Use of a fluorescein derivative of phosphatidylethanolamine as a pH probe at water/lipid interfaces.

A fluorescent indicator for the determination of pH in the vicinity of water/lipid interfaces was produced by the covalent linkage of fluoresceinisothiocyanate to Escherichia coli phosphatidylethanolamine. When embedded in monolayers spread on an air/water interface, its apparent pK was shown to be only slightly affected by the nature of the polar headgroups or the packing density of the host phospholipids. Its properties were not affected by the ion content of the subphase. For small unilamellar vesicles, its properties were only affected when localized in the inner layer. This probe could therefore be of value in the study of proton fluxes along biological membranes.

Uncoupled glycerol distribution as the origin of the accumulation of 3-hydroxypropionaldehyde during the fermentation of glycerol by enterobacter agglomerans CNCM 1210

Batch fermentation of glycerol to 1,3-propanediol (1,3PPD) by Enterobacter agglomerans CNCM 1210 showed the lethal accumulation of 3-hydroxypropionaldehyde (3-HPA) when performed under initial substrate content higher than 40 g/L. Assigned to the inhibition by the NAD/NADH ratio of the 3-HPA converting enzyme: 1,3PPD dehydrogenase, intracellular assays were conducted in an attempt to identify the metabolic mechanisms involved in the increase of that ratio. An overflow metabolism through the 1,3PPD formation pathway was established, while a catabolic limitation in the oxidative branch at the level of glyceraldehyde-3-phosphate dehydrogenase occurred. Uncoupled activities of synthesis and consumption of reducing equivalents are thus suspected to provoke the increase of the NAD/NADH ratio and the subsequent accumulation of 3-HPA. Copyright 1998 John Wiley & Sons, Inc.

Modulation of Carbon and Electron Flow in Clostridium acetobutylicum by Iron Limitation and Methyl Viologen Addition.

The metabolic flexibility of Clostridium acetobutylicum during growth on glucose with methyl viologen addition (1 mM) and/or iron limitation was examined in batch cultures at pH 5.5. The physiological effects of iron limitation and methyl viologen addition are additive, suggesting that they have different and complementary sites of action.

Modulation of metabolism of Clostridium acetobutylicum grown in chemostat culture in a three-electrode potentiostatic system with methyl viologen as electron carrier.

The metabolism of Clostridium acetobutylicum was manipulated in chemostat culture at pH 5 and 6.5 in a three-electrode potentiostatic system with methyl viologen (MV) as the electron carrier. When a constant potential was applied at pH 5, the broth redox potential continuously decreased and, simultaneously, a high increase in the reduced MV concentration (MV(+.)) and the specific rate of butanol production was observed while butyric acid was taken up. A linear relationship was reported between the specific rate of NAD(P)(+) reduction by ferredoxin-NAD(P)(+) reductase and the broth redox potential, as long as the growth rate was not affected. To reach a steady state in glucose limited culture, a control system of the redox potential was required. However, it seems that C. acetobutylicum is able to adapt its metabolism when the broth redox potential was regulated at low value. On the other hand, at pH 6.5, the current generated by the electrochemical device had no effect either on broth redox potential and MV(+.) concentration or on the metabolism.

Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool.

Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular NADH/NAD ratio (I. Vasconcelos, L. Girbal, and P. Soucaille, J. Bacteriol. 176:1443-1450, 1994). We report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high NADH/NAD ratios. Partial substitution of glucose by pyruvate while maintaining glycerol concentration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures. However, glycerol-sufficient cultures had a constant flux through the glyceraldehyde-3-phosphate dehydrogenase and a constant NADH/NAD ratio. A high substitution of glucose by pyruvate [P/(G+P) value of 0.67 g/g] provided a carbon-limited culture with butanol and butyrate as the major end products. In this alcohologenic culture, the induction of the NADH-dependent butyraldehyde and the ferredoxin-NAD(P) reductases and the higher expression of alcohol dehydrogenases were related to a high NADH/NAD ratio and a low intracellular ATP concentration. In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities decreased with the intracellular ATP concentration, suggesting a transcriptional regulation of these two genes, which are arranged in an operon (K. A. Walter, R. V. Nair, R. V. Carry, G. N. Bennett, and E. T. Papoutsakis, Gene 134:107-111, 1993).

Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol.

The metabolism of Clostridium acetobutylicum was manipulated, at neutral pH and in chemostat culture, by changing the overall degree of reduction of the substrate, using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids, and the intracellular enzymatic pattern indicated the absence of butyraldehyde dehydrogenase activity and very low levels of coenzyme A-transferase, butanol, and ethanol dehydrogenase activities. In contrast, cultures grown on mixtures of glucose and glycerol produced mainly alcohols and low levels of hydrogen. The low production of hydrogen was not associated with a change in the hydrogenase level but was correlated with the induction of a ferredoxin-NAD reductase and a decreased level of NADH-ferredoxin reductase. The production of alcohols was related to the induction of a NAD-dependent butyraldehyde dehydrogenase and to higher expression of NAD-dependent ethanol and butanol dehydrogenases. The coenzyme A-transferase was poorly expressed, and thus no acetone was produced. These changes in the enzymatic pattern, obtained with cultures grown on a mixture of glucose and glycerol, were associated with a 7-fold increase of the intracellular level of NADH and a 2.5-fold increase of the level of ATP.

Physiologic Mechanisms Involved in Accumulation of 3-Hydroxypropionaldehyde during Fermentation of Glycerol by Enterobacter agglomerans.

When grown in 700 mM glycerol within the pH range 6.0 to 7.5, anaerobic pH-regulated cultures of Enterobacter agglomerans exhibited an extracellular accumulation of 3-hydroxypropionaldehyde (3-HPA). This phenomenon, which causes fermentation cessation, occurred earlier when pH was low. In contrast, substrate consumption was complete at pH 8. Levels of glycerol-catabolizing enzymes, i.e., glycerol dehydrogenase and dihydroxyacetone kinase for the oxidative route and glycerol dehydratase and 1,3-propanediol dehydrogenase for the reductive route, as well as the nucleotide pools were determined periodically in the pH 7- and pH 8-regulated cultures. A NAD/NADH ratio of 1.7 was correlated with the beginning of the production of the inhibitory metabolite. Further accumulation was dependent on the ratio of glycerol dehydratase activity to 1,3-propanediol dehydrogenase activity. For a ratio higher than 1, 3-HPA was produced until fermentation ceased, which occurred for the pH 7-regulated culture. At pH 8, a value below 1 was noticed and 3-HPA accumulation was transient, while the NAD/NADH ratio decreased. The low rate of glycerol dissimilation following the appearance of 3-HPA in the culture medium was attributed to the strong inhibitory effect exerted by 3-HPA on glycerol dehydrogenase activity.

Transmembrane pH of Clostridium acetobutylicum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased.

Evidence is reported here that alkalinization of Clostridium acetobutylicum cytoplasm involves hydrogenase activity. A decrease of in vivo hydrogenase activity is accompanied by intracellular accumulation of protons leading to a negative (interior acidic) pH gradient. However, the organism is able to maintain a constant proton motive force by interconverting chemical and electrical potentials.

Physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome.

A physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome was constructed. The macrorestriction map for CeuI, EagI, and SstII was created by ordering the 38 restriction sites by one- and two-dimensional pulsed-field gel electrophoresis (PFGE) and by using an original strategy based on the CeuI enzyme and indirect end labelling by hybridization on both sides of the CeuI sites with rrs (16S RNA) and 3' rrl (23S RNA) probes. The circular chromosome was estimated to be 4.15 Mb in size, and the average resolution of the physical map is 110 kb. The chromosome contains 11 rrn loci, which are localized on 44% of the chromosome in a divergent transcriptional orientation regarding the presumed location of the replication origin. In addition to these 11 rrn operons, a total of 40 identified genes were mapped by hybridization experiments with genes from C. acetobutylicum and from various other clostridia as probes. The genetic map of C. acetobutylicum was compared to that of the three other endospore-forming bacteria characterized so far: Bacillus subtilis, Clostridium beijerinckii, and Clostridium perfringens. Parodoxically, the chromosomal backbone of C. acetobutylicum showed more similarity to that of B. subtilis than to those of the clostridia.

Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824.

A 2.8-kbp DNA region of Clostridium acetobutylicum ATCC 824 containing the putative hydrogenase gene (hydA) was cloned and sequenced. The 1,745-bp hydA encodes a 64,415-Da protein and presents strong identity with the [Fe] hydrogenase genes of Desulfovibrio and Clostridium species. The level of the putative hydA mRNA was high in cells from an acidogenic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate-limited continuous culture. These results were in agreement with the hydrogenase protein level, indicating that expression of hydA is regulated at the transcriptional level. Primer extension analysis identified a major transcriptional start site 90 bp upstream of the hydA start codon. The position of a putative rho-independent transcription terminator immediately downstream of the termination codon is in agreement with the size of the hydA transcript (1.9 kb) determined by Northern (RNA) blot experiments and confirms that the gene is transcribed as a monocistronic operon. Two truncated open reading frames (ORFs) were identified downstream and upstream of hydA and in opposite directions. The amino acid sequence deduced from ORF2 presents strong identity with ortho phosphoribosyl transferases involved in pyrimidine synthesis. The amino acid sequence deduced from ORF3 presents no significant similarity to any sequence in various available databases.

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.

Partitioning of pristinamycins in aqueous two-phase systems: A first step toward the development of antibiotic production by extractive fermentation.

The partitioning of pristinamycins was studied in dextran and polyethylene glycol (PEG) aqueous two-phases systems. Pristinamycins partitioned preferentially into the PEG-rich top phase. The partition coefficient was independent of molar mass of PEG and dextran and of antibiotic concentration, but, increased exponentially with the tieline length of the system. Partition of pristinamycins was greatly improved when fatty acids esters of PEG were mixed with PEG. In such mixtures, the partition of coefficient increased up to a value of 24, dependent on the carbon chain length of fatty acids and the modified PEG concentrations. Moreover, in such system, the two groups of pristinamycins, I and II, were extracted in accordance with their hydrophobicity. Recovery of pristinanamycins produced by Streptomyces pritinaespiralis in a fermentation broth was achieved with a dextran/PEG system. Cells were confined into the bottom phase and pristinamycins partitioned in the top phase. However, due to binding of the pristinamycins to the cells, the partition coefficient was slightly lower than of pure antibiotics solutions. (c) 1994 John Wiley & Sons, Inc.

Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan.

An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824. The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp. The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1. Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline. The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain. The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall.

Autolysis of Clostridium acetobutylicum ATCC 824.

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.

3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species.

Glycerol fermentation by Enterobacter agglomerans revealed that both growth and 1,3-propanediol production ceased after consumption of about 430 mM glycerol, irrespective of the initial glycerol content. This phenomenon was assigned to the production of 3-hydroxypropionaldehyde, which was identified by proton nuclear magnetic resonance and which showed a bacteriostatic effect. The accumulation during glycerol fermentation was also observed with two other enterobacterial species, i.e., Klebsiella pneumoniae and Citrobacter freundii.

Evidence for conduction of protons along the interface between water and a polar lipid monolayer.

Movements of H+ along the polar heads of phospholipids spread in monolayers were compared to movements of H+ in the aqueous subphase. The probe for detecting H+ movement along the monolayer was a pH-sensitive fluorescein chromophore covalently bound to the head group of phosphatidylethanolamine. The behavior of this probe was not affected by the electrical properties of the lipid/water interface. Lateral diffusion of H+ along the phospholipid/water interface was then studied by acid-jump experiments in which advantage was taken of the large size of the monolayer. H+ was injected a few centimeters away from the probe observation area. The time needed for H+ diffusion to the probe was monitored by the change in the fluorescence signal, fluorescein being nonfluorescent in an acid medium. Diffusion of H+ in the bulk phase was monitored by the fluorescence change of water-soluble fluorescein isothiocyanate. Diffusion along the lipid monolayer was found to be 20 times faster than in the bulk water phase and required a structured monolayer in order to occur, as revealed by variation of the molecular area occupied by the lipid molecules. The molecular basis of rapid H+ transfer along the lipid monolayer may be the existence of a hydrogen-bond network along the polar heads, capable of supporting a rapid "hop and turn" of H+.

Purification and characterization of acidolysin, an acidic metalloprotease produced by Clostridium acetobutylicum ATCC 824.

Acidolysin an extracellular protease produced by Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography with a recovery of 91%. The enzyme was a monomeric protein with a molecular weight of 44,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an acidic isoelectric point of 3.3. Acidolysin was very sensitive to metal-chelating agents and phosphoramidon and was unaffected by sulfhydryl reagents. It was shown to be a calcium- and zinc-containing protease. It exhibited optimal activity against Azocoll at pH 5 and 45 degrees C. It was stable at low pH and heat labile above 50 degrees C. It exhibited specificity toward peptide bonds formed by the amino group of hydrophobic amino acids (isoleucine, leucine, and phenylalanine) and its NH2-terminal amino acid sequence showed a high degree of similarity with that of Bacillus subtilis neutral metalloprotease A. Acidolysin is the first phosphoramidon-sensitive, acidic zinc metalloprotease reported.

The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain.

Degeneration is the process whereby Clostridium acetobutylicum ATCC 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. Two degenerate mutants (M5 and DG1) of C. acetobutylicum ATCC 824 do not contain the four genes (ctfA, ctfB, adc, and aad) for acetone and butanol formation. Strain ATCC 824 contains a 210-kb plasmid (pSOL1) which is absent in M5 and DG1. pSOL1 carries the four acetone and butanol formation genes. A restriction map of pSOL1 was constructed by using ApaI, SmaI, SstII, and NarI digestions. M5 and DG1 could be complemented for acetone and butanol formation by expressing the corresponding genes (ctfA, ctfB, and adc for acetone; aad for butanol) on the plasmid. Degeneration of this strain thus appears to be the result of pSOL1 loss.

Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures.

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.