Jan R. Andreesen

Introduction to the Physiology and Biochemistry of the Genus Clostridium

The genus Clostridium was created by Prazmowski in 1880. Four criteria presently classify an organism as a Clostridium: (1) the ability to form endospores; (2) restriction to an anaerobic energy metabolism; (3) the inability to carry out a dissimilatory reduction of sulfate; and (4) the possession of a gram-positive cell wall, which may react gram-negative. These criteria are met by an otherwise diverse assembly of microorganisms, and the genus Clostridium has grown to be one of the largest genera among prokaryotes. A total of 83 species are listed in Bergey’s Manual of Systematic Bacteriology (Cato et al., 1986). Since this list was compiled, a number of new species have been described, while others, such as C. tetanomorphum and C. cylindrosporum, have been omitted (see Chapter 1). In this chapter the span of properties found among the Clostridia will be outlined. Additional information on the general taxonomy, the general properties of Clostridia, and clostridial fermentations may be found in a number of recent reviews (Barker, 1961, 1978, 1981; Wood, 1961; Thauer et al., 1977; Gottschalk and Andreesen, 1979; Gottschalk et al., 1981; Booth and Mitchell, 1987).

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate

An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H2-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO2. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO2, H2 and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H2, formate or e.g. valine were added as H-donors. In pure culture on alanine under N2, strain al-2 grew very poorly and produced H2 up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H2-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H2S, CH4 or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (≥0.1 μM); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO2. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO2 and H2. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.

Clostridium formicoaceticum nov. spec. isolation, description and distinction from C. aceticum and C. thermoaceticum.

ZusammenfassungClostridium formicoaceticum wird als eine neue Art beschrieben. Es vergärt Fructose und eine Reihe von Hexon- und Hexuronsäuren zu Acetat und Formiat. Während des Wachstums wird hauptsächlich Acetat gebildet; größere Mengen von Formiat erscheinen unter den Gärungsendprodukten erst in der stationären Wachstumsphase. C. formicoaceticum ist mesophil. Es unterscheidet sich von C. aceticum durch sein Unvermögen, mit Wasserstoff und Kohlendioxid zu wachsen. Weiterhin kann molekularer Wasserstoff auch in Gegenwart einer organischen Energiequelle von C. formicoaceticum nicht für die Acetatsynthese genutzt werden.SummaryA new species—Clostridium formicoaceticum—is described. It ferments fructose and several hexonic and hexuronic acids to acetate and formate. During active growth acetate is the main product of the fermentation. Considerable quantities of formate appear among the fermentation products in the stationary growth phase. C. formicoaceticum is mesophilic. It differs from C. aceticum in its inability to grow on hydrogen plus carbon dioxide. Furthermore, hydrogen gas does not stimulate acetate production even in the presence of organic energy sources.

[39] Formate dehydrogenase, a selenium-tungsten enzyme from Clostridium thermoaceticum

Publisher Summary This chapter describes the purification procedure of formate dehydrogenase, a selenium–tungsten enzyme from Clostridium thermoaceticum. Formate dehydrogenase of C. thermoaceticum catalyzes the reversible reaction. C. thermoaceticum is a thermophilic anaerobic bacterium that ferments sugars to acetate as the only product. It is a strictly anaerobic bacterium, and it fails to grow in the presence of small amounts of oxygen. The only known naturally occurring electron acceptor is NADP, and the enzyme should perhaps be named formate: NADP oxidoreductase. The physiological role of the enzyme is to catalyze the reduction of CO 2 to formate. C. thermoaceticum (DSM 521) is maintained in agar-stab cultures or in liquid broth cultures. The buffer used in the purification of the formate dehydrogenase from C. thermoaceticum contains thioglycolate and iron. These substrates interfere with the assay of protein if the biuret method or the method by Lowry et al . is used. Therefore, protein is assayed by precipitating with trichloroacetic acid and by determining the turbidity after 10 minutes at 400 nm.

Reductive cleavage of sarcosine and betaine by Eubacterium acidaminophilum via enzyme systems different from glycine reductase

The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO2 or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [75Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.

Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation

The formate dehydrogenases of Clostridium acidiurici and of C. cylindrosporum coupled the oxidation of formate with the reduction of viologen dyes. The basal activity level was about 0.85 μmoles/min s mg of protein for both species. The level of formate dehydrogenase of C. acidiurici increased 12-fold when 10-7 M tungstate and selenite were present during growth. Molybdate exerted no effect. On the other hand, molybdate and selenite were required to increase the formate dehydrogenase of C. cylindrosporum, and tungstate exhibited an antagonistic effect in this organism.Growth on hypoxanthine generally depended on the addition of bicarbonate. Supplementation with tungstate and selenite accelerated growth of C. acidiurici and increased again the level of formate dehydrogenase. The addition of both, molybdate and selenite was necessary to initiate growth of C. cylindrosporum and to form an active formate dehydrogenase.The differences in the requirement for metal ion supplementation to form high levels of formate dehydrogenase and their involvement in hypoxanthine degradation can be used to differentiate between C. acidiurici and C. cylindrosporum.

Some properties of formate dehydrogenase, accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum

Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S0.5 values were 0.9×10-4 M for formate and 5.8×10-3 M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70°C, whereas the activity was stable only up to 50°C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10-6 M selenite and 10-7 M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. 185W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. 185W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.

Tungsten, a component of active formate dehydrogenase from Clostridium thermoaceticum

Formate dehydrogenase from Clostridium thermoaceticum catalyzes the reaction: CO? + NADPH + HCOO+ NADP+. The activity of the enzyme in growing cells is enhanced when selenite and molybdate are added together to the growth medium [ 11. Tungstate replaces and is better than molybdate, and “Se-selenite is incorporated into the protein fraction with the formate dehydrogenase activity [ 11. It was concluded that the formate dehydrogenase may be a metalloenzyme containing both selenium and tungsten or molybdenum. In this communication we report that ‘as W-tungsten is incorporated into the active enzyme fraction. In addition, a second protein fraction with a mol. wt of about 60 000 was found labelled with radioactive tungsten.

The effect of ferrous ions, tungstate and selenite on the level of formate dehydrogenase inClostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation

In a medium containing a trace element solution and 10-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10-4 M tungstate and 10-5 M selenite in addition.Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide.The formate dehydrogenase catalyzed also the reduction of CO2 to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO2 exchange could be observed with cell-free extracts ofC. formicoaceticum.Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate.

Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini

The enzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini could be purified to homogeneity by means of anion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and chromatography on hydroxylapatite. During enrichment procedures both enzymes showed a significant loss in specific activity. The molecular weight of nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase was determined to be about 300,000 and 120,000, respectively. They were highly substrate specific and transferred electrons only to artificial acceptors of high redox potential. The Km for their specific substrates was about 1.0 mM for both enzymes, and their pH optimum was determined to be 7.5. For nicotinate dehydrogenase a content of 8.3 mol iron, 1.5 mol acid-labile sulfur, 2.0 mol flavin, and 1.5 mol molybdenum per mol of enzyme was determined. Both enzymes contained FAD and Fe/S center. After inhibition by KCN, thiocyanate was detected, and subsequently the initial nicotinate dehydrogenase activity was restored by the addition of Na2S indicating the presence of cyanolyzable sulfur. 6-Hydroxynicotinate dehydrogenase seemed to contain the same type of constituents as determined for nicotinate dehydrogenase. A partial immunological identity of the enzymes could be shown by antibodies raised against nicotinate dehydrogenase.