Nickel dependence of factor F430 content in Methanobacterium thermoautotrophicum

Factor F₄₃₀ is a yellow compound of unknown structure present in methanogenic bacteria. It has recently been shown to contain nickel. In this communication the influence of the nickel concentration in the growth medium on the factor F₄₃₀ content of Methanobacterium thermoautotrophicum and on the nickel content of factor F₄₃₀ was studied. It was found: (1) The content of factor F₄₃₀ in the cells was strongly dependent on the nickel concentration of the growth medium. Cells grown on media with 2.5 M NiCl₂ contained 28 times as much factor F₄₃₀ per g as those grown on media with 0.075 M NiCl₂; (2) factor F₄₃₀ was synthesized in nickel deprived cells only upon the addition of nickel Nickel uptake paralleled factor F₄₃₀ synthesis; (3) independent of the nickel concentration in the growth medium, the extinction coefficient at 430 nm of factor F₄₃₀ per mol nickel was always near 22,500 cm^-1 (mol Ni)^-1. These findings indicate that nickel is an essential component of factor F₄₃₀.Dedicated to Professor Otto Kandler on the occasion of his 60th birthday     

Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ

Superoxide dismutase (SOD) was purified from cells of the strict anaerobic methanogenic archaeon Methanobrevibacter arboriphilus strain AZ. The four-step purification procedure resulted in enzyme with specific activity of 3970 units/mg and yield of 22%. It was shown that the SOD is a Fe-containing homotetramer composed of subunits of 21.2 kD each. Sodium azide (13.5 mM), unlike KCN, inhibits the activity of the SOD. Hydrogen peroxide (0.5 mM) inactivates the enzyme, which is consistent with the properties of the known Fe-containing SODs from methanogenic Archaea.     

两种鸣禽发声控制核团体积的比较研究

用焦油紫染色,图像分析及统计学方法比较研究了两种鸣禽栗巫鸟和燕雀发声控制核团的体积差异．结果表明,在发声活动中起重要作用的核团前脑ＨＶｃ、ＲＡ 和Ｘ区的体积存在显著的种间差异：发声技巧较高的雄性栗巫鸟的相应核团均相对大于雄性燕雀;而在发声中作用较小的ｎⅫｔｓ及雌性鸣禽的相应所有核团均无显著种间差异． 这一结果表明,发声能力的高低与前脑发声核团的体积直接相关．     

The superficial antigenic mosaic of Methanobrevibacter smithii

A panel of six different monoclonal antibodies (6A to F) was generated using Methanobrevibacter smithii strain PS as immunogen. The antibodies were characterized and calibrated by standard techniques and with a novel application of the slide immunoenzymatic assay (SIA) for determination of the l-chain type of the monoclonal antibody molecule. Five (and possibly six) determinants were identified with the antibodies. Each antibody recognized one determinant exclusively, except for antibodies 6B and 6F which might recognize the same determinant, although some data suggest that antibody 6F recognizes a sixth determinant different from the other five. The determinant for antibody 6A involves Glu, Lys and Orn. It is most likely located in the region of the peptide moiety of pseudomurein which is typical of strain PS. The six antibodies reacted with whole bacterial cells unfixed or formalinized and/or heat-fixed, but did not react with the other M. smithii reference strain ALI, or with any other reference methanogen tested. However, the antibodies did react with a number of isolates from human feces considered M. smithii from morphologic, physiologic and immunologic information, and were instrumental for grouping the isolates.Abbreviations PBS phosphate buffered saline - SIA slide immunoenzymatic assay - IIF indirect immunofluorescence     

Mutagenicity of Intermediates Produced in the Early Stage of the Browning Reaction of Triose Reductone with Nucleic Acid Related Compounds on Bacterial Tests

Two types of reductive intermediates, linear and tricyclic forms, isolated from browning mixtures of triose reductone (TR) with guanine and its derivatives showed evident mutagenicity on Salmonella typhimurium TA 100 without S-9 mixture. The linear intermediates, N²-(3-oxo-2-hydroxypropenyl) compounds of guanine, guanosine, 2′(3′)-guanylic acid and 5′-guanylic acid were more effective than the tricyclic one, l, N²-(2-hydroxypropenylidene)guanine, though they were far less active than 4-nitroquinoline-N-oxide. No acceleration in mutagenicity was observed with Cu^{2 +} and other metal ions. The reaction mixtures of TR and nucleic acid bases were also mutagenic on TA 100. Intermediates of TR with guanine and its derivatives did not have a lethal effect in Recassays with Bacillus subtilis.     

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.     

Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters

AIMS: The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. METHODS AND RESULTS: PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage-contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222-bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml(-1). CONCLUSIONS: The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage-contaminated water but not in uncontaminated marine water or bovine-contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage-polluted coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.     

Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

Molecular evolution and functional divergence of UDP-hexose 4-epimerases

UDP-glucose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) and/or the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) in sugar metabolism. GalEs belong to the short-chain dehydrogenase/reductase superfamily, use a conserved ‘transient keto intermediate’ mechanism and have variable substrate specificity. GalEs have been classified into three groups based on substrate specificity: group 1 prefers UDP-Glc/Gal, group 3 prefers UDP-GlcNAc/GalNAc, and group 2 has comparable activities for both types of the substrates. The phylogenetic relationship and structural basis for the specificities of GalEs revealed possible molecular evolution of UDP-hexose 4-epimerases in various organisms. Based on the recent advances in studies on GalEs and related enzymes, an updated view of their evolutional diversification is presented.     

Defective formation and/or utilization of carbon monoxide in H2/CO2 fermenting methanogens dependent on acetate as carbon source

Autotrophic methanogens reduce CO₂ to CO and assimilate CO in a carbonylation reaction. Heterotrophic species were found not to form CO and/or to incorporate CO into cell matiral. The absence of CO formation correlated with the absence of carbon monoxide dehydrogenase activity. The heterotrophic Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanococcus voltae and Methanospirillum hungatei (strain GP 1) were investigated.