Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl₂, and MgCl₂. A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.     

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.     

Extracellular glycosyl hydrolase activity of the Clostridium strains producing acetone, butanol, and ethanol

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% wheat flour medium amounted to 12.7–15 g/l with butanol constituting 51.0–55.6%. Activities of these strains towards xylan, β-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acetobutylicum 6, C. acetobutylicum 7, and C. acetobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824 in lab-scale butch cultures. It was demonstrated that starch in the medium could be partially substituted with plant biomass.     

Changing proportions of DNA components in Euglena gracilis

The ratios of satellite deoxyribonucleic acid components to chromosomal deoxyribonucleic acid in Euglena gracilis Z were measured by analytical density gradient ultracentrifugation. Chloroplast deoxyribonucleic acid with a buoyant density of 1.685 g/cm³ exhibited a constant ratio to chromosomal deoxyribonucleic acid during exponential growth and increased twofold as the culture reached the end of the exponential growth phase. The quantity of a satellite deoxyribonucleic acid with a buoyant density of 1.691 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but increased to approximately equal the quantity of chloroplast deoxyribonucleic acid as the culture approached the end of the exponential growth phase. The quantity of a deoxyribonucleic acid component with a buoyant density of 1.700 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but represented approximately one-third of the total deoxyribonucleic acid as the culture entered the stationary phase of growth.     

Enhanced butanol production and reduced autolysin activity after chloramphenicol treatment of Clostridium acetobutylicum ATCC 824

Summary Release of autolysin during the late exponential growth phase of Clostridium acetobutylicum resulted in early lysis of the culture and reduction of solvent formation. A simple and effective way of reducing autolysin activity and increasing solvent production is partial inhibition of protein synthesis with chloramphenicol (CAP). The extracellular autolytic activity in the culture, determined by following loss of turbidity of washed clostridial cells in 0.04m sodium phosphate buffer at 37° C, was decreased by 40% after CAP treatment. This caused an extension of cell viability by 12 h and an increase in butanol production by 30%. The optimal time of CAP addition was 12 h of incubation, and the optimal antibiotic concentration was 120 g/ml. The effects of CAP on the fermentation are due to the inhibition of protein synthesis leading to a decrease in autolysin level in the culture. The results obtained provide economic advantages for industrial production of solvents by minimizing autolysin activity and maximizing solvent yield during the critical solvent-producing phase. Correspondence to: R. W. Traxler     

Selection of an Asporogenous Strain of Clostridium acetobutylicum in Continuous Culture Under Phosphate Limitation

Based on the observation that cells of Clostridium acetobutylicum unable to store granulose do not initiate sporulation, a staining procedure was developed for the detection of asporogenous mutants. By application of this procedure it was shown that an asporogenous strain of C. acetobutylicum was selected in continuous culture under phosphate limitation.     

Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer, Clostridium butyricum VPI 3266, and an Engineered Strain, Clostridium acetobutylicum DG1(pSPD5)

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH₂ flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD⁺ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.     

An hypothetical structure for an intermolecular electron transfer complex of cytochromes c and b5.

The arrangement of base-sequences in the chloroplast deoxyribonucleic acid from Euglena gracilis Z was investigated by analyzing the bimodal profile of chloroplast deoxyribonucleic acid in an alkaline CsCl density gradient. The three main fractions in the alkaline gradient contain both single-stranded self-complementary base-sequences and base-sequences that are not self-complementary. Four single-stranded self-complementary deoxyribonucleic acid components were isolated from alkaline CsCl preparative density gradients. A fifth component was derived by annealing two fractions containing deoxyribonucleic acid that was not self-complementary. The five fractions corresponded to the five components of total chloroplast deoxyribonucleic acid that exhibited differential thermal denaturation. The results indicate that these five components are not interspersed throughout the genome in small segments but that they exist as relatively large segments. This implies that the chloroplast deoxyribonucleic acid contains tandemly arranged segments differing in base-composition.     

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

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.     

Membrane H+ Conductance of Clostridium thermoaceticum and Clostridium acetobutylicum: Evidence for Electrogenic Na+/H+ Antiport in Clostridium thermoaceticum

H⁺ conductance in de-energized cells of Clostridium thermoaceticum and Clostridium acetobutylicum was determined from the rate of realkalinization of the medium after an acid pulse. In both organisms, cell membrane proton permeability was increased by fermentation end products and ionophores. In C. thermoaceticum, H⁺ conductance was increased by Na⁺ ions compared with K⁺ as counterions. In these cells, addition of Na⁺, but not K⁺, elicited efflux of H⁺; H⁺ efflux was stimulated by SCN⁻ and decreased by various ionophores. We concluded that C. thermoaceticum possesses an electrogenic Na⁺/H⁺ antiporter. In contrast, C. acetobutylicum cells did not have an electrogenic Na⁺/H⁺ antiporter.     

Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition

In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology.     

Protoplast formation and leakage of intramembrane cell components: induction by the competence activator substance of pneumococci.

Treatment of pneumococci with activator (a protein that induces bacterial "competence" to absorb deoxyribonucleic acid molecules and undergo genetic transformation) can cause either protoplast formation or leakage of intracellular components to the medium depending on postincubation conditions. The leaked intracellular components include nucleoside phosphates, beta-galactosidase, deoxyribonuclease, autolysin, and hemolysin. Leakage and protoplast formation are induced by the electrophoretically pure activator, and these phenomena require the same conditions as induction of competence for genetic transformation, namely, genetic capacity for competence, protein synthesis, incorporation of choline, and the optimal pH for activation. It is suggested that the activator protein accelerates a normal process of transport (leakage) of autolysin molecules into the periplasmic space. The activity of these autolysin molecules from within would then unmask deoxyribonucleic acid binding sites located on the plasma membrane.     

The ald Gene, Encoding a Coenzyme A-Acylating Aldehyde Dehydrogenase, Distinguishes Clostridium beijerinckii and Two Other Solvent-Producing Clostridia from Clostridium acetobutylicum

The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M_r of 51,353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD⁺ binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.     

Production by Clostridium acetobutylicum ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.     

Effects of nutritional enrichment on the production of acetone-butanol-ethanol (ABE) by Clostridium acetobutylicum

Clostridium acetobutylicum is an industrially important organism that produces acetone-butanol-ethanol (ABE). The main objective of this study was to characterize the effects of increased cell density on the production of ABE during the phase transition from acidogenesis to solventogenesis in C. acetobutylicum. The increased ABE productivity of C. acetobutylicum was obtained by increasing the cell density using a newly designed medium (designated C. a cetobutylicum medium 1; CAM1). The maximum OD₆₀₀ value of C. acetobutylicum ATCC 824 strain obtained with CAM1 was 19.7, which is 1.8 times higher than that obtained with clostridial growth medium (CGM). The overall ABE productivity obtained in the CAM1-fermetation of the ATCC 824 strain was 0.83 g/L/h, which is 1.5 times higher than that (0.55 g/L/h) obtained with CGM. However, the increased productivity obtained with CAM1 did not result in an increase in the final ABE titer, because phase transition occurred at a high titer of acids.     

Metabolite analysis of Clostridium acetobutylicum: Fermentation in a microbial fuel cell

Microbial fuel cells (MFCs) were used to monitor metabolism changes in Clostridium acetobutylicum fermentations. When MFCs were inoculated with C. acetobutylicum, they generated a unique voltage output pattern where two distinct voltage peaks occurred over a weeklong period. This result was markedly different to previously studied organisms which usually generate one sustained voltage peak. Analysis of the fermentation products indicated that the dual voltage peaks correlated with glucose metabolism. The first voltage peak correlated with acidogenic metabolism (acetate and butyrate production) and the second peak with solventogenic metabolism (acetone and butanol production). This demonstrates that MFCs can be applied as a novel tool to monitor the shift from acid production to solvent production in C. acetobutylicum.     

Effects of acetic and formic acid on ABE production by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> and <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

The effect of acetic acid and formic acid on acetone-butanol-ethanol (ABE) production by solventogenic Clostridia was investigated. The ABE concentration in Clostridium acetobutylicum was found to have increased slightly on addition of 3.7 ∼ 9.7 g/L acetic acid, but was found to have drastically reduced in the presence of 11.7 g/L acetic acid. However, the solvent production of C. beijerinckii was not affected by addition of acetic acid in the range of 3.7 ∼ 11.7 g/L. C. acetobutylicum was more vulnerable to formic acid than C. beijerinckii. In C. acetobutylicum, the total ABE production decreased to 77% on addition of 0.4 g/L formic acid and 25% with 1.0 g/L formic acid. The total ABE production by C. acetobutylicum was also noted to have decreased from 15.1 to 8.6 g/L when 8.7 g/L acetic acid and 0.4 g/L formic acid co-existed. The solvent production by C. beijerinckii was not affected at all under the tested concentration range of formic acid (0.0 ∼ 1.0 g/L) and co-presence of acetic acid and formic acid. Therefore, C. beijerinckii is more favorable than C. acetobutylicum when the ABE is produced using lignocellulosic hydrolysate containing acetic and formic acid.     

The Influence of Syringomycin on Ribonucleic Acid Synthesis

Syringomycin, a wide-spectrum antibiotic produced by strains of Pseudomonas syringae which cause bacterial canker of peach, was able to bind to salmon sperm and calf thymus deoxyribonucleic acid but not to calf thymus histone; it also inhibited ribonucleic acid polymerase activity. These abilities to bind to deoxyribonucleic acid and to inhibit ribonucleic acid polymerase were inactivated when the phytotoxic and antibiotic properties of syringomycin were inactivated.     

Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation

During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.     

Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co²⁺ and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAΔ1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmAΔ1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.