Reductive dechlorination of perchloroethylene and the role of methanogens

Abstract Perchloroethylene (PCE) was reductively dechlorinated to trichloroethylene in a 10% anaerobic sewage sludge. About 80% of the initially added PCE (300 nmol) was dechlorinated within three weeks. The calculated rates were 250 nM and 445 nM · day⁻¹ during the first and second weeks of incubation, respectively. The depletion of PCE varied in sludges obtained from different sources.
The role of methanogenesis in the dechlorination of PCE was evaluated by inhibiting the methanogens by addition of bromoethane sulfonic acid, a potent methanogenic inhibitor. Dechlorination of PCE was significantly inhibited in sludges amended with the inhibitor. Almost 41–48% less PCE was dechlorinated in sludges containing 5 mM BESA, indicating a relation between the two processes (methanogenesis and dechlorination). Direct proof that methanogens can transform chlorinated aliphatic compounds was obtained using axenic cultures of acetate-cleaving methanogens. Methanosarcina sp , originally isolated from a chlorophenol degrading consortium, showed significantly higher dechlorinating activity as compared to Ms. mazei . Based on these studies and other recently reported observations, it appears that methanogens/methanogenesis play an important role in the anaerobic dechlorination of chlorinated aliphatics such as PCE.     

Crystal structures and enzymatic properties of three formyltransferases from archaea: environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N(5)-formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate-reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98 degrees C) has recently been solved at 1.65 A resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37 degrees C) and from Archaeoglobus fulgidus (growth temperature optimum 83 degrees C) at 1.9 A and 2.0 A resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was -32 for the M. kandleri enzyme, only -8 for the M. barkeri enzyme, and -11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3-diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3-diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting-out salts.     

Thermophilic methanogenic Archaea in compost material: occurrence, persistence and possible mechanisms for their distribution to other environments

Since compost is widely used as soil amendment and the fact that during the processing of compost material high amounts of microorganisms are released into the air, we investigated whether compost may act as a carrier for thermophilic methanogens to temperate soils.

All eight investigated compost materials showed a clear methane production potential between 0.01 and 0.98 μmol CH₄ g dw⁻¹ h⁻¹ at 50 °C. Single strand conformation polymorphism (SSCP) and cloning analysis indicated the presence of Methanosarcina thermophila, Methanoculleus thermophilus, and Methanobacterium formicicum.

Bioaerosols collected during the turning of a compost pile showed both a highly similar SSCP profile compared to the corresponding compost material and clear methane production during anoxic incubation in selective medium at 50 °C. Both observations indicated a considerable release of thermophilic methanogens into the air.

To analyse the persistence of compost-borne thermophilic methanogens in temperate oxic soils, we therefore studied their potential activity in compost and compost/soil mixtures, which was brought to a meadow soil, as well as in an agricultural soil fertilised with compost. After 24 h anoxic incubation at 50 °C, all samples containing compost showed a clear methanogenic activity, even 1 year after application.

In combination with the in vitro observed resilience of the compost-borne methanogens against desiccation and UV radiation we assume that compost material acts as an effective carrier for the distribution of thermophilic methanogens by fertilisation and wind.     

Temporal change of composition and potential activity of the thermophilic archaeal community during the composting of organic material

To date, composting has been regarded as an aerobic process but it has been shown that composting piles are often sources of atmospheric methane. In order to gain a more comprehensive view on the diversity of methanogenic Archaea in compost, gas chromatographical methods and molecular cloning were used to study relationships of thermophilic archaeal communities and changes in methane production potential during compost maturation. According to the thermophilic methane production potential, wide differences could be detected between differently aged compost materials. In material derived from 3- and 4-week-old piles, low and no thermophilic methane production potential, respectively, was observed at 50 degrees C. Material from a 6-week-old pile showed the maximum methane production. With compost maturation, the production slowly decreased again with 6 weeks, 8 weeks, and mature compost showing an optimum methane production potential at 60 degrees C. At 70 degrees C, only 6-week-old material showed a comparable high production of methane. The 16S rRNA-based phylogenetic surveys revealed an increase of archaeal diversity with compost maturation. In the 6-week-old material, 86% of the sequences in the archaeal 16S rRNA library had the highest sequence similarities to Methanothermobacter spp. and the remaining 14% of the clones were related to Methanosarcina thermophila. Quantification of methanogens in 6-week-old material, on the basis of the methane production rate, resulted in values of about 2x10(7) cells per gram fresh weight. In 8-week-old and mature compost material, the proportion of sequences similar to Methanothermobacter spp. decreased to 34% and 0%, respectively. The mature compost material showed the highest variation in identified sequences, although 33% could be assigned to as yet uncultured Archaea (e.g. Rice cluster I, III, and IV). Our results indicate that compost harbours a diverse community of thermophilic methanogens, with changing composition during the maturation process, presumably due to altered pile conditions. Likewise, compost may act as a potential carrier for thermophilic methanogens in temperate soils because it is widely used as a soil amendment.     

Identification of replication origins in archaeal genomes based on the Z-curve method

The Z-curve is a three-dimensional curve that constitutes a unique representation of a DNA sequence, i.e., both the Z-curve and the given DNA sequence can be uniquely reconstructed from the other. We employed Z-curve analysis to identify one replication origin in the Methanocaldococcus jannaschii genome, two replication origins in the Halobacterium species NRC-1 genome and one replication origin in the Methanosarcina mazei genome. One of the predicted replication origins of Halobacterium species NRC-1 is the same as a replication origin later identified by in vivo experiments. The Z-curve analysis of the Sulfolobus solfataricus P2 genome suggested the existence of three replication origins, which is also consistent with later experimental results. This review aims to summarize applications of the Z-curve in identifying replication origins of archaeal genomes, and to provide clues about the locations of as yet unidentified replication origins of the Aeropyrum pernix K1, Methanococcus maripaludis S2, Picrophilus torridus DSM 9790 and Pyrobaculum aerophilum str. IM2 genomes.     

Tetramethylammonium:coenzyme M methyltransferase system from Methanococcoides sp.

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens. Received: 7 April 1998 / Accepted: 26 June 1998     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Effect of a corrinoid on <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> DNA synthesis

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B₁₂ group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. barkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeal cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.