Comparative bioenergetics of sulfate reduction in Desulfovibrio and Desulfotomaculum spp.

Extracts of Desulfotomaculum nigrificans, Desulfotomaculum orientis, and Desulfotomaculum ruminis exhibit low levels of inorganic pyrophosphatase but were found to have high levels of pyrophosphate:acetate phosphotransferase. Conversely, extracts of Desulfovibrio gigas, Desulfovibrio vulgaris, and Desulfovibrio desulfuricans Norway 4 were shown to have high levels of inorganic pyrophosphatase but negligible amounts of pyrophosphate:acetate phosphotransferase. Both enzymes are reductant activated and appear to have an analogous function in removing pyrophosphate formed during the activation of sulfate. Conservation of the bond energy of pyrophosphate in Desulfotomaculum eliminates the necessity for invoking electron-transfer-coupled phosphorylation to account for the growth of these bacteria on lactate plus sulfate. Relative growth yields of Desulfovibrio vulgaris and Desulfotomaculum orientis on lactate plus sulfate indicate that the latter does not carry out significant electron-transfer-coupled phosphorylation in this mode of growth.     

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone reduces corrosion from Desulfotomaculum orientis

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming and biofilm formation of Gram-positive bacteria (Ren et al., 2002, Lett Appl Microbiol 34: 293-299). In the present study, the Gram-positive sulphate-reducing bacterium (SRB), Desulfotomaculum orientis, was used to study the inhibition of mild steel corrosion due to the addition of furanone. The weight loss from batch coupon experiments incubated with 40 microg x ml(-1) furanone was reduced fivefold compared with samples that lacked furanone. Analysis of the metal surface with environmental scanning electron microscopy further confirmed the protection afforded by the addition of furanone. In agreement with the corrosion inhibition, most probable number (MPN) analysis showed that 20 and 40 microg x ml(-1) furanone inhibited 58% and 96% of the D. orientis growth respectively. Hence, furanone has the potential to inhibit microbial-induced corrosion related to Gram-positive bacteria.     

The presence and activity of Desulfotomaculum spp in sulphate-limited freshwater sediments

Abstract Addition of pyrophosphate stimulated sulphate uptake and utilization of volatile fatty acids by anaerobic freshwater sediments. Population estimates of sulphate-reducing bacteria demonstrated that a larger number were capable of utilizing acetate then lactate. Estimates increased when pyrophosphate was added to the medium and decreased by the same degree when the sediment was heated. Addition of molybdate to the sulphate-limited sediments stimulated rather than inhibited methanogenesis. The results suggest that the sediment contains a metabolically active population of acetate-utilizing members of the genus Desulfotomaculum .     

5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum.

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14C-labeled uracil or FU by the two strains was compared, the wild type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype.     

Description of Desulfotomaculum nigrificans subsp. salinus as a new species, Desulfotomaculum salinum sp. nov

This study focused on the physiological, chemotaxonomic, and genotypic characteristics of two thermophilic spore-forming sulfate-reducing bacterial strains, 435T and 781, of which the former has previously been assigned to the subspecies Desulfotomaculum nigrificans subsp. salinus. Both strains reduced sulfate with the resulting production of H2S on media supplemented with H2 + CO2, formate, lactate, pyruvate, malate, fumarate, succinate, methanol, ethanol, propanol, butanol, butyrate, valerate, or palmitate. Lactate oxidation resulted in acetate accumulation; butyrate was oxidized completely, with acetate as an intermediate product. Growth on acetate was slow and weak. Sulfate, sulfite, thiosulfate, and elemental sulfur, but not nitrate, served as electron acceptors for growth with lactate. The bacteria performed dismutation of thiosulfate to sulfate and hydrogen sulfide. In the absence of sulfate, pyruvate but not lactate was fermented. Cytochromes of b and c types were present. The temperature and pH optima for both strains were 60-65 degrees C and pH 7.0. Bacteria grew at 0 to 4.5-6.0% NaCl in the medium, with the optimum being at 0.5-1.0%. Phylogenetic analysis based on a comparison of incomplete 16S rRNA sequences revealed that both strains belonged to the C cluster of the genus Desulfotomaculum, exhibiting 95.5-98.3% homology with the previously described species. The level of DNA-DNA hybridization of strains 435T and 781 with each other was 97%, while that with closely related species D. kuznetsovii 17T was 51-52%. Based on the phenotypic and genotypic properties of strains 435T and 781, it is suggested that they be assigned to a new species: Desulfotomaculum salinum sp. nov., comb. nov. (type strain 435T = VKM B 1492T).     

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.     

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov.

An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H₂/CO₂. Lactate and pyruvate were oxidized completely to CO₂ with sulfate as electron acceptor. Sulfate was required for growth on H₂/CO₂, and both acetate and sulfide were produced from H₂/CO₂-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H₂/CO₂, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min^–1 [mg protein]^–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization

The 8-hydroxy-5-deazaflavin (coenzyme F420) reducing hydrogenase from the obligate anaerobe Methanobacterium thermoautotrophicum delta H has been purified 41-fold to apparent homogeneity. The major active enzyme form is a high molecular weight aggregate of Mr ca. 800,000, composed of three subunits, alpha (Mr 47K), beta (Mr 31K), and gamma (Mr 26K). The hydrogenase is purified aerobically in reversibly inhibited form, and conditions for anaerobic reductive activation with H2, high salt, thiols, and electron acceptors have been defined. The minimal species transferring electrons from H2 to coenzyme F420 appears to be an alpha beta delta (Mr 115K) complex. The tightly associated redox cofactors per 115K species are 0.6-0.7 nickel atom, 0.8-0.9 flavin adenine dinucleotide (FAD), and 13-14 iron atoms in iron-sulfur centers. The subunits have been separated by denaturing gel electrophoresis, which has permitted determination of amino acid composition, subunit N-terminal sequencing, and preparation of subunit-directed antibodies. There is iron associated with the alpha-subunit, but placement of the nickel and FAD has not been established.     

Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum.

Cultures of Methanobacterium thermoautotrophicum were supplemented with 13C-labeled acetate or pyruvate, and the labeling pattern of the corrinoid, factor III, was established by 13C NMR spectroscopy. Complete 13C signal assignments were obtained by two-dimensional NMR experiments. The labeling pattern of factor III was analyzed by comparison with those of amino acids and nucleosides. The corrin ring system is derived from eight molecules of glutamate. The aminopropanol moiety is derived in a hitherto unknown pathway from pyruvate by reductive amination. The heterocyclic ring of hydroxybenzimidazole shares the labeling pattern of the imidazole ring of purines. The remaining four carbon atoms of the carbocyclic ring show the labeling signature of a carbohydrate with two of the carbons introduced from acetate and two from C-1 of pyruvate. However, erythrose can be ruled out as the specific precursor on the basis of a detailed investigation of aromatic amino acids indicating that erythrose 4-phosphate is obtained by reductive carboxylation of a triose precursor and not by the pentose phosphate cycle.     

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum.

The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the epsilon-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.     

delta 4-3-Oxosteroid 5 beta-reductase. Structure and function.

delta 4-3-Oxosteroid 5 beta-reductase catalysing reduction of delta 4-3-oxosteroids to give A/B cis-conformation was intraperitoneally injected into BALB/c strain mice with Ribi adjuvant. Monoclonal antibody specific for this enzyme was prepared from their spleen cells. Using this monoclonal antibody as a probe the enzyme was further purified using reversed phase liquid chromatography to determine amino-acid sequence protein-chemically. Attempts to determine the N-terminal amino acid failed, indicating that the N-terminal amino acid is blocked. The protein was therefore subjected to digestion with lysyl endopeptidase after alkylating with iodoacetate. The peptides thus formed were isolated and purified by reversed-phase high-performance liquid chromatography and their amino-acid sequences were determined. Using antibodies and oligonucleotides as probes a cDNA which contained a 978 bp long open reading frame encoding 326 amino-acid residues (Mr 37376) was isolated from rat liver cDNA libraries and the entire sequence of the protein was deciphered from its nucleotide sequence. The COS cells transfected with this cDNA revealed a versatile activity to reduce varied kinds of delta 4-3-oxosteroids, i.e. 7 alpha-hydroxy-4-cholesten-3-one, androstenedione and cortisone as postulated by Okuda and Okuda (1984, J. Biol. Chem. 259, 7519-7524) and Furuebisu et al. (1987, Biochim. Biophys. Acta 912, 110-114. With a newly established immunoblotting assay method several tissues and organs were surveyed and it was found that the enzyme exists only in the liver and there is an apparent difference between sexes as to the content of this enzyme. However, there was little if any difference in the amount of mRNAs between both sexes, which may indicates that the sexual difference of rat liver cytosol 5 beta-reductase is due to a posttranslational modification and/or degradation.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.     

Assay of adenosine 5'-P1-tetraphospho-P4-5'-adenosine and adenosine 5'-P1-tetraphospho-P4-5'-guanosine in Physarum polycephalum and other eukaryotes. An isocratic high-pressure liquid-chromatography method.

A5'pppp5'A has been proposed to serve as a molecular signal that triggers DNA replication. When published methods proved to be inadequate for the assay of A5'pppp5'A in Physarum polycephalum by h.p.l.c. (high-pressure liquid chromatography), a set of purification procedures was developed that allowed assay of as little as 2pmol of A5'pppp5'A. A5'pppp5'A was purified from cellular extract by covalent boronate chromatography, treated with alkaline phosphatase to hydrolyse residual mononucleotides and analysed by isocratic ion-exchange h.p.l.c. The analysis was facilitated by a pre-column switching procedure that allowed early-eluted species to be diverted from the analytical column. By using this procedure A5'pppp5'A has been detected in Physarum polycephalum (1.4 pmol/mg of protein), Saccharomyces cerevisiae (3.6 pmol/mg of protein) and rat liver (3.3 pmol/mg of protein). In each case a minor peak was also seen, which was identified as A5'pppp5'G. The identity of both peaks was confirmed by co-elution with standards on isocratic and gradient h.p.l.c. and treatment with enzymes, including a dinucleoside polyphosphate pyrophosphohydrolase from Physarum polycephalum.     

Occurrence and biosynthesis of 5-aminoimidazole-4-carboxamide ribonucleotide and N-(beta-D-ribofuranosyl)formamide 5'-phosphate in Methanobacterium thermoautotrophicum delta(H).

5-Aminoimidazole-4-carboxamide ribonucleotide (ZMP) and N-(beta-D-ribofuranosyl)formamide 5'-phosphate (FAR-P) have been identified as products of the metabolism of ATP and 5-phospho-alpha-D-ribosyl diphosphate by Methanobacterium thermoautotrophicum delta(H), a member of the domain Archaea. Evidence indicates that the first three steps in the pathway to the formation of these compounds are the same as the first three steps of histidine biosynthesis and lead to the generation of pro-phosphoribosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'-proFAR). The 5'-proFAR then undergoes hydrolysis to ZMP and FAR-P. The reaction was detected by an unexpected high concentration of ZMP in cell extracts of M. thermoautotrophicum delta(H).     

Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.

A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO₄2· H₂O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the ɛ-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed. Received: 22 October 1997 / Accepted: 16 June 1997     

4-hydroxy-N-methylproline analogues in Melaleuca spp.

4-hydroxy-N-methylproline and 4-hydroxy-N,N′-dimethylproline have been identified in the aqueous extracts of Melaleuca spp. Characterization of the two compounds is discussed.