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1.
Environ Microbiol ; 15(5): 1275-89, 2013 May.
Article in English | MEDLINE | ID: mdl-22568606

ABSTRACT

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the 'Candidatus Scalindua' species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K.stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm.


Subject(s)
Aquatic Organisms/genetics , Aquatic Organisms/metabolism , Genome, Bacterial , Metagenome , Nitrogen Cycle , Planctomycetales/genetics , Planctomycetales/metabolism , Aquatic Organisms/classification , Nitrite Reductases/metabolism , Oceans and Seas , Oxidation-Reduction , Planctomycetales/classification , Quaternary Ammonium Compounds/metabolism , RNA, Ribosomal, 16S/genetics , Water Microbiology
2.
Nature ; 479(7371): 127-30, 2011 Oct 02.
Article in English | MEDLINE | ID: mdl-21964329

ABSTRACT

Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N(2)) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N(2)H(4)). Here we show that N(2)H(4) is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N(2)H(4). We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N(2)H(4) synthesis and its oxidation to N(2). These results present a new biochemical reaction forging an N-N bond and fill a lacuna in our understanding of the biochemical synthesis of the N(2) in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.


Subject(s)
Anaerobiosis , Quaternary Ammonium Compounds/metabolism , Ammonia/metabolism , Atmosphere/chemistry , Bacteria, Anaerobic/metabolism , Biocatalysis , Hydrazines/metabolism , Nitrate Reductase/metabolism , Nitric Oxide/biosynthesis , Nitric Oxide/metabolism , Nitrites/metabolism , Nitrogen Cycle , Nitrogen Fixation , Oxidation-Reduction , Quaternary Ammonium Compounds/chemistry
3.
Methods Enzymol ; 486: 89-108, 2011.
Article in English | MEDLINE | ID: mdl-21185432

ABSTRACT

Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite under anoxic conditions. The anammox process is currently used to remove ammonium from wastewater and contributes significantly to the loss of fixed nitrogen from the oceans. In this chapter, we focus on the ecophysiology of anammox bacteria and describe new methodologies to grow these microorganisms. Now, it is possible to enrich anammox bacteria up to 95% with a membrane bioreactor that removes forces of selection for fast settling aggregates and facilitates the growth of planktonic cells. The biomass from this system has a high anaerobic ammonium oxidation rate (50 fmol NH(4)(+) · cell(-1) day(-1)) and is suitable for many ecophysiological and molecular experiments. A high throughput Percoll density gradient centrifugation protocol may be applied on this biomass for further enrichment (>99.5%) of anammox bacteria. Furthermore, we provide an up-to-date list of commonly used primers and introduce protocols for quantification and detection of functional genes of anammox bacteria in their natural environment.


Subject(s)
Bacteria, Anaerobic/growth & development , Bacteriological Techniques , Quaternary Ammonium Compounds/metabolism , Anaerobiosis , Bacteria, Anaerobic/genetics , Biomass , Bioreactors , Centrifugation, Density Gradient , In Situ Hybridization, Fluorescence , Nitrites/metabolism , Oligonucleotide Probes , Oxidation-Reduction , Polymerase Chain Reaction , RNA, Ribosomal, 16S/analysis
4.
Environ Microbiol ; 10(11): 3120-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18462401

ABSTRACT

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.


Subject(s)
Bacteria, Anaerobic/isolation & purification , Bacteria, Anaerobic/metabolism , Nitrogen/metabolism , Seawater/microbiology , Acetic Acid/metabolism , Bacteria, Anaerobic/genetics , Bacteria, Anaerobic/ultrastructure , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Formates/metabolism , Genes, rRNA , Hydrazines/metabolism , Iron/metabolism , Lipids/analysis , Manganese/metabolism , Microscopy, Electron, Transmission , Molecular Sequence Data , Nitrates/metabolism , Oxidation-Reduction , Phylogeny , Propionates/metabolism , Quaternary Ammonium Compounds/metabolism , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Sweden
5.
Appl Environ Microbiol ; 73(3): 740-9, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17142379

ABSTRACT

In nature, H2- and CO2-utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth, Methanothermobacter thermautotrophicus was cultured in a fed-batch fermentor and in a chemostat under different 80% H(2)-20% CO2 gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (pH2). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (YCH4 max), notably approximately 3 and 7 g (dry weight) of methane formed mol-1, for growth under low (pH2 < 12 kPa)- and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the "specific maintenance" requirements or, more likely, the degree of proton leakage and proton slippage processes. At low pH2 values, the "specific maintenance" diminished and the specific growth yields approached YCH4 max, indicating that growth and methanogenesis became fully coupled.


Subject(s)
Autotrophic Processes , Hydrogen/metabolism , Methane/metabolism , Methanobacteriaceae/growth & development , Carbon Dioxide/metabolism , Carbon Dioxide/pharmacology , Culture Media , Fermentation , Hydrogen/pharmacology , Methanobacteriaceae/drug effects , Methanobacteriaceae/metabolism
6.
Int J Biol Macromol ; 39(1-3): 88-94, 2006 Aug 15.
Article in English | MEDLINE | ID: mdl-16580723

ABSTRACT

The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. The anammox pathway of nitrogen conversion can only be elucidated after the responsible proteins have been purified and characterised. The anammox bacteria have a complex cell envelope consisting of protein and lipopolysaccharide and they grow in dense cell aggregates. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by gel formation. It was demonstrated that protein-protein (i.e. disulfide formation) as well as protein-polysaccharide interaction caused this gel formation in extracts. Cell extract gelled upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. The present work describes the potential causes for gel formation in anammox cell extracts. Optimized protocols for sample preparation for polyacrylamide gel electrophoresis and ion exchange chromatography are presented. High-resolution gel electrophoresis of the cell extract was achieved after clarification from polymeric substances with denaturating phenol extraction and the purification of a 10 kDa cytochrome c is presented as an example.


Subject(s)
Bacteria, Anaerobic/chemistry , Bacterial Proteins/isolation & purification , Cytochromes c/isolation & purification , Ammonia/metabolism , Bacteria, Anaerobic/growth & development , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Wall/chemistry , Cell Wall/metabolism , Cytochromes c/metabolism , Lipopolysaccharides/chemistry , Oxidation-Reduction
7.
Mol Microbiol ; 62(6): 1618-30, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17427286

ABSTRACT

Methanothermobacter thermautotrophicus is a methanogenic Gram-positive microorganism with a cell wall consisting of pseudomurein. Currently, no information is available on extracellular pseudomurein biology and so far only two prophage pseudomurein autolysins, PeiW and PeiP, have been reported. In this paper we show that PeiW and PeiP contain two different N-terminal pseudomurein cell wall binding domains. This finding was used to identify a novel domain, PB007923, on the M. thermautotrophicus genome present in 10 predicted open reading frames. Three homologues were identified in the Methanosphaera stadtmanae genome. Binding studies of fusion constructs of three separate PB007923 domains to green fluorescent protein revealed that it also constituted a cell wall binding domain. Both prophage domains and the PB007923 domain bound to the cell walls of Methanothermobacter species and fluorescence microscopy showed a preference for the septal region. Domain specificities were revealed by binding studies with other pseudomurein-containing archaea. Localized binding was observed for M. stadtmanae and Methanobrevibacter species, while others stained evenly. The identification of the first pseudomurein cell wall binding domains reveals the dynamics of the pseudomurein cell wall and provides marker proteins to study the extracellular pseudomurein biology of M. thermautotrophicus and of other pseudomurein-containing archaea.


Subject(s)
Cell Wall/metabolism , Methanobacteriaceae/metabolism , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Peptidoglycan/metabolism , Amino Acid Sequence , Binding Sites , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Methanobacteriaceae/enzymology , Methanobacteriaceae/genetics , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , N-Acetylmuramoyl-L-alanine Amidase/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid
8.
Microbiology (Reading) ; 151(Pt 5): 1697-1705, 2005 May.
Article in English | MEDLINE | ID: mdl-15870477

ABSTRACT

Coenzyme F420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F420-dependent hydrogenase. The standard free-energy change at pH 7 of F420 reduction was determined to be -15 kJ mol(-1), irrespective of the temperature (25-65 degrees C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F420 is a useful probe to assess in situ hydrogen concentrations in H2-metabolizing methanogens.


Subject(s)
Hydrogen/metabolism , Methane/metabolism , Riboflavin/analogs & derivatives , Riboflavin/metabolism , Acetates , Culture Media , Fermentation , Hydrogen-Ion Concentration , Methanobacteriaceae/growth & development , Methanobacteriaceae/metabolism , Methanol/metabolism , Oxidation-Reduction , Riboflavin/chemistry , Thermodynamics
9.
Eur J Biochem ; 270(1): 66-75, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12492476

ABSTRACT

The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2-dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2). Formyl-methanofuran synthesis is an endergonic reaction (DeltaG degrees ' = +16 kJ.mol-1). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DeltaG') were found to range between +10 and +35 kJ.mol-1 depending on the pH2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions. Heterodisulfide reduction (DeltaG degrees ' = -40 kJ.mol-1) was associated with free energy changes as high as -55 to -80 kJ.mol-1. The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow pH2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.


Subject(s)
Aldehyde Oxidoreductases/metabolism , Archaeal Proteins/metabolism , Methanobacteriaceae/metabolism , Oxidoreductases/metabolism , Phosphothreonine/analogs & derivatives , Cell Division , Disulfides/chemistry , Disulfides/metabolism , Energy Metabolism , Furans/metabolism , Hydrogen-Ion Concentration , Mesna/chemistry , Mesna/metabolism , Methanobacteriaceae/growth & development , Microbiological Techniques , Phosphothreonine/chemistry , Phosphothreonine/metabolism , Protons , Sodium/metabolism
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