Substrate specificity of soluble methane monooxygenase. Mechanistic implications.

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO(2). Rates up to 0.4 mumol min(-1) mg of wet cells(-1) were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO(2) reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 mumol min(-1) mg of protein(-1) (35 degrees C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was "ping-pong," indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO(2) to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO(2) do not possess a CO-oxidizing system.

Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

A new anaerobic, mesophilic, spore-forming cellulolytic bacterium is described Cellulose is cleared within 24 to 48 h around colonies formed in cellulose agar roll tubes Cells stain gram negative and are nonmotile rods which form oblong spores either centrally or subterminally in a clostridial swelling Colonies are irregular with an opaque edge and a center devoid of both vegetative cells and spores Cellulose, xylan, pectin, cellubiose, glucose, maltose, galactose, sucrose, lactose, and mannose serve as substrates for growth H/sub 2/, CO/sub 2/, acetate, butyrate, formate, and lactate are produced during fermentation of cellulose or cellobiose The temperature and pH for optimum growth are 37/sup 0/C and 70, respectively The DNA composition is 26 to 27 mol% guanine plus cytosine This bacterium resembles Clostridium lochhheadii in morphological and some biochemical characteristics but is not identical to it The name Clostridium cellulovorans sp nov is proposed The type strain is 743B (ATCC 35296)

https://aem.asm.org/content/aem/48/1/88.full.pdf

Isolation and Characterization of an Anaerobic, Cellulolytic Bacterium, Clostridium cellulovorans sp. nov

Anaerobic digestion of glucose with separated acid production and methane formation

Strains of Clostridium thermoaceticum were tested for H2- and CO-dependent growth in a defined medium containing metals, minerals, vitamins, cysteine-sulfide, CO2-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditions (the defined medium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively. Product stiochiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2----CH3COOH + 0.1 cell C + 0.3 unrecovered C and 6.8CO----CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced significantly higher acetate concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 values for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the YCO value for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source utilized for growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the Vmax values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 micromoles of H2 oxidized per min mg of protein, respectively; the Vmax values for CO dehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 micromoles of CO oxidized per min per mg of protein, respectively.

Characterization of the H2- and CO-dependent chemolithotrophic potentials of the acetogens Clostridium thermoaceticum and Acetogenium kivui.

Eine neue, spezifische Farbreaktion ermoglicht den Nachweis der Ameisensaure und des Formiats ohne vorherige Reduktion zu Formaldehyd. Der Nachweis beruht auf der roten Farbung, die man durch Reaktion von Citronensaure bzw. Aconitsauren mit Ameisensaure oder Formiat in Gegenwart von Essigsaureanhydrid, i-Propanol oder einer geeigneten Acetamid-i-Propanol-Losung und einer kleinen Menge Alkali erhalt. Die bei Verwendung von Citronensaure erzeugte rote Farbe zeigt ein Lichtabsorptionsmaximum bei 515 nm. Weniger als 20 μg Formiat/ml konnen nachgewiesen werden.

E. Lang

Papers

Spezifische Farbreaktion zum direkten Nachweis der Ameisensäure