Methanobacterium formicicum,an endosymbiont of the anaerobic ciliateMetopus striatus McMurrich

The Gram-positive methanogenic endosymbiont of the sapropelic ciliateMetopus striatus was isolated and identified asMethanobacterium formicicum. In the ciliate cell the methanogens are in close association with microbody-like organelles. No mitochondria could be detected. The nature of the microbodies and the physiological background of the observed association are discussed.     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus quennerstedt

Epifluorescence microscopy revealed the presence of a methanogenic bacterium as an endosymbiont in the sapropelic marine ciliate Metopus contortus. The in situ methanogenic activity of the symbiont could be demonstrated. The isolated endosymbiont was an irregular, disc-shaped bacterium with a diameter of 1.6–3.4 m. It had a generation time of 7 or 12 hours on growth on H₂/CO₂ or formate, respectively. The temperature range for growth was between 16 and 36°C with an optimum at 32°C. The optimal pH range for growth was 6.8 to 7.3. Salts, with an optimum concentration of 0.25 M, and tungsten were required for growth. The mol% G+C was 38.7%. The cell envelope consisted of proteins and a glycoprotein with an apparent molecular weight of 110,000. Morphology, antigenic relationship and the G+C content established the isolate MC1 as a new species of the genus Methanoplanus, and the name Methanoplanus endosymbiosus is proposed.Abbreviations G+C Guanine+cytosine - SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2-ethane) sulfonic acid     

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.     

Monoxenic culture of the anaerobic ciliate Trimyema compressum Lackey

Sapropelic ciliates from anoxic mud samples were enriched and cultivated in monoculture together with natural food bacteria growing on cellulose. The ciliates lacked cytochrome oxidase and contained bluish fluorescent endosymbionts. One of the anaerobic ciliates, Trimyema compressum, contained methanogenic bacteria as was shown by methane formation. During continued cultivation, T. compressum gradually lost its endosymbionts. With SEM microscopy no episymbiotic bacteria could be detected.From enrichment cultures of T. compressum, anaerobic bacteria were isolated in pure culture. One of the strains, a Bacteroides spec., proved capable of serving as food bacteria, thus allowing establishment of monoxenic T. compressum cultures. These cultures exhibited a requirement for sterols as growth factors. The doubling time of this ciliate was 13 h at 28°C. The highest yield obtained was 2100 cells/ml.Dedicated to Holger W. Jannasch on the occasion of his 60th birthday     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F₄₂₀-reducing formate dehydrogenase and coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F₄₂₀-dependent formate dehydrogenase activity or F₄₂₀-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST Phosphate-buffered saline containing 0.1% (v/v) Triton X-100     

Monoxenic cultivation of the anaerobic amoeboflagellate Psalteriomonas lanterna and isolation of the methanogenic endosymbiont

Abstract The endosymbiont of the anaerobic amoeboflagellate Psalteriomonas lanterna was isolated and identified as Methanobacterium formicicum , a H₂/CO₂- and formate-utilizing methanogen. A monoxenic culture of P. lanterna was established in a complex liquid medium with Pseudomonas fluorescenss as food bacterium.     

Fermentation products of the anaerobic ciliate Trimyema compressum in monoxenic cultures

A new monoxenic culture of the anaerobic ciliate Trimyema compressum was established on the purple nonsulfur bacterium Rubrivivax gelatinosus. Compared with the first monoxenic culture of T. compressum on a Bacteroides species no significant difference in growth rate was observed. Using both monoxenic cultures the fermentation products of the ciliate, acetate, lactate, formate and succinate were quantified. Ammonium was another product of the ciliates metabolism. The amount of the endproducts was dependent on the amount of bacteria consumed. The ratio of carbon consumed to ammonium formed was 8.7:1 indicating incomplete degradation of nitrogenous compounds of the bacterial biomass. Under microoxic conditions no effect of oxygen on the yield of the ciliate was observed but the amount of acetate and lactate was 25% lower. Cells of T. compressum with Methanobacterium formicicum as deliberately introduced endosymbiont did not form significantly different fermentation products, however, instead of formate methane was detected as product. The yield of the ciliate was not affected. It is concluded that methanogenic bacteria deliberately introduced into the cytoplasm of T. compressum as endosymbionts are pure commensalists.     

A comparison of two strains of the anaerobic ciliate Trimyema compressum

Two strains of the anaerobic ciliate Trimyema compressum, isolated from different habitats, were compared. The cytoplasm of the ciliates contained hydrogenosome-like microbodies and methanogenic bacteria; the latter were lost during continued cultivation. In addition both strains harbored a non-methanogenic endosymbiont, which was lost in strain K. The ciliates lacked cytochromes, cytochrome oxidase and catalase but contained superoxide dismutase. Hydrogenase activity could be demonstrated only in strain N. In monoxenic culture strain K needed sterols as growth factors. The cells of both strains reacted similarly with respect to oxygen tolerance (up to 0.5 mg O₂/l), inhibition of growth by cyanide and azide, and resistance to antimycin A. Only cells of strain N showed growth inhibition by chloramphenicol. It is concluded that Trimyema compressum is an anaerobic, microaerotolerant organism, its microbodies show more resemblance to hydrogenosomes than to mitochondria.     

Functional relationship between protein-bound and free factor F430 in Methanobacterium

Factor F430 is a nickel porphinoid present in all methanogenic bacteria. It is a component of methyl-CoM reductase to which it is tightly but not covalently bound. Evidence is presented that in Methanobacterium thermoautotrophicum grown on nickel sufficient medium only approximately 30% of total F430 is associated with methyl-CoM reductase and that 70% is present in a non-bound, free form. When such cells were transferred to a nickel deficient medium the bacteria continued to grow although synthesis of total F430 stopped. During growth in the absence of nickel the amount of total F430 per 1 culture remained constant and that per g cells decreased. The ratio of free F430 to bound F430, however, changed. Free F430 was converted into the protein-bound form until almost all of the free F430 had disappeared. The kinetics of labelling with ⁶³Ni of free and bound F430 agreed rather well with that calculated for a precursor-product relationship between free and bound F430.Dedicated to Professor H. G. Schlegel on the occasion of his 60th birthday     

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.     

Selectivity of food bacteria for the growth of anaerobic ciliate Trimyema compressum

The anaerobic ciliate Trimyema compressum was cultivated on various food bacteria. Significant growth was observed when Lactobacillus sp., Escherichia coli, Enterobacter aerogenes, Desulfovibrio vulgaris, Methanoculleus bourgense, or Pelobacter propionicus cells were fed to the ciliates. The highest cell yield which we obtained was ca. 9,000 cells/ml when feeding D. vulgaris. However, no growth of the ciliates was observed on the culture with Clostridium novyi, Propionibacterium sp., Desulfobulbus propionicus, Methanobrevibacter arboriphilicus, Methanobacterium sp., Methanosarcina barkeri, or Methanothrix soehngenii cells. The ciliates produced acetate and methane as major end products in any cultures and small amounts of propionate, butyrate and hydrogen were also detected in some cultures. Physiological studies on the food bacteria which we tested indicated that the growth of T. compressum depended on the bacterial species, but there was no apparent correlation between the digestibility and the basic properties of those bacteria (i.e. size of the bacteria, gram-staining properties, susceptibility to the known lytic enzymes, Archaea or Bacteria).     

Acetyl CoA,a central intermediate of autotrophic CO2 fixation in Methanobacterium thermoautotrophicum

The pathway of autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum has been investigated by long term labelling of the organism with isotopic acetate and pyruvate while exponentially growing on H₂ plus CO₂. Maximally 2% of the cell carbon were derived from exogeneous tracer, 98% were synthesized from CO₂. Since growth was obviously autotrophic the labelled compounds functioned as tracers of the cellular acetyl CoA and pyruvate pool during cell carbon synthesis from CO₂. M. thermoautotrophicum growing in presence of U-¹⁴C acetate incorporated ¹⁴C into cell compounds derived from acetyl CoA (N-acetyl groups) as well as into compounds derived from pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), hexosephosphates (galactosamine), and pentosephosphates (ribose). The specific radioactities of N-acetylgroups and of the three amino acids were identical. The hexosamine exhibited a two times higher specific radioactivity, and the pentose a 1.6 times higher specific radioactivity than e.g. alanine. M. thermoautotrophicum growing in presence of 3-¹⁴C pyruvate, however, did not incorporate ¹⁴C into cell compounds directly derived from acetyl CoA. Those compounds derived from pyruvate, dicarboxylic acids and hexosephosphates became labelled. The specific radioactivities of alanine, aspartate and glutamate were identical; the hexosamine had a specific radioactivity twice as high as e.g. alanine.The finding that pyruvate was not incorporated into compounds derived from acetyl CoA, whereas acetate was incorporated into derivatives of acetyl CoA and pyruvate in a 1:1 ratio demonstrates that pyruvate is synthesized by reductive carboxylation of acetyl CoA. The data further provide evidence that in this autotrophic CO₂ fixation pathway hexosephosphates and pentosephosphates are synthesized from CO₂ via acetyl CoA and pyruvate.     

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.     

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     

Degradation of food compounds and growth response on different food quality by the anaerobic ciliate Trimyema compressum

The biochemical composition of two food bacteria was examined on which monoxenic cultures of Trimyema compressum grew with different yields. The food bacteria were the saccharolytic fermenting bacterium Bacteroides WoCb15 and the purple nonsulfur bacterium Rubrivivax gelatinosus. Differences in composition of bacterial biomass concerned mainly the carbohydrate content. By different culture conditions for R. gelationsus and pasteurization of carbohydrate-rich cells, we were able to feed the ciliate with food mixtures of different carbohydrate content. Dry mass yields of the ciliate reached a maximum with mixtures of 80% carbohydrate-rich pasteurized cells plus 20% carbohydratepoor living cells. In the absence of degradable carbohydrate, energy metabolism depended on protein as substrate. Utilization of protein was incomplete, large amounts were converted into soluble compounds that accumulated in the culture medium. The ciliate consumed storage carbohydrate of living or pasteurized food bacteria equally well, while growth with short generation times was still dependent on a certain percentage of living bacteria as source of native protein. Lipids, nucleic acids and denatured proteins were not degradable by the ciliate. Consequences for the fermentative metabolism of Trimyema compressum are discussed.     

Nutrition and factors limiting the growth of a methanogenic bacterium (Methanobacterium thermoautotrophicum)

The purification of Methanobacterium thermoautotrophicum from a culture contaminated with a heterotrophic organism is described. A defined inorganic medium under H₂/CO₂ (80:20 v/v) has been developed to support growth of M. thermoautotrophicum up to a concentration of at least 1.7 g dry weight/l. In a conventional medium iron and nitrogen sources were found to be growth-limiting factors. Throughout most of the culture period the rate of transfer of hydrogen or carbon dioxide from gas to liquid was the factor which controlled the growth rate.The growth yields of bacteria were in the range 0.6–1.6 g dry weight/mole CH₄.Abbreviation TGP thioglycollate peptone medium     

Sodium dependence of growth and methane formation in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was found to require sodium for growth and for CO₂ reduction to methane. The dependence of the rate of growth and methane formation on the sodium concentration was hyperbolic with an apparent K _s for sodium of approximately 1 mM. The findings indicate that sodium has a specific function in the energy metabolism of this bacterium.     

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.