Nitrogen fixation by Methanobacterium formicicum

Abstract: Methanobacterium formicicum utilized molecular nitrogen as the sole nitrogen source for growth as monitored by methane production and culture turbidity measurements. The rate of methane production by the bacteria was correlated to nitrogen gas concentrations. In the absence of nitrogen gas or any other nitrogen source, the bacteria completely stopped growing. The presence of selenium and molybdenum in the culture medium was vital for the growth of the bacteria under nitrogen fixing conditions.     

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.     

7-methylpterin in methanogenic bacteria

Abstract A blue fluorescent compound was extracted and purified from cells of Methanobacterium thermoautotrophicum . The compound was identified as 7-methylpterin on the basis of its (physico-) chemical properties and by comparison with 7-methylpterin prepared by organic synthesis. The compound is present in all methanogenic bacteria studied so far and it provides methanogenic bacteria the characteristic blue fluorescence observed upon fluorescence microscopy.     

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.     

九株嗜热产甲烷菌的特性

从处理生活废水的厌氧污泥床的4个样品中,分离、纯化了9株嗜热的、利用H_2/CO_2和甲酸盐产甲烷的细菌。它们在细胞形态和生理特性上基本一致。细胞为直或略弯的杆状,两端钝圆,0.3～0.4×1～3μm;单个、成对,或多个相联,可达10μm以上。革兰氏阳性,不运动。细胞和菌落在荧光显微镜下呈现产甲烷菌所特有的绿色荧光。化能自养。生长温度30～75℃以下,最适为55～65℃。生长pH5.8～9.0,最适为6.9～7.6。菌株602B_3DNA中G+C含量为44.6mol％。将该菌株鉴定为嗜热甲酸甲烷杆菌的不同菌株:Methanobacterium thermoformicicum 602B_3     

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.     

耐低温甲烷杆菌SHB的分离鉴定与生长特性分析

利用亨盖特厌氧操作技术,从承德塞罕坝湿地污泥中分离出1株耐低温的产甲烷菌SHB。该菌株宽约0.31μm,长约2.87μm,细杆,形成长丝。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16～40℃,最适pH为6.0～9.0,能在0%～5%盐浓度范围内生长。该菌株对青霉素、链霉素、罗红霉素、利福平、新霉素有抗性,对氯霉素、四环素、卡那霉素、庆大霉素敏感。经生理、生化及16S rDNA分析,确定该菌株与甲烷杆菌属(Methanobacterium sp.)的亲缘关系最近。该菌株的生长温度和pH范围都很宽,在沼气生产方面有潜在的应用前景。     

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.     

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.     

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 .     

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.     

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.     

Methanobacterium thermoalcaliphilum spec. nov., a new moderately alkaliphilic and thermophilic autotrophic methanogen

An autotrophic moderately alkaliphilic and thermophilic nonmotile rod-shaped methanogen was isolated from a biogas plant. The isolate grows only on H₂+CO₂ and requires yeast extract. Growth optimum is at 60°C with a generation time of 4 h. In the absence of substrates complete lysis occurs. The pH range for growth is 7.5–8.5. Growth was also observed at pH values above 9.0. The DNA base composition is 38.8 mol% G+C. According to its physiological properties the nameMethanobacterium thermoalcaliphilum is proposed.Abbreviations G+C Guanine+cytosine     

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 .     

嗜热甲酸甲烷杆菌602B_3与拟杆菌5G—102的混合培养

研究了嗜热甲酸甲烷杆菌(Methanobacterium thermoformicicum)602B_3与发酵糖蜜产生C0_2和H_2的拟杆菌(Bacteroides sp.)5G-102的人工可配伍性。确定了它们混合培养的适宜培养基组分为(g/L):CaC0_3 50.0,NaHCO_3 4.0,Na_2S 0.16,NH_4Cl 1.07,K_2HPO_4 1.04,MgCl_2 0.19,糖蜜40(V/V),自来水IL,起始pH 6.8～7.2。混合培养物在60℃培养48h,甲烷产量达到最高,约45mmol/L。人工混合培养成功为进一步研究其在提高石油采收率工艺中的可用性提供了前提。     

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.     

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     

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     

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.