Phosphoroclastic Reaction Research Articles

Impact of anaerobiosis strategy and bioreactor geometry on the biochemical response of Clostridium butyricum VPI 1718 during 1,3-propanediol fermentation

The impact of anaerobiosis strategy on 1,3-propanediol production during cultivation of Clostridium butyricum VPI 1718 in different size bioreactors was studied. In batch trials with N2 gas infusion, the fermentation was successfully accomplished, regardless of initial glycerol concentration imposed and bioreactor geometry. However, in the absence of N2 continual sparging, significant variations concerning the biochemical response of the strain were observed. Specifically, at 1-L bioreactor, the absence of N2 infusion at high initial glycerol concentration induced lactate dehydrogenase activity and thus lactic acid synthesis, probably due to partial blockage of phosphoroclastic reaction caused by insufficient self-generated anaerobiosis environment. During fed-batch cultivation with continual N2 sparging, the strain produced ∼71gL−1 of 1,3-propanediol, whereas under self-generated anaerobiosis, 1,3-propanediol pathway was evidently restricted, as only 30.5gL−1 of 1,3-propanediol were finally produced. Apparently, N2 infusion strategy paired with bioreactor geometry can alter the biochemical behavior of the particular strain.

Anaerobic purification and crystallization to improve the crystal quality: ferredoxin II from Desulfovibrio gigas

Sulfate-reducing bacteria (SRB), which are strict anaerobes, contain an electron-transfer chain from pyridine nucleotides to molecular oxygen. This unique enzymatic equipment allows the bacterium to produce ATP when exposed to air from the degradation of internal reserves of polyglucose. Ferredoxin II (Fd II) is a small electron-transfer protein isolated from the strict anaerobic sulfate-reducing bacterium Desulfovibrio gigas. The protein contains 58 amino acids and an iron-sulfur cluster. The cluster [3Fe-4S] spontaneously undergoes conversion to [4Fe-4S] when it is used as an electron mediator in the phosphoroclastic reaction. The iron-sulfur geometries and interconversion mechanism appear to have physiological significance between the oxidized and reduced states. Crystallization of Fd II in an anaerobic environment was achieved at a higher resolution of 1.37 A and the differences between the anaerobic and aerobic structures will reveal the unique iron-storage function and electron-transfer mechanism of ferredoxin II from D. gigas.

Effect of nutrient limitation on product formation during continuous fermentation of xylose with Thermoanaerobacter ethanolicus JW200 Fe(7).

Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1). Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1). Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1). The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.

Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium.

Continuous cultures, under cellobiose sufficient concentrations (14. 62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a q(cellobiose) of 2.57 mmol g cells(-1) h(-1) was attained whereas the highest value obtained on complex media was 0.68 mmol g cells(-1) h(-1) (Payot et al. 1998. Microbiology 144:375-384). On a synthetic medium at D = 0.035 h(-1) under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H(2)/CO(2) ratio was found equal to 1. At a higher dilution rate (D = 0.115 h(-1)), there was no lactate production and the pathways toward ethanol and NADH-ferredoxin-hydrogenase contributed to balance the reducing equivalents; in this case a H(2)/CO(2) ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady-state concentration of NADH and NAD(+) pools from 11.8 to 22.1 micromol (g cells) (-1), (ii) in the intracellular NADH/NAD(+) ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h(-1), 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium.

The influence of H2, CO2 and dilution rate on the continuous fermentation of acetone-butanol

The effect of product gases, H2 and CO2, on solvent production was studied using a continuous culture of alginate-immobilized Clostridium acetobutylicum. Initially, in order to find the optimum dilution rate for aceton--butanol production in this system, fermentations were carried out at various dilution rates. With 10% H2 and 10% CO2 in the sparging gas, a dilution rate of 0.07 h−1 was found to maximize volumetric productivity (0.58 g·l−1·h−1), while the maximum specific productivity of 0.27 g−·h−1 occured at 0.12 h−1. Continuous cultures with vigorous sparging of N2 produced only acids. It was concluded that in the case of continuous fermentation H2 is essential for good solvent production, although good solvent production is possible in an H2-absent environment in the case of batch fermentations. When the fermentation was carried out at atmospheric pressure under H2-enriched conditions, the presence of CO2 in the sparging gas did not slow down glucose metabolism; rather it changed the direction of the phosphoroclastic reaction and as a result increased the butanol/acetone ratio.

Role of hydrogenase 1 of clostridium pasteurianum in the reduction of metronidazole

Competition studies between the phosphoroclastic reaction and the metronidazole reduction reaction using dialyzed crude cell-free extracts of Clostridium pasteurianum which were essentially devoid of Hydrogenase 1 activity demonstrated that this enzyme plays an important role in the reduction of metronidazole. To determine further the exact function for Hydrogenase 1 in the reduction of the drug, this enzyme was highly purified from C. pasteurianum. Metronidazole reduction activity copurified with Hydrogenase 1 specific activity throughout the purification procedure. Drug reduction required the presence of an electron carrier and could not be accomplished by the enzyme alone. Ferredoxin, and also the low potential electron carrier dyes, methyl and benzyl viologen, and the flavin coenzymes, FAD and flavin mononucleotide (FMN), could couple the reduction of metronidazole. Hydrogenase 1 activity and its metronidazole reduction activity were inactivated irreversibly in the presence of oxygen. Metronidazole could be reduced only by an electron carrier-Hydrogenase 1 mechanism or directly by sodium dithionite.

Effect of pyruvate on glucose metabolism in Clostridium acetobutylicum

Pyruvate effects on the metabolism of Clostridium acetobutylicum during glucose fermentation were studied. After addition to the culture medium, the pyruvate was rapidly used, provoking several changes in the metabolic pattern of the bacteria. When pyruvate addition occurred early in the fermentation, the glucose utilization decreased and the solventogenic phase was not induced. When pyruvate was added during solventogenesis, glucose consumption was slightly affected and the cells fermented both substrates simultaneously: however, the acidogenic phase started again to the detriment of solvent formation. Usually, during the solvent phase, the cells remetabolized acetic and butyric acids into solvents, but when pyruvate was added, the utilization of acids was stopped and the specific rates of acetate and butyrate formation increased immediately. The acidogenic growth phase was characterized by high levels of acetate and butyrate kinase which dropped during the solvent phase. Addition of pyruvate limited the down shift of these two enzymes and the levels of the activities remained constant during the course of the fermentation. Conversely, the acetoacetate decarboxylase, which is characteristic of the solvent phase, decreased sharply in the presence of pyruvate. The fact that the specific rate of glucose consumption was not decreased by the pyruvate metabolism, a cosubstrate, proves that the phosphoroclastic reaction is not a limiting step. Furthermore, the pyruvate utilization represented a promising approach to obtain useful data on the intracellular compounds implicated in the mechanism for switching from the acidogenic to the solventogenic phase.

Isolation and characterization of a rubredoxin and a two-(4Fe-4S) ferredoxin from Thermodesulfobacterium commune

Two non-heme iron proteins, ferredoxin and rubredoxin, have been isolated from the thermophilic non-sporeforming sulfate-reducer Thermodesulfobacterium commune. In most respects, the rubredoxin is similar to the corresponding mesophile proteins from other anaerobic bacteria but it differs by its higher content of proline residues. Native ferredoxin from T. commune is a dimer comprising two identical subunits of approx. 7000 molecular weight. Its absorption spectrum exhibits two maxima at 385 nm (29 500 M −1 · cm −1) and 280 nm (36 100 M −1 · cm −1) and its A 385 A 280 absorbance ratio is 0.82. The protein contains 8 atoms each of iron and labile sulfur per molecule. The absorption at 385 nm and the content of iron of the protein are in agreement with the presence of two (4Fe-4S) clusters in T. commune ferredoxin. Its amino acid composition shows the presence of six cysteine residues and is characterized by the absence of histidine and a high content of aromatic residues. The N-terminal amino acid sequence of T. commune ferredoxin has been established. The comparison of amino acid sequences shows that it presents more homology with the one-(4Fe-4S) ferredoxin from Clostridium thermoaceticum than with the two-(4Fe-4S) ferredoxin from Desulfovibrio desulfuricans Norway. T. commune ferredoxin shows thermal stability and retains its full activity in the phosphoroclastic reaction after treatment at 70°C. The protein exhibits sensitivity towards oxygen at 25°C.

Role of the phosphoroclastic reaction of Clostridium pasteurianum in the reduction of metronidazole.

To demonstrate the importance of electron siphoning by the metronidazole reductase system from reduced ferredoxin to the mechanism of action of the drug in Clostridium pasteurianum, the effects of the reduction of metronidazole on the phosphoroclastic reaction were studied. Metronidazole concentrations between 0.5 and 5 mM caused a significant increase in acetyl phosphate production by the phosphoroclastic reaction compared to the control system without metronidazole. When this enzymatic reaction was assayed by standard manometric techniques under nitrogen gas, two simultaneous effects of electron siphoning were demonstrated: (i) the electrons from reduced ferredoxin were initially consumed for the reduction of metronidazole instead of being evolved as H2 via the ferredoxin-linked hydrogenase and (ii) phosphoroclastic activity was stimulated, with augmented production of CO2 and acetyl phosphate. This work further supports the notion of preferential scavenging of electrons away from ferredoxin-linked enzymatic reactions by metronidazole reductase(s) in C. pasteurianum.

Interconversion from 3Fe into 4Fe clusters in the presence of Desulfovibrio gigas cell extracts

Desulfovibrio gigas ferredoxin II (FdII) contains a single 3Fe cluster [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V., and Münck, E. (1980) J. Biol. Chem. 255, 3242-3244]. In the oxidized state the protein exhibits an intense electron paramagnetic resonance (EPR) signal at g = 2.02. Upon one-electron reduction the center becomes EPR silent. In the presence of D. gigas crude cell extracts, devoid of acidic electron carriers and supplemented with pyruvate and FdII, an EPR signal typical of reduced [4Fe-4S] centers is obtained. The appearance of this signal correlates with the beginning of stimulation of the phosphoroclastic reaction as judged by the production of H2. These results, supported by the occurrence of easy chemical conversion of the 3Fe cluster of D. gigas ferredoxin into 4Fe structures [Moura, J.J.G., Moura, I., Kent, T.A., Lipscomb, J.D., Huynh, B.H., LeGall, J., Xavier, A.V., and Münch, E. (1982) J. Biol. Chem. 257, 6259-6267], suggest that cluster conversion takes place in conditions close to the situation in vivo. This cluster interconversion is discussed in the context of some of the relevant metabolic pathways of Desulfovibrio spp.

Characterization of a new type of ferredoxin from Desulfovibrio africanus

A new ferredoxin designated ferredoxin III has been isolated from Desulfovibrio africanus grown on media high in iron. Native ferredoxin III is a dimer constituted by two identical subunits of approx. 7500. It is distinguished from the two other ferredoxins (I and II) isolated from this microorganism by its amino acid composition, N-terminal sequence, spectral properties and ironsulfur content. The amino acid composition of D. africanus ferredoxin III is typical of ferredoxins with an excess of acidic over basic residues and the absence of histidine and arginine residues. The absorption spectrum of ferredoxin III exhibits two maxima, at 408 nm ( ϵ = 58.5 · 10 3 M −1 · cm −1) and 285 nm ( ϵ = 82 · 10 3 M −1 · cm −1), with a shoulder at 305 nm ( ϵ = 75 · 10 3 M −1 · cm −1). Its A 408/A 285 absorbance ratio is 0.78. Ferredoxin III contains approx. 12–13 atoms each of iron and labile sulfur. This is in agreement with the high value of the extinction coefficient at 408 nm, which is slightly higher than 3-fold that of one [4Fe-4S] cluster. However, the number of cysteine residues of the protein (six residues), which is about the half that of iron atoms, is indicative of the presence of a new type of iron-sulfur cluster in ferredoxin III. The protein is unstable in a low ionic strength environment; the addition of neutral salts stabilizes the protein conformation. The data on the biological activity of ferredoxin III as compared to the two other ferredoxins from D. africanus show that the three iron-sulfur proteins function with equal effectiveness as electron carrier in the phosphoroclastic reaction and the H 2-sulfite reductase system.

Comparative studies of two ferredoxins from Desulfovibrio desulfuricans Norway

Two ferredoxins isolated from Desulfovibrio desulfuricans Norway have been purified and characterized. The less acidic, designated as ferredoxin I, contains four iron atoms, four acid-labile sulfur groups and six cysteine residues per molecule. Ferredoxin II is more acidic and abundant than ferredoxin I, but is very unstable to O2. Ferredoxin I and ferredoxin II differ according to amino acid composition but are homologous with respect to their N-terminal amino acid sequence. The absorption spectra of the two ferredoxins are similar to those of other Desulfovibrio species. Both proteins appear to be dimers of identical 6000-dalton subunits. Their activity was tested in two types of reaction in the electron transfer chain (phosphoroclastic reaction and sulfite reductase activity). The isolation of two different ferredoxins from the same organism, Desulfovibrio, has been reported in Desulfovibrio africanus but the significance of two ferredoxins functioning in the same electron transfer chain is not yet understood.

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A 278 A 490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A 385 A 283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology. The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.

Structural control of the redox potentials and of the physiological activity by oligomerization of ferredoxin

Fig. 1. Two relevant electron pathways in Desulphovibrio sp., the phosphoroclastic reaction and sulphite reductase system. The central role of cytochrome cs and the coupling effect of ferredoxin are schematized. Since the trithionate pathway, which involves the existance of sulphur intermediates during the reduction of sulphite to sulphide [ 161, is still under controversy, the global value of g’, from SO:to S*is given here. This value has been computed from the data in [ 17-201. Abbreviations: PDH, pyruvate dehydrogenase; Cyt cl, cytochrome cJ (4 haems); Fd, (4 Fe, 4 S) ferredoxin; Hase, hydrogenase 3x (4 Fe, 4 S).

Bisulfite reductase of Desulfovibrio vulgaris: explanation for product formation.

Bisulfite reductase, purified from Desulfovibrio vulgaris, was coupled with the pyruvate phosphoroclastic reaction. Moderate to low reducing conditions resulted in the formation of trithionate; however, when the concentration of reductant was high, a mixture of trithionate and thiosulfate was formed. Sulfide was also a detectable product, but only when the concentration of bisulfite was low. Flavodoxin mediated native coupling between bisulfite reductase and the phosphoroclastic reaction. A model for bisulfite reductase activity is proposed.

The selective toxicity of antimicrobial nitroheterocyclic drugs.

Summary: Three antimicrobial nitroimidazole drugs (metronidazole, dimetridazole, and tinidazole) inhibit a range of Clostridia and the protozoan Trichomonas vaginalis; they have an identical site and mode of action as specific electron acceptors from the pyruvate phosphoroclastic reaction. Analogues of the drugs are compared and the structural requirements for activity explained. The nitrofuran (nitrofura-zone) probably has a different mechanism of action.

The structure of a clostridial flavodoxin, an electron-transferring flavoprotein. 3. An interpretation of an electron-density map at a nominal resolution of 3.25 Angstrom.

Within the last five years, several low molecular weight flavoproteins have been isolated from microorganisms. The first of these proteins was purified from Clostridium pasteurianum grown in limiting concentrations of iron (Knight and Hardy, 1966) and was named flavodoxin since it substituted for the non-heme iron protein, ferredoxin, in ferredoxin-dependent reactions (Knight and Hardy, 1966; 1967). Subsequently flavodoxins have been isolated from other strict anaerobes including Peptostreptococcus elsdenii (Mayhew and Massey, 1969), Clostridium MP, a strain closely related to C. pasteurianum (Mayhew, 1971a), and two species of Desulfovibrio (Dubourdieu and Le Gall, 1970), and also from E. coli grown under aerobic conditions (Vetter and Knappe, 1971). All the flavodoxins contain a single FMN and no other prosthetic groups or metals have been detected (Knight and Hardy, 1966; Mayhew and Massey, 1969). They can replace ferredoxins as electron carriers in the phosphoroclastic reaction of pyruvate, the fixation of nitrogen, the photosynthetic...

The mode of action of metronidazole against Trichomonas vaginalis.

Summary: Metronidazole (1-β-hydroxyethyl-2-methyl-5-nitroimidazole) inhibited the evolution of hydrogen gas in Trichomonas vaginalis before it inhibited carbon dioxide evolution. Evidence is presented that the phosphoroclastic reaction of the clostridial type was the major mechanism by which both gases were evolved, and it is postulated that metronidazole inhibits, directly or indirectly, the hydrogenase component of the system. A possible mechanism of action is discussed.

Requirement for coenzyme A in the phosphoroclastic reaction of anaerobic bacteria.

Various bacteria which degrade pyruvate by the phosphoroclastic reaction were examined with respect to the role of coenzyme A (CoA) in this reaction. The strictly anaerobic bacteria, which cleave pyruvate by the phosphoroclastic reaction characteristic of Clostridia, required catalytic levels of CoA for the CO(2)-pyruvate exchange and acetoin-forming portions of the phosphoroclastic reaction. These reactions were reversibly inhibited by the CoA analogue, desulfo-CoA. In contrast, using cell-free extracts of bacteria which degrade pyruvate by the coliform phosphoroclastic reaction (pyruvate formate-lyase), no requirement for CoA could be observed for the formate-pyruvate exchange reaction. It is suggested that CoA serves a regulatory function in the early portion of the clostridal type of phosphoroclastic reaction.

Purification and Characterization of Flavodoxin from Peptostreptococcus elsdenii

Abstract Flavodoxin has been crystallized from Peptostreptococcus elsdenii grown at low iron concentrations. The properties of this protein are similar to those of flavodoxin from Clostridium pasteurianum. It has a molecular weight of about 15,000 and contains 1 molecule of flavin mononucleotide per molecule of protein. It does not contain labile sulfide or iron. It lacks the amino acid histidine. Ferredoxin and flavodoxin are interchangeable in the oxidation of pyruvate by extracts of P. elsdenii from media either high or low in iron, and in the phosphoroclastic reaction of C. pasteurianum. On a molar basis flavodoxin is somewhat less effective than ferredoxin in these systems. Flavodoxin is reduced by light irradiation in the presence of EDTA and an intermediate having a spectrum characteristic of flavin neutral semiquinone is generated. The rate and extent of reduction are pH-dependent. The spectrum of the semiquinone is unaffected at high pH and by high concentrations of urea. The extinction coefficient of oxidized flavodoxin at 445 mµ is 10,200 m-1 cm-1. The extinction coefficient of flavodoxin semiquinone at 580 mµ is 4,500 m-1 cm-1.