found from its parent strain, C. acetobutylicum ATCC 824 (data not shown).



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  		FIG. 1. Fatty acid composition of C. acetobutylicum ATCC 824 during batch fermentation (pH 5.0). The values (percentages of total fatty acids identified) are the means of two independent experiments. Symbols: {blacksquare}, 16:0; •, 16:1; {blacktriangleup}, 17cyc; {blacktriangledown}, 18:1; {diamondsuit}, 19cyc.

Cloning and arrangement of cfa gene from C. acetobutylicum.
A putative cfa gene was identified based on a homology search of a partial genome sequence available from Genome Therapeutics Corp. and cloned by PCR into the shuttle vector, as shown in Fig. 2. The recently published whole genome sequence (37) confirmed the unique choice. A CFA synthase (GenBank accession no. AAK78853) was encoded, with high homology, as indicated by PSI-Blast (3a), with the CFA synthase of E. coli (51) (44% identity, 59% similarity). The CFA synthase in C. acetobutylicum also showed high homology with the cyclopropane mycolic acid synthases and CFA synthase homologs in Mycobacterium tuberculosis, 32 to 34% identity with CMAS-1 (GenBank accession no. AAA75624), CMAS-2 (AAC43488), MMAS-2 (AAC44617), and PcaA (CAA17425), which are required for introduction of the cyclopropane ring of mycolic acids, a major component of the cell envelope of M. tuberculosis. The CFA synthase in C. acetobutylicum is highly conserved with the cofactor SAM binding sites and the cyclopropanation reaction sites of the cyclopropane mycolic acid synthases (25) (data not shown).



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  		FIG. 2. marR-like and cfa gene organization in C. acetobutylicum. Two stem-loop structures exist upstream (-9.6 kcal/mol) and downstream (-27.0 kcal/mol) of the operon. The lines represent the DNA fragments used in the construction of the plasmids for overexpression. Carets at the beginning of the lines indicate the ptb promoter. pSOS84-cfa was constructed in the vector pSOS84 with an erythromycin resistance determinant, and other plasmids were constructed in the vector encoding thiamphenicol resistance. pJcfa and pJPTBcfa were constructed in the vector pJIR750. pPTBMR and pPTBcfa were constructed in the newly developed vector pSC12.

The gene preceding cfa encodes a protein (GenBank accession no. AAK78852) with high homology to members of the MarR family, e.g., 31% identity with SlyA of E. coli (CAA09442) and 25% identity with MarR of E. coli (A47072). The search of the Pfam database (http://www.sanger.ac.uk) confirmed that this protein contained a MarR domain. The MarR family is a group of bacterial regulatory factors, including activators and repressors (1), with a winged-helix DNA binding motif (3). MarR family proteins are involved in regulation of a great range of physiological processes, including resistance to multiple antibiotics, organic solvents, and oxidative stress (2, 55); organic hydroperoxide resistance (46); phenolic compound degradation (14); virulence to plants (47); and sporulation (8).

The gene upstream of the marR-like gene encodes a putative transporter (GenBank accession no. AAK78851), predicted to be an integral membrane protein. Located downstream of cfa is a gene encoding a probable amino acid ABC transporter component (GenBank accession no. AAK78854), exhibiting high homology with Bacillus subtilis YxeN (50% identity).

It seems that the marR-like gene and cfa gene form an independent operon, because there was only a 24-bp space between them, and stem-loop structures existed upstream (-9.6 kcal/mol) and downstream (-27.0 kcal/mol) of these two genes. This gene organization around cfa was not found in other microbial genomes, including the unfinished genome sequences in GenBank.

Complementation of E. coli cfa mutants by cfa gene from C. acetobutylicum. (i) Growth rate in ethanol and propanol.
E. coli strains that lack CFA poorly survive repeated freezing and thawing (17), exposure to 20% ethanol (17), or exposure to pH 3 (9). To confirm the function of the cloned cfa gene, the growth rate of cultures exposed to ethanol and propanol was measured. Plasmid pSOS84-cfa was transformed into the CFA-deficient mutant strains E. coli GI121 and GI76-13 (17). The control strain DH10B, negative control plasmids pSOS84, pBR322, and pIMP1, and positive control plasmid pGI6 (overexpressing the cfa gene of E. coli) were also examined.

As shown in Table 2, pSOS84-cfa could complement the CFA mutant strain GI76-13 as did as the native E. coli cfa plasmid pGI6 when the strains were exposed to ethanol and propanol. Without antibiotic, the strain harboring pSOS84-cfa had the same growth rate as pGI6 in 5% ethanol but a lower rate in 2.5% propanol, although still higher than that of the strains bearing its parent vector, pIMP1. With antibiotics, pSOS84-cfa grew comparably to pGI6 in both ethanol and propanol and had a much shorter doubling time than the host containing pIMP1.


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  		TABLE 2. Exponential-phase doubling time of E. coli strainsa

(ii) Fatty acid analysis of complemented strains.
Fatty acid analysis of the complemented strains confirmed the activity of C. acetobutylicum cfa. Consistent with the previous report (17), E. coli cfa-deficient strains GI121 and GI76-13 containing the vector pBR322 did not produce any CFA (17cyc and 19cyc) (Table 3). Introduction of plasmid pSOS84-cfa or pGI6, harboring the cfa gene from C. acetobutylicum and E. coli, respectively, into both deficient strains induced a corresponding shift in the fatty acid composition of the cell, with both strains exhibiting restored levels of 17cyc and 19cyc (Table 3).


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  		TABLE 3. Effect of cfa-bearing plasmids on E. coli fatty acid compositiona

Overexpression of cfa gene in wild-type C. acetobutylicum ATCC 824.
To test whether the pSOS84-cfa construct also functioned in C. acetobutylicum, it was transformed into the wild-type strain. The fatty acid composition of early-log-phase cells clearly showed that the construct highly increased the CFA content. The overexpression strain converted 27% of 16:1 into 17cyc and 91% of 18:1 into 19cyc, while the wild-type strain did not produce detectable levels (<0.1%) of CFAs at this early-log-phase stage of growth (Table 4). The efficiency of conversion of 16:1 and 18:1 reflected the substrate specificity of the CFA synthase.


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  		TABLE 4. Effect of overexpression of cfa on C. acetobutylicum ATCC 824 fatty acid compositiona

The influence of overexpression of the cfa gene on solvent formation was investigated. The cfa-overexpressing strain C. acetobutylicum ATCC 824(pJcfa) and the control strain, C. acetobutylicum ATCC 824(pJIR750), were cultured in static flasks without pH control, and the solvent and fatty acid composition were analyzed (Fig. 3). As expected, the CFA content was considerably higher in the early log phase; 17cyc and 19cyc in C. acetobutylicum ATCC 824(pJcfa) constituted 6.3% and 9.5%, respectively, of total fatty acids, but in the control strain these components constituted only 1.3% and 3.7%, respectively. In terms of the conversion rate from unsaturated fatty acids into CFAs, the values for C. acetobutylicum ATCC 824(pJcfa) in the early log phase were 51% for 17cyc and 100% for 19cyc, while those of the control strain were only 8% and 31%, respectively. At the onset of stationary phase (24 h of culture), the CFAs of the overexpression and control strains both reached a similar peak level.



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  		FIG. 3. (Panels 1) Product concentration and optical density profiles for static flask fermentation (pH uncontrolled) with C. acetobutylicum ATCC 824(pJcfa) (A) and 824(pJIR750) (B). Symbols: {blacksquare}, ethanol; •, acetone; {blacktriangleup}, acetate; {blacktriangledown}, butanol; {diamondsuit}, butyrate; {circ}, optical density at 600 nm. The values are the means of five independent experiments. (Panels 2) Corresponding fatty acid composition of the cells. The values (percentages of total fatty acids identified) are the means of two independent experiments. Symbols: {blacksquare}, 16:0; •, 16:1; {blacktriangleup}, 17cyc; {blacktriangledown}, 18:1; {diamondsuit}, 19cyc; {circ}, optical density at 600 nm.

The final concentration of solvent product in the cfa-overexpressing strain was quite low; butanol was present at about 20% of the level found in the control strain, and acetone was also reduced by a similar fraction. The butanol and acetone concentrations in C. acetobutylicum ATCC 824(pJcfa) were 28.8 mM and 3.9 mM, respectively, while in the control strain they were 152.7 mM and 26.1 mM, respectively (Fig. 3 [1]). In the wild-type strain, acid products (butyrate and acetate) usually reach a peak in the acidogenic phase and then decrease as solvent production commences (27, 33, 39). However, the acid products of the cfa-overexpressing strain C. acetobutylicum ATCC 824(pJcfa) remained unaltered after they reached the peak value.

Overexpression of cfa gene in butyrate kinase-deficient strain C. acetobutylicum PJC4BK.
During the acid production phase in C. acetobutylicum, butyrate is produced from butyryl phosphate, catalyzed by butyrate kinase (23, 27). Recently, the butyrate kinase (buk) gene of C. acetobutylicum ATCC 824 was inactivated, and the butyrate formation pathway of this organism was altered (16). The resulting strain, C. acetobutylicum PJC4BK, was genetically characterized and shown to produce a reduced level of butyrate (16) and an increased level of solvents (butanol, acetate, and ethanol) during fermentation experiments at pH >=5.0 (21). To investigate the fatty acid content of this solvent-overproducing strain and examine the effect of cfa overexpression, plasmid pJcfa and the vector pJIR750 were transformed into this strain.

There were minimal differences in fatty acid composition between C. acetobutylicum PJC4BK containing the vector (Fig. 4 [2]B) and the wild-type strain, C. acetobutylicum ATCC 824, containing the vector (Fig. 3[2]B). Similar to the wild-type strain, C. acetobutylicum PJC4BK containing the cfa plasmid exhibited a higher CFA level during the early growth phase than C. acetobutylicum PJC4BK containing the control plasmid (Fig. 4[2]A). However, contrary to the wild-type strain, solvent production did not decrease much; the final concentrations of butanol and acetone in C. acetobutylicum PJC4BK(pJcfa) were 97.1 mM and 13.5 mM, respectively, while in C. acetobutylicum PJC4BK(pJIR750) the levels were 128.0 mM and 18.7 mM, respectively (Fig. 4[1]).



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  		FIG. 4. (Panels 1) Product concentration and optical density profiles for static flask fermentation (pH uncontrolled) with C. acetobutylicum PJC4BK(pJcfa) (A) and PJC4BK(pJIR750) (B). Symbols: {blacksquare}, ethanol; •, acetone; {blacktriangleup}, acetate; {blacktriangledown}, butanol; {diamondsuit}, butyrate; {circ}, optical density at 600 nm. The values are the means of five independent experiments. (Panels 2) Corresponding fatty acid composition of the cells. The values (percentages of total fatty acids identified) are the means of two independent experiments. Symbols: {blacksquare}, 16:0; •, 16:1; {blacktriangleup}, 17cyc; {blacktriangledown}, 18:1; {diamondsuit}, 19cyc; {circ}, optical density at 600 nm.

Effect of separate overexpression of cfa and marR-like genes.
In the constructs pSOS84-cfa and pJcfa, there were actually two genes, the marR-like gene and the cfa gene, both under control of the ptb promoter. Because the marR-like gene could encode a functional protein, the decrease in solvent production in pSOS84-cfa- and pJcfa-bearing strains might be due to direct or indirect effects of the marR-like gene. Therefore, a new set of plasmids with independent overexpression of the marR-like gene, pPTBMR, and the cfa gene, pPTBcfa, were constructed and transformed into wild-type and C. acetobutylicum pJC4BK strains.

Overexpression of the cfa gene alone, as in strains containing plasmids bearing both the marR-like gene and the cfa gene, resulted in higher CFA content at early log phase in both the wild type and C. acetobutylicum PJC4BK strain (Fig. 5 [2]A and Fig. 6 [2]A) and correlated with a substantial decrease in solvent production in the wild type (Fig. 5[1]A) and a modest decrease in C. acetobutylicum PJC4BK (Fig. 6[1]A). Therefore, this experiment confirmed that the results shown in Fig. 3 and Fig. 4 are the effects of the cfa gene.



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  		FIG. 5. (Panels 1) Product concentration and optical density profiles for static flask fermentation (pH uncontrolled) with C. acetobutylicum ATCC 824(pPTBcfa) (A), 824(pPTBMR) (B), and 824(pSC12) (C). Symbols: {blacksquare}, ethanol; •, acetone; {blacktriangleup}, acetate; {blacktriangledown}, butanol; {diamondsuit}, butyrate; {circ}, optical density at 600 nm. The values are the means of five independent experiments. (Panels 2) Corresponding fatty acid composition of the cells. The values (percentages of total fatty acids identified) are the means of two independent experiments. Symbols: {blacksquare}, 16:0; •, 16:1; {blacktriangleup}, 17cyc; {blacktriangledown}, 18:1; {diamondsuit}, 19cyc; {circ}, optical density at 600 nm.



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  		FIG. 6. (Panels 1) Product concentration and optical density profiles for static flask fermentation (pH uncontrolled) with C. acetobutylicum PJC4BK(pPTBcfa) (A), PJC4BK(pPTBMR) (B), and PJC4BK(pSC12) (C). Symbols: {blacksquare}, ethanol; •, acetone; {blacktriangleup}, acetate; {blacktriangledown}, butanol; {diamondsuit}, butyrate; {circ}, optical density at 600 nm. The values are the means of five independent experiments. (Panels 2) Corresponding fatty acid composition of the cells. The values are the means of two independent experiments. Symbols: {blacksquare}, 16:0; •, 16:1; {blacktriangleup}, 17cyc; {blacktriangledown}, 18:1; {diamondsuit}, 19cyc; {circ}, optical density at 600 nm.

Overexpression of the marR-like gene alone in C. acetobutylicum ATCC 824 and C. acetobutylicum JC4BK resulted in a significantly decreased CFA content over the whole growth period (Fig. 5[2]B and 6[2]B). In the early log phase, no 17cyc or 19cyc was found in C. acetobutylicum ATCC 824(pPTBMR), whereas in the control strain C. acetobutylicum ATCC 824(pSC12), they constituted 1.1% and 2.6%, respectively, of total fatty acids. Upon the onset of stationary phase, 17cyc and 19cyc in C. acetobutylicum ATCC 824(pPTBMR) constituted 0.0% and 2.4%, respectively, of total fatty acids, whereas in the control strain they constituted 3.9% and 14.4%, respectively. For C. acetobutylicum PJC4BK, at early log phase, no 17cyc or 19cyc was found in C. acetobutylicum PJC4BK(pPTBMR), whereas in the control strain C. acetobutylicum PJC4BK(pSC12), 17cyc and 19cyc constituted 0.5% and 2.2%, respectively, of total fatty acids. At the onset of stationary phase, the levels of 17cyc and 19cyc in C. acetobutylicum JC4BK (pPTBMR) were 0.0% and 1.8%, respectively, whereas in the vector-bearing strain, the levels were 4.4% and 12.1%, respectively.

cfa-overexpressing strain is more resistant to butanol and butyric acids.
Butanol (90 mM) was added to early-log-phase cultures of the cfa-overexpressing strain C. acetobutylicum ATCC 824(pJPTBcfa) (OD600 = 0.487) and the wild-type strain containing the vector, 824(pJIR750) (OD600 = 0.318). After 100 min, the cfa-overexpressing strain continued to grow (OD600 = 0.727) while the vector-containing wild-type strain showed little growth (OD600 = 0.416), suggesting that cfa overexpression in C. acetobutylicum resulted in cells that were more resistant to butanol at this early stage of growth.

However, butyric acid resistance appears to be more complicated. An early-exponential-phase culture was subjected to acid treatment. The cfa-overexpressing strain C. acetobutylicum ATCC 824(pJPTBcfa) (OD600 = 0.358) and the control strain, C. acetobutylicum 824(pJIR750) (OD600 = 0.303), were brought to 30 mM butyric acid. After 100 min, the OD600 of the cfa-overexpressing strain reached a higher value (0.533) than that of the control strain (0.372). Therefore, it seems that the cfa-overexpressing strain was more resistant to butyric acid, based on the continued growth just after the shock. However, over a long period of time (24 to 48 h), the wild-type strain adapted and grew to a higher density. This phenomenon may be due to the ability of the wild-type strain to counteract the toxic effect of acids by more vigorous solvent production.


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DISCUSSION
 
Overexpression of cfa did not cause loss of plasmid pSOL1.
As shown in Fig. 3 and Fig. 5, strains C. acetobutylicum ATCC 824(pJcfa) and ATCC 824(pJPTBcfa) overexpressed cfa and exhibited decreased solvent production. Similarly, a degenerate mutant of C. acetobutylicum, M5 (10), did not produce solvents. In the latter case, the lower solvent production resulted from the loss of a 192-kb plasmid, pSOL1 (11), which encodes most of the solvent genes. To test whether cfa overexpression caused an increased tendency to lose the pSOL1 plasmid, the {alpha}-amylase activity of a number of colonies of the culture was examined. Since {alpha}-amylase (GenBank accession no. AAK76913) is encoded by a gene on pSOL1, its presence would indicate the presence of a functional pSOL1 plasmid, as demonstrated in studies of strain degeneration in C. acetobutylicum ATCC 824 (43). The colonies of the cfa-overexpressing strain C. acetobutylicum ATCC 824(pJPTBcfa) showed the same {alpha}-amylase halo activity as the wild type, while the C. acetobutylicum M5 strain did not, suggesting that a major loss of pSOL1 was not the explanation for the reduced solvent production.

Overexpression of cfa increased acid and solvent resistance.
In considering possible explanations for how early expression of cfa would lead to a reduction in solvent production, one hypothesis is that the increase in CFAs produced by overexpression of cfa can partially offset the toxic effect of the butyric and acetic acids produced, so the cells do not shift as much metabolism to solvent formation as part of a stress feedback response.

In the early log phase, the cfa-overexpressing strain was more resistant to butyric acid than the wild type. However, over a long period of time (24 to 48 h), the wild-type strain adapted and became more acid resistant. Considering that the wild-type strain produced more solvent than the cfa-overexpressing strain under normal growth conditions (Fig. 2 and 4), it seems that the higher CFA level may inhibit some mechanism of stationary-phase acid adaptation directly or indirectly related to solvent production. The increase in acid resistance caused by overexpression of cfa is consistent with the report that early-stationary-phase cultures of cfa-deficient E. coli strains are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3 (9). Recently CFA was also found to be related to stationary-phase acid tolerance in Salmonella enterica serovar Typhimurium (unpublished data, inferred from the annotation of GenBank accession no. AF417203 that cyclopropane fatty acid is related to stationary-phase acid tolerance in S. enterica serovar Typhimurium).

Overexpression of cfa may reduce the phosphorylated Spo0A level or suppress the activity of an unknown solvent regulator.
Another hypothesis is that overexpression of cfa may result in an increase in CFA content, which decreases the membrane fluidity; thus, a signal involved in inducing solvent production could be altered, leading to a reduction in the level of active (phosphorylated) Spo0A or another required regulator, which therefore dampens the induction of solventogenesis. The hypothesis is based on the following findings. (i) Spo0A is a positive regulator of solvent genes, such as adhE (aad) (48) and adc (42). (ii) Overexpression of cfa in the butyrate kinase-deficient mutant C. acetobutylicum PJC4BK did not inhibit solvent production as much as in the wild type. It was proposed that butyryl phosphate in C. acetobutylicum PJC4BK may accumulate and act like acetyl phosphate (21) and therefore could be a factor in determining levels of phosphorylated Spo0A. Acetyl phosphate, in E. coli and other bacteria, may act as an agent that can phosphorylate regulatory proteins of the two-component signal transduction system, resulting in modification of gene expression (29). (iii) In Bacillus subtilus, the phosphoryl group was shown to be transferred from the autophosphorylating sensor kinase, KinA or KinB, to the response regulator Spo0F, subsequently to Spo0B, and finally to Spo0A (7). Although KinA is a cytoplasmic kinase, KinB is a membrane protein, and its activation requires KapB, a lipoprotein (12). Therefore, membrane properties may affect the abundance of phosphorylated Spo0A, and observation of unsaturated fatty acid inhibition of KinA has been reported (45). The phosphorelay system in C. acetobutylicum appears to be different from that of B. subtilis, as indicated by the absence of orthologues of the kinA, kinB, spo0F, and spo0B genes (37); however, some kind of phosphorelay system involving lipoproteins or membrane-bound sensors may exist in C. acetobutylicum.

Another possibility is that the increased membrane rigidity caused by the early overexpression of cfa may suppress the activity of another unknown transcriptional regulator of the solvent genes in addition to Spo0A, as proposed by Thormann et al. (48). Harris et al. also noted that a spo0A disruption did not completely abolish solvent gene expression and solvent production (the butanol level in the mutant was 7.6% of that in the parent strain) (22).

Unique organization of the marR-like gene and the cfa gene in C. acetobutylicum ATCC 824.
In C. acetobutylicum, the cfa gene and marR-like gene appear to form an independent operon. The marR-like gene product may act to reduce expression of the cfa gene directly or indirectly, based on the observation that the presence of the marR-like gene on a plasmid resulted in reduced CFA levels (Fig. 5 and 6). This gene organization around cfa genes was not found in other microbial genomes, including the unfinished genome sequences in GenBank, particularly in other clostridial species, i.e., C. perfringens, Clostrifium botulinum A, or Clostridium difficile.

The marR-like gene in C. acetobutylicum ATCC 824.
The marR-like gene in C. acetobutylicum belongs to the MarR protein family, based on homology comparison. The majority of the MarR family members are transcriptional repressors (1). Although the gene organization of the marR-like and cfa genes in one operon has not been found in other sequences deposited in GenBank, it was found that marR family genes are associated with genes specifying lipid metabolism. There is an apparent operon consisting of a marR homolog and fabH. The fabH gene encodes the 3-ketoacyl-acyl carrier protein-synthase III, which is proposed to initiate fatty acid synthesis. The marR homolog-fabH gene arrangements are found in C. acetobutylicum (the MarR homolog has GenBank accession no. AAK81502, the corresponding nucleotide sequence has GenBank accession no. AE007868), C. perfringens (BAB80773 and AP003189), Lactococcus lactis subsp. lactis (AAK04868 and AE006310), Streptococcus pyogenes MGAS8232 (AAL98346 and AE010089), and Streptococcus pyogenes M1GAS (AAK34498 and AE006603). The nucleotide spacing between the two genes in these organisms is only 0 to 5 bp and could imply a role for the marR homolog in regulation of fabH expression.

Although some factors, such as acid end products, nutrient limitation, and pH, have been considered triggering agents for solvent production (27, 33, 39), the detailed molecular mechanism is still unclear. The significant solventogenesis suppression caused by the early expression of CFA provides another factor to investigate and incorporate into a complete picture of the switch from acidogenesis to solventogenesis.


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ACKNOWLEDGMENTS
 
We thank L. M. Harris and E. T. Papoutsakis for some clostridial cell samples used in preliminary experiments and J. E. Cronan, Jr., for the cfa-deficient E. coli strain and E. coli cfa plasmid.

This work were supported by U.S. Department of Agriculture grant 00-35504-9269, National Science Foundation grant BES-0001288, and R. A. Welch Foundation grants C-1268 (G.N.B.) and C-1372 (F.B.R.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005. Phone: (713) 348-4920. Fax: (713) 348-5154. E-mail: gbennett{at}bioc.rice.edu. Back


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Applied and Environmental Microbiology, May 2003, p. 2831-2841, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2831-2841.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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