Characterization of Aspergillus sydowii (Thom et Church), a fungal pathogen of Caribbean sea fan corals

In the Caribbean, the fungus Aspergillus sydowii is currently causing an epizootic among sea fan corals (Gorgonia spp.). To elucidate potential factors that may have facilitated the emergence of this disease, we characterized and compared temperature requirements, susceptibility to coral crude extracts, and metabolic profiles of pathogenic (marine) and non-pathogenic (terrestrial) strains of A. sydowii. Growth of all A. sydowii strains were observed at all temperatures tested (22–36 °C) with an optimum of approximately 30 °C. Sea fan crude extracts inhibited growth of A. sydowii but were less effective at higher temperatures. Thus, temperature is likely to have a strong influence on the dynamics of the Gorgonia–Aspergillus interaction by promoting the growth of the pathogen while reducing the efficacy of host resistance. Metabolically, marine A. sydowii strains pathogenic to sea fans were distinct from non-pathogenic terrestrial strains.     

Purification and characterization of NADP-dependent 7β-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52

An NADP-dependent 7β-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7β-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (K_m) value of 0.05 mM for 3α,7β-dihydroxy-5β-cholanic acid whereas higher values were obtained with 3α,7β-dihydroxy-5β-cholanoyl glycine (0.20 mM), and 3α,7β-dihydroxy-5β-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and has a K_m value of 0.30 mM. A molecular weight of 64 000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.     

Interactive effects of vitamins A and K3 on laying performance,egg quality,tibia attributes and antioxidative status of aged Roman Pink laying hens

Extending laying cycle is a tendency in hen breeding, but egg quality declines as laying hens age. The present study was conducted to investigate the interactive effects of vitamins A and K₃ on laying performance, egg and tibia quality, and antioxidative status of aged Roman Pink laying hens. In a 3 × 3 factorial arrangement, 1 080 87-week-old laying hens were allocated to nine groups with eight replicates in each group. Deficient, adequate and excess vitamins A (0, 7 000 and 14 000 IU/kg) and K₃ (0, 2.0 and 4.0 mg/kg) were supplemented into a basal diet with 1 320 IU/kg of vitamin A and 0.5 mg/kg of vitamin K₃. After 2 weeks of adaption to basal diet, hens were fed corresponding diets for 8 weeks. Vitamins A and K₃ did not significantly affect the laying performance. However, they showed interactive effects on yolk ratio at week 93 as well as tibia weight and diameter (P < 0.05), and hens fed deficient vitamins A and K₃ had the highest yolk ratio and tibia weight, but the lowest tibia diameter. Compared with deficient addition, adequate or excess vitamins A and K₃ increased yolk color at weeks 93 and 97 (P < 0.05). Compared with hens fed deficient or excess vitamins, hens fed adequate vitamins A and K₃ had higher eggshell strength at week 93 or 97 (P < 0.05). Increasing vitamin A elevated plasma total superoxide dismutase (T-SOD) activity and decreased hepatic glutathione peroxidase (GSH-Px) activity (P < 0.05). Excess vitamin K₃ increased hepatic T-SOD activity (P < 0.05). Vitamins A and K₃ exhibited interaction on the activities of antioxidative enzymes in eggshell gland (P < 0.05), and adequate or excess vitamins A and K₃ increased the activities of GSH-Px, T-SOD and catalase (CAT). Adequate and excess vitamin A up-regulated the mRNA expression of GSH-Px1, GSH-Px3 and SOD1 in eggshell gland (P < 0.05). Vitamins A and K₃ showed interactive effects on CAT mRNA expression in eggshell gland (P < 0.05) and hens fed adequate vitamins A and K₃ had the highest CAT mRNA levels. In conclusion, dietary addition of vitamins A and K₃ improved the eggshell quality and yolk color as well as antioxidative status in eggshell gland of aged laying hens. Adequate vitamins A and K₃ showed beneficial effects and excess levels did not exhibit superior effects.     

Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h^–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low K_m values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher K_m values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V_max values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high K_m for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde. Received: 31 August 1995 / Accepted: 4 December 1995     

Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B₁₂) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998     

Sulfite formation by wine yeasts

The enzyme catalyzing the reduction of sulfite by reduced benzyl viologen (BVH) was partially purified and characterized from two strains of wine yeasts, a sulfite-producing strain and a non-producing strain.Both enzymes showed corresponding features in pH-optima, optima of buffer and benzyl viologen concentrations.The enzymes did not catalyze the reduction of nitrite by reduced viologen dyes, but the reduction of sulfite was uncompetitively inhibited by nitrite. Compounds of sulfur metabolism such as sulfate, thiosulfate, cysteine, serine and methionine did not influence the activity of either of the enzymes. The main differences between the two enzymes exist in the specific activities in crude extracts, the K _m -values for sulfite, substrate inhibition rates, and localization in different fractions during (NH₄)₂SO₄ precipitation. The specific activity in crude extracts of the sulfite-producing strain (0.052 moles S^2- x min^-1 x mg^-1) was about three fold higher than that of the non-producing strain (0.0179 moles S^2- x min^-1 x mg^-1). On the other hand the sulfite-producing strain had a higher K _m -value for sulfite (2×10^-3 M) and was more strongly inhibited by the substrate than the non-producing strain (6×10^-3 M).     

Characterization and Regulation of Sulfur Reductase Activity in Thermotoga neapolitana

The growth of the hyperthermophilic, anaerobic bacterium Thermotoga neapolitana is stimulated by elemental sulfur by an unknown mechanism. We detected hydrogen-dependent sulfur reductase (sulfhydrogenase) and polysulfide dehydrogenase activities in cell extracts of this organism, demonstrating that it has at least two pathways for sulfidogenesis. Hydrogen-dependent sulfur reductase and hydrogenase activities are catalyzed by the purified hydrogenase of Thermotoga maritima, and this enzyme was called the sulfhydrogenase (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). Cells grown without elemental sulfur or cystine had 1.3 to 3.3 times higher sulfhydrogenase activities than those grown with either of these sources of sulfane sulfur. Hydrogenase activity was 2 to 5 times higher. Polysulfide dehydrogenase was up to 48-fold more active in cell extracts than the sulfhydrogenase. The activity of polysulfide dehydrogenase was approximately twofold higher when cells were grown in the presence of elemental sulfur. Its activity was oxygen labile in crude extracts, and it appears to be a cytoplasmic enzyme. Polysulfide was preferred over elemental sulfur as an electron acceptor (K_m = 0.15 mM) and was more active with NADH (K_m = 0.03 mM) than NADPH (K_m = 0.41 mM). Growth in the presence of elemental sulfur appeared to slightly increase the activity of polysulfide dehydrogenase and slightly decrease both activities of sulfhydrogenase (hydrogenase and polysulfide reductase), while growth without elemental sulfur had the opposite effects. The greater activity of polysulfide dehydrogenase and its apparent regulation indicate that it is the more physiologically important means of polysulfide reduction.     

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity are evaluated. IC₅₀ of nobiletin and hesperidin is 1.49 mM and 16.08 mM, respectively and their inhibition mechanism is competitive type with K_i = 2.82 mM and noncompetitive with K_i = 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC₅₀ values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition on melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.5% at 31.25 μg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE give efficacious antiproliferation effects on B16 mouse melanoma cell with IC₅₀ values 88.6 μM and 62.96 μg/mL, respectively, by the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.     

Growth and nutritional characteristics of cowpea rhizobia

Summary Twenty-five slow-growing strains of cowpea rhizobia were examined for growth and nutritional characteristics. Growth and nutritional data of these isolates were surprisingly homogeneous given their proposed genetic diversity. Most strains tested were capable of anaerobic growth in the presence of nitrate and all were found capable of autotrophic growth in a defined atmosphere of CO₂ and H₂ with oxygen or nitrate as terminal electron acceptors. These isolates grew heterotrophically with various carbohydrates and organic acids. Nitrogen utilization was consistent with that of other slow-growing rhizobia. Medium composition strongly affected the final pH of the culture. Cowpea rhizobia generally did not require vitamins; those requiring vitamins exhibited good growth when biotin was supplemented to the medium.     

Crude Extracts and Secondary Metabolites of Epichloë bromicola against Phytophthora infestans

Potato late blight caused by Phytophthora infestans is still one of the main factors limiting potato production. Epichloë spp. can provide host plants with various resistances, which makes them show great potential in the biological control of diseases. In this study, we explored the potential biological activity of crude extracts of 20 strains of Epichloë bromicola to control P. infestans. The crude extracts of strains 1 and 8 showed significant antifungal activity with an inhibition rate of 88 % and 81 %, respectively, and showed different effects on the mycelium morphology of P. infestans observed by scanning electron microscopy. Moreover, the two crude extracts demonstrated an interesting therapeutic and protective effect on potato late blight, and none of the extracts had an adverse effect against zebrafish embryos. A total of 13 metabolites were isolated from the crude extract of strain 8, and these tested compounds showed a weak antifungal effect and the inhibition rate was less than 80 %. These findings suggested that strains 1 and 8 have potential for biocontrol of late potato blight.     

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species,the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied.Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for K_m, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent K_m and V_max values for NADH of the total LDH activity in MRM were followed.It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.The investigation were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

Cytochromes in free-living rhizobia

Difference spectra of the crude cell-free extracts of 22 strains of fast-growing and slow-growing rhizobia, including members of the cowpea group, reveal the presence of cytochromes c, b, aa₃, o, and a soluble CO-reactive hemoprotein P-428 in all of them. No strict correlation between the growth properties of the strains and their cytochrome contents are observed. All the pigments are present in varying quantities at all stages of growth ofRhizobium meliloti SU 216 andBradyrhizobium species (Lupinus) RL3001. A progressive increase in the level of the pigments is observed with the progress of bacterial growth until an optimum concentration is reached, whereupon the level tends to decrease. However, the ratio of cytochrome c to cytochrome b increases linearly throughout the growth period. Cytochromes b, c552, aa₃, and o are particle bound, whereas cytochrome c550 and P-428 are soluble.     

A thermo-stable dehalogenase in the extracts fromPseudomonas sp (strain 19S)

Summary A thermo-stable dehalogenase was demonstrated in the crude extracts fromPseudomonas sp (19S). The ability of the enzyme to catalyze the dehalogenation of various halogen-substituted organic acids was investigated and the highest activity was found with monochloroacetate. The enzyme followed Michaelis-Menten kinetics, and the K_m for monochloroacetate was 0.2mM. Maximum activity was found at pH 10.5 and 60°C. The enzyme activity in the cell-free extract was unaffected by EDTA or by Mn, Zn, or Cu ions, but was dramatically reduced by HgCl₂ (70%) and Pb (NO₃)₂ (80%).     

Identification of hydroxypyruvate and glyoxylate reductases in maize leaves

At least two hydroxypyruvate reductases (HPRs), differing in specificity for NAD(P)H and (presumably) utilizing glyoxylate as a secondary substrate, were identified by fractionation of crude maize leaf extracts with ammonium sulfate. The NADH-preferring enzyme, which most probably represented peroxisomal HPR, was precipitated by 30 to 45% saturated ammonium sulfate, while most of the NADPH-dependent activity was found in a 45 to 60% precipitate. The HPRs had similar low K_ms for hydroxypyruvate (about 0.1 millimolar), regardless of cofactor, while affinities of glyoxylate reductase (GR) reactions for glyoxylate varied widely (K_ms of 0.4-12 millimolar) depending on cofactor. At high hydroxypyruvate concentrations, the NADPH-HPR from the 30 to 45% precipitate showed negative cooperativity with respect to this reactant, having a second K_m of 6 millimolar. In contrast, NADPH-HPR from the 45 to 60% precipitate was inhibited at high hydroxypyruvate concentrations (K₁ of 3 millimolar) and, together with NADPH-GR, had only few, if any, common antigenic determinants with NADH-HPR from the 30 to 45% fraction. Both NADPH-HPR and NADPH-GR activities from the 45 to 60% precipitate were probably carried out by the same enzyme(s), as found by kinetic studies. Following preincubation with NADPH, there was a marked increase (up to sixfold) in activity of NADPH-HPR from either crude or fractionated extracts. Most of this increase could be attributed to an artefact resulting from an interference by endogeneous NADPH-phosphatase, which hydrolyzed NADPH to NADH, the latter being utilized by the NADH-dependent HPR. However, in the presence of 15 millimolar fluoride (phosphatase inhibitor), preincubation with NADPH still resulted in over 60% activation of NADPH-HPR. The NADPH treatment stimulated the V_max of the reductase but had no effect on its K_m for hydroxypyruvate. Enzyme distribution studies revealed that both NADH and NADPH-dependent HPR and GR activities were predominantly localized in the bundle sheath compartment. Rates of NADPH-HPR and NADPH-GR in this tissue (over 100 micromoles per hour per milligram of chlorophyll each) are in the upper range of values reported for leaves of C₃ species.     

The effects of temperature and pH on the apparent Michaelis constant of glutathione reductase from maize (Zea mays L.)

The interacting effects of temperature and pH on the kinetics of glutathione reductase from maize have been studied in detail. The apparent K_m for oxidized glutathione (GSSG) measured with desalted crude extracts increased in an exponential manner with rising temperature as a single variable. Increasing pH as a single variable also resulted in higher values of apparent K_m for GSSG. When pH was allowed to vary with temperature, a curve which combined the pH and temperature responses was observed. Temperature had the stronger influence and this combined curve was displaced from the temperature curve due to the effect of pH. The pH to which the assay buffer was adjusted at 30°C had an influence on the pattern of the results in this type of experiment. The response of apparent K_m for NADPH, and of apparent K_m for GSSG using partially-purified extracts, were also examined. The variation with temperature, at constant pH, was again exponential. The pattern of change of apparent K_m with temperature is strongly dependent on experimental conditions. Affinity/temperature relationships deduced from such data would only reflect enzyme function in vivo if the physiological environment could be reproduced exactly in the assay mixture.     

Rice callus extracts for enhancing skin wound healing

Enhanced cell migration in the course of wound healing is required to repair damaged skin. We investigated the effects of rice callus extracts on the migration of skin keratinocytes. Rice callus extracts were obtained by using three different methods: pressurized hot water, crude ethanol, and liquid-liquid extractions. The extract obtained by using crude ethanol extraction was more effective in the migration of skin cells than that obtained by pressurized hot water extraction (PHWE). The crude ethanol extract (CEE) was further partitioned by performing liquid-liquid extraction. As phenolics are inhibitory compounds affecting cell migration, we analyzed the total phenolic content of the rice callus extracts. The level of phenolics in the n-hexane partitioned extract (n-HPE) of CEE was lower than that in all other partitioned extracts. The n-HPE was most effective in enhancing cell migration. We analyzed wound healingrelated factors including platelet derived growth factor B (PDGF-B), transforming growth factor beta 1 (TGF-β1), heparin-binding epidermal growth factor (HB-EGF), and fibroblast growth factor 2 (FGF-2) after the treatment of n-HPE. Most of the expressions of cell migration-related growth factors increased, but HB-EGF dramatically increased (6.5-fold) in keratinocytes treated with n-HPE. The results indicate that n-HPE contains more stimulating growth factors or proteins and less cell migration inhibiting factors than the other tested extracts; thus, n-HPE treatment produced the greatest enhancement of cell migration.     

Antimicrobial activity in sub-Arctic marine invertebrates

In the marine environment, any living or non-living surface is exposed to bacterial colonization. Many invertebrate species in temperate, tropical and Antarctic regions have demonstrated chemical defences against the formation of microbial films. In the present study, the antimicrobial activity of sub-Arctic invertebrates was investigated for the first time. Crude extracts of abundant invertebrates belonging to several taxonomic groups were tested for their inhibitory effects on the growth of five sympatric phylogenetically diverse bacterial strains. Six out of 18 (33%) crude extracts inhibited bacterial growth at natural extract concentrations. The crude extract of the sponge Haliclona viscosa inhibited growth of all five bacterial strains, suggesting the presence of metabolites with broad-spectrum activity. Three active compounds were isolated from H. viscosa having antibacterial properties similar to those of the crude extract. Our data indicate that antibacterial secondary metabolites are present in sub-Arctic marine invertebrates but are less abundant than in temperate, tropical or Antarctic species.     

Metabolism of Dichloromethane by the Strict Anaerobe Dehalobacterium formicoaceticum

The metabolism of dichloromethane by Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [¹³C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO₂ from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 μM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane. D. formicoaceticum exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited by N,N′-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F₀F₁-type ATP synthase.