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.     

Evidence for the occurrence of autolytic enzymes in Methanobacterium wolfei

Cultures of the pseudomurein-containing archaebacterium Methanobacterium wolfei regularly lysed a short while after the energy source H₂ was exhausted, or when H₂ in growing cultures was replaced by N₂. During lysis of cells, the DNA was released into the culture medium.No intact cell wall sacculi of lysed cells could be detected, but a soluble fragment of the pseudomurein was isolated and characterized.The lysate of Methanobacterium wolfei was used to lyse other species of the genus Methanobacterium. Since no phages were detected, autolytic enzymes probably are responsible for cell lysis.     

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 .     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.     

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     

The role of hydrogen mass transfer for the growth kinetics of Methanobacterium thermoautotrophicum in batch and chemostat cultures

Hydrogen concentration was determined in batch and chemostat cultures of Methanobacterium thermoautotrophicum, both in the headspace and in the medium using mass spectrometry. The calculated dissolved hydrogen concentration in the medium as derived from the headspace hydrogen concentration when equilibrium conditions between gas and liquid phase were assumed, was ten times higher than the experimentally determined hydrogen concentration. Variation of the partial pressure of hydrogen resulted in different values for substrate affinity for hydrogen (K_s) and yield (Y) of the cells. Upon hydrogen limitation, K_s decreased while the yield coefficient for hydrogen increased, indicating a change in the affinity of the cells towards hydrogen. Received 15 November 1996/ Accepted in revised form 21 July 1997     

一株河流污泥中产甲烷杆菌的生物学特性研究

目的:筛选产甲烷菌并对其进行鉴定和特性分析.方法:利用亨盖特厌氧操作技术从河流污泥中分离出的一株产甲烷杆菌.通过形态特征、生理生化、16S rRNA基因序列分析确定菌株的分类地位,利用气相色谱仪测定甲烷含量.结果:该菌株杆状,产芽孢,极端严格厌氧,宽约0.36μm,长约3.20μm,有时观察成链状,在液体培养的过程中,聚集成絮状沉积在管底.能够利用H2/CO2和甲钠盐生长,不利用甲醇、三甲胺、乙酸和二级醇类.最适生长温度范围为37℃,最适pH为7.5 ～8.0,能在0～1％盐浓度范围内生长.结论:菌株AG的最适温度较高,最适pH偏碱,在沼气发酵中具有重要应用前景.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Utilization of mercapto-2-ethanol as medium reductant for determination of the metabolic response of methanogens towards inorganic sulfur compounds

Abstract Mercapto-2-ethanol was examined as a nontoxic and non-metabolizable reducing agent for growth of methanogens. It was used as a medium reductant to prove that Methanobacterium thermoautotrophicum and Methanobacterium strain ivanov grew with either sulfide or elemental sulfur as the sole source of nutrient sulfur but not with sulfate, thiosulfate, sulfite or dithionite. The later inorganic sulfur sources, except sulfate, were potent inhibitors of growth and methanogenesis at 5 mM. The practical utility of mercapto-2-ethanol as a reducing agent and the toxicity of inorganic sulfur sources on metabolic activity of the methanogens are discussed.     

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei.

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes. The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.     

Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum

The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoautotrophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calculated molar ratios of the amino acids and the percentages of the N-or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.     

The role of prophage for genome diversification within a clonal lineage of Lactobacillus johnsonii: characterization of the defective prophage LJ771

Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE (“antitoxin”)/pemK (“toxin”) gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5′ end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.     

Monitoring growth of the methanogenic archaea Methanobacterium formicicum using an electronic nose

Growth of the methanogenic archaea, Methanobacterium formicicum, in pure culture was monitored by analysing samples from the gas phase with an array of chemical gas sensors (an `electronic nose'). Analyses of the methane and protein formation rates were used as independent parameters of growth, and the data obtained from the electronic nose were evaluated using principal component analysis (PCA). We found that different growth phases can be distinguished with the electronic nose followed by PCA evaluation. The fast response of the sensors in combination with the high correlations with other parameters measuring growth show that the electronic nose can be a useful tool to rapidly determine methanogenic growth.     

Thermodynamic and kinetic analysis of the H2 threshold for Methanobacterium bryantii M.o.H

H₂ thresholds, concentrations below which H₂ consumption by a microbial group stops, have been associated with microbial respiratory processes such as dechlorination, denitrification, sulfate reduction, and methanogenesis. Researchers have proposed that observed H₂ thresholds occur when the available Gibbs free energy is minimal (ΔG ≈ 0) for a specific respiratory reaction. Others suggest that microbial kinetics also may play a role in controlling the thresholds. Here, we comprehensively evaluate H₂ thresholds in light of microbial thermodynamic and kinetic principles. We show that a thermodynamic H₂ threshold for Methanobacterium bryantii M.o.H. is not controlled by ΔG for methane production from H₂ + HCO₃⁻. We repeatedly attain a H₂ threshold near 0.4 nM, with a range of 0.2–1 nM, and ΔG for methanogenesis from H₂ + HCO₃⁻ is positive, +5 to +7 kJ/mol-H_2, at the threshold in most cases. We postulate that the H₂ threshold is controlled by a separate reaction other than methane production. The electrons from H₂ oxidation are transferred to an electron sink that is a solid-phase component of the cells. We also show that a kinetic threshold (S _min) occurs at a theoretically computed H₂ concentration of about 2400 nM at which biomass growth shifts from positive to negative.