Isolation and characterization of the carbohydrate moiety bound to N-7-mercaptoheptanoyl-O-threonine phosphate

Abstract Component B ( N -7-mercaptoheptanoyl-threonine- O -3-phosphate) (HS-HTP) which is an absolute requirement in the methylcoenzyme M methylreductase reaction was found to be part of a complex UDP-disaccharide when isolated carefully from cell-free supernant of Methanobacterium thermoautotrophicum . The site of attachment of HS-HTP to the UDP-disaccharide was through a carboxylic-phosphoric anhydride linkage of the C-6 mannosaminuronic acid to the phosphate group in HS-HTP. This bond is quite labile and this may account for the fact that the intact molecule, called methyl reducing factor (MRF) was not isolated previously. The structure of MRF was determined by combined fast atom bombardment mass spectrometry and ¹H-, ¹³C-, and ³¹P-NMR spectroscopy and assigned as: uridine 5'-[ N -7-mercaptoheptanoyl- O -3-phosphothreonine(2-acetamido-2-deoxy- β -mannopyranuronosyl)acid anhydride]-(1 → 4)- O -2-acetamido-2-deoxy α -glucopyranosyl diphosphate.     

Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost

Abstract During the first stage of the preparation of mushroom compost oxygen is believed to be readily available. However we measured methane in the evoking air above the compost piles and were able to isolate thermophilic methanogenic bacteria from this compost. The isolates grow only on H₂ and CO₂ as energy and carbon source and do not require complex factors for growth. On the basis of nutritional and morphological characteristics these methanogens were identified as strains of Methanobacterium thermoautotrophicum .     

Tripolyphosphatase from Methanobacterium thermoautotrophicum (strain ΔH)

Abstract A low-melecular-mass polyphosphatase (tripolyphosphatase, PPPi) from the archaeon Methanobacterium thermoautotrophicum (strain ΔH) was purified 340-fold and characterized. The tripolyphosphatase showed an optimal activity at pH 9.7 (at 60°C). Though the highest activities were measured with tripolyphosphate, tetrapolyphosphate (57%), phosphate glass type 5 (41%) and phosphate glass type 15 (20%) could also be used as substrates. However, tripolyphosphatase was unable to use pyrophosphate. The enzyme was dependent on the presence of Mg²⁺. In the presence of 2 mM PPPi, an optimal activity was found at 6 mM Mg²⁺. The K _m for PPPi was estimated at 0.37 mM. In addition, the enzyme was inhibited by KF (50% at 6 mM) and appeared to be very heat stable: after an incubation of 2 h at 85°C about 85% of the activity was still present.     

Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10–15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala: Glu: GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M 1) alanine is replaced by threonine, however. Neutral sugars and-in some strains-additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or d-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three l-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min.The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.Non Standard Abbreviations SDS sodium dodecylsulfate - EDTA ethylenediaminetetra acetic acid - DNP dinitrophenyl Dedicated to Prof. Dr. Adolf Butenandt on the occasion of his 75th birthday     

Sensitivity of methanogenic bacteria from paddy soil to oxygen and desiccation

Abstract The effects of combinations of desiccation and exposure to O₂ were studied in pure cultures of Methanosarcina barkeri strain Fusaro and in a new Methanosarcina strain and a new Methanobacterium strain which were both isolated from dry oxic paddy soil. Incubation of bacterial suspensions under air for 200 min resulted in a decreased potential to produce CH₄, but not in a decreased viability. The inhibitory effect of O₂ slightly increased with increased salt concentration. Desiccation of bacterial suspensions under N₂ resulted in reduction of viability to 10% and of potential CH₄ production to 0.6%. Desiccation of bacterial suspensions under air resulted in a larger decrease of both viability (0.5%) and potential CH₄ production (0.03%). This decrease was smaller at rapid compared to slow desiccation. Survival and potential CH₄ production were further inhibited when the suspension was dried in the presence of sand grains or glass beads coated with FeS or FeNH₄PO₄. However, survival and potential CH₄ production increased dramatically in the presence of pyrite (FeS₂) grains. Then, as much as 10% of the initial methanogenic population survived oxic desiccation. This relatively good resistance is in agreement with observations that methanogens in rice fields survive the periods when the paddy soil is dry and oxic.     

Calcium ion uptake by Methanobacterium thermoautotrophicum: Evidence for a carrier-mediated uptake system

Abstract Cell suspensions of Methanobacterium thermoautotrophicum took up ⁴⁵Ca²⁺ in a temperature-dependent, Ca²⁺-saturable and Co²⁺-sensitive process. The accumulation of ⁴⁵Ca²⁺ was lower in the cells energized by CO₂+ H₂ than in those under non-energized conditions. The accumulated Ca²⁺ were, in part, released by the divalent cations ionophore A23187 in the presence of EGTA while the uptake of Ca²⁺ was accelerated by the addition of A23187 to the medium containing Ca²⁺. The results indicate the presence of a carrier-mediated Ca²⁺ uptake in the Methanobacterium thermoautotrophicum membrane which is compensated by an energy-dependent and outward-directed Ca²⁺ transport.     

Stimulation of the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in cell-free extracts of Methanobacterium thermoautotrophicum by the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate

The conversion of formaldehyde to methylcoenzyme M in cell-free extracts of Methanobacterium thermoautotrophicum was stimulated up to 10-fold by catalytic amounts of the heterodisulfide (CoM-S-S-HTP) of coenzyme M and 7-mercaptoheptanoylthreonine phosphate. The stimulation required the additional presence of ATP, also in catalytic concentrations. ATP and CoM-S-S-HTP were mutually stimulatory on the methylcoenzyme M formation and it was concluded that the compounds were both involved in the reductive activation of the methyltetrahydromethanopterin: coenzyme M methyltransferase. Micromolar concentrations of benzyl viologen or cyanocobalamin inhibited the formaldehyde conversion; these compounds, however, strongly stimulated the reduction of CoM-S-S-HTP. The results described here closely resemble observations made on the activation and reduction of CO₂ to formylmethanofuran indicating that this step and the reductive activation of the methyltransferase are controlled by some common mechanism.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - MFR methanofuran - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP the heterodisulfide of HS-CoM and HS-HTP - BES 2-bromoethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - CN-Cbl cyanocobalamin - HO-Cbl hydroxycobalamin - HBI 5-hydroxybenzimidazole - DMBI 5,6-dimethylbenzimidazole     

Oxidation of sulfide to thiosulfate by Microcoleus chtonoplastes

Sulfide oxidation and product formation was studied in the cyanobacterium Microcoleus chtonoplastes. Anoxygenic photosynthesis was induced in the presence of sulfide and light. It was demonstrated that thiosulfate was the only product of the 3(3,4-dichlorophenyl)1,1-dimethylurea (DCMU)-insensitive photo-oxidation of sulfide. The affinity of this system for sulfide was shown to be very low.Oxygenic photosynthesis continued in the presence of sulfide. After an induction period of 3 h, oxygenic and anoxygenic photosynthesis were shown to be operating simultaneously.The ecological importance of sulfide oxidation and thiosulfate production by M. chtonoplastes is discussed in the context of laminated microbial ecosystems, where cyanobacteria and purple sulfur bacteria coexist.     

Evidence for a defective prophage on the chromosome of Methanobacterium wolfei

Abstract Evidence shows the presence on the chromosome of Methanobacterium wolfei of a defective prophage which, by DNA-DNA hybridization, is closely related to the virulent archaeophage ψM1 of Methanobacterium thermoautotrophicum Marburg. Partial sequencing of a M. wolfei 16S rRNA gene and phylogenetic analysis indicated that this organism is more closely related to other representatives of the genus Methanobacterium than to M. thermoautotrophicum Marburg. The chromosomal region of M. wolfei encoding the putative prophage was found to be deleted for two non-contiguous segments of the phage ψM1 genome and thus encompassed only 80 to 90% of the ψM1 DNA. The prophage region was mapped to a 30 kb restriction fragment on the physical map of the M. wolfei chromosome. A randomly chosen DNA fragment was cloned from phage ψM1 DNA, as was its homologous counterpart from the chromosome of M. wolfei . The 126-bp region present in both clones exhibited 100% sequence identity.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

5,6,7,8-Tetrahydromethanopterin-dependent enzymes involved in methanogenesis

Abstract In the process of methanogenesis, 5,6,7,8-tetrahydromethanopterin (H₄MPT) is the carrier of the C₁ unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5-formyl- and 10-formyl-H₄MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5- and 10-formyl derivatives then yields 5,10-methenyl-H₄MPT, which is reduced in two subsequent coenzyme F₄₂₀-dependent reactions to 5-methyl-H₄MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H₄MPT-dependent reactions are discussed.     

Reduction potential characterization of methanogen factor 390

Abstract During oxidative stress, Methanobacterium thermoautotrophicum derivatizes the two-electron carrier, coenzyme F₄₂₀, to form factor 390 (F₃₉₀). Two methods were used to estimate the reduction potential of this chromophore. Oxidative titration of reduced F₃₉₀ by potassium ferricyanide in the presence of either NADH or a redox indicator dye yielded an estimate of −320 mV for the reduction potential. A sulfite dissociation constant of 11 mM was measured which correlates to a reduction potential of −310 mV when compared to other 5-deazaflavins and nicotinamides. Thus, the F₃₉₀ reduction potential is within a useful working range for the microorganism.     

Characterization of the Superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg

Abstract A gene ( sod ) encoding Superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identity was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod , separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium .     

Transformation of tetra- and trichloromethane to CO2 by anaerobic bacteria is a non-enzymic process

Abstract Degradation of tetrachloromethane was examined in the three strictly anaerobic bacteria, Acetobacterium woodii, Desulfobacterium autotrophicum and Methanobacterium thermoautotrophicum When incubated under anaerobic conditions in reduced buffer, suspensions of each organism degraded CCl₄ by both reductive and substitutive mechanisms. The products formed included less-highly chlorinated methanes and CO₂. Cell-free extracts of A. woodii degraded tetrachloromethane in a manner similar to that in whole cells but at a lower rate (63 vs. 110 μkat/kg of protein). When M. thermoautotrophicum or A. woodii was autoclaved, reductive dechlorination was partly abolished, whereas substitutive dechlorination was retained. Trichloromethane was oxidized to CO₂ by both native and autoclaved cells of A. woodii . Halomethanes are thus degraded anaerobically by reductive, substitutive and oxidative mechanisms.     

The coupling between catabolism and anabolism of Methanobacterium thermoautotrophicum in H2- and iron-limited continuous cultures

The aim of the present work was to investigate whether uncoupling of catabolism from anabolism, which was often observed in heterotrophic microorganisms under energy-sufficient growth conditions, also occurs in the autotrophic bacterium Methanobacterium thermoautotrophicum. For this purpose, M. thermoautotrophicum was cultivated in continuous cultures that were limited by the trace element iron. The influences of both dilution rate and iron supply rate on the coupling between anabolism and catabolism were investigated. As compared to continuous cultures of M. thermoautotrophicum limited by the energy substrate H₂, a 5-fold decrease in the biomass concentration and a 3-fold decrease in H₂, CO₂, and CH₄ conversion rates were observed in iron-limited cultures. However, the specific substrate and product conversion rates increased as compared to the values determined in energy-limited cultures. Thus, iron limitation provoked an uncoupling of catabolism from anabolism. At a dilution rate of 0.096 h⁻¹ and at an iron concentration of 17 μM in the feed, the specific H₂ consumption rate was 100% higher than the rate determined under H₂-limiting conditions, whereas at a dilution rate of 0.168 h⁻¹, the values differed only by 5%. Uncoupling of catabolism from anabolism also increased dramatically when the iron supply rate was lowered but the dilution rate was kept constant. Thus, the extent of uncoupling is a function of both the dilution rate and the iron supply rate. It was found that the specific consumption rate of H₂ increased in parallel with the partial pressure of H₂ in the culture medium. This suggested that the catabolic activity of M. thermoautotrophicum was not stringently controlled at the enzymatic level and can be considerably stimulated by the excess of H₂ in the medium. Hypotheses as to the fate of the excess energy derived from uncoupled catabolism are discussed, but the physiological reason for the partial uncoupling between catabolism and anabolism remains yet to be clarified.     

Thermodynamic analysis of growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum, an anaerobic archaebacterium using methanogenesis as the catabolic pathway, is characterized by large heat production rates, up to 13 W g⁻¹, and low biomass yields, in the order of 0.02 C‐mol mol⁻¹ H₂ consumed. These values, indicating a possibly “inefficient” growth mechanism, warrant a thermodynamic analysis to obtain a better understanding of the growth process. The growth‐associated heat production (Δ_rH) and the growth‐associated Gibbs energy dissipation per mol biomass formed (Δ_rG) were −3730 kJ C‐mol⁻¹ and −802 kJ C‐mol⁻¹, respectively. The Gibbs energy change found in this study is indeed unusually high as compared to aerobic methylotrophes, but not untypical for methanogens grown on CO₂. It explains the low biomass yield. Based on the information available on the energetic metabolism and on an ATP balance, the biomass yield can be predicted to be approximately in the range of the experimentally determined value. The fact that the exothermicity exceeds vastly even the Gibbs energy change can be explained by a dramatic entropy decrease of the catabolic reaction. Microbial growth characterized by entropy reduction and correspondingly by unusually large heat production may be called entropy‐retarded growth. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 74–81, 1999.