RESUMEN
Anaerobic ammonium oxidation (anammox) has a major role in the Earth's nitrogen cycle and is used in energy-efficient wastewater treatment. This bacterial process combines nitrite and ammonium to form dinitrogen (N2) gas, and has been estimated to synthesize up to 50% of the dinitrogen gas emitted into our atmosphere from the oceans. Strikingly, the anammox process relies on the highly unusual, extremely reactive intermediate hydrazine, a compound also used as a rocket fuel because of its high reducing power. So far, the enzymatic mechanism by which hydrazine is synthesized is unknown. Here we report the 2.7 Å resolution crystal structure, as well as biophysical and spectroscopic studies, of a hydrazine synthase multiprotein complex isolated from the anammox organism Kuenenia stuttgartiensis. The structure shows an elongated dimer of heterotrimers, each of which has two unique c-type haem-containing active sites, as well as an interaction point for a redox partner. Furthermore, a system of tunnels connects these active sites. The crystal structure implies a two-step mechanism for hydrazine synthesis: a three-electron reduction of nitric oxide to hydroxylamine at the active site of the γ-subunit and its subsequent condensation with ammonia, yielding hydrazine in the active centre of the α-subunit. Our results provide the first, to our knowledge, detailed structural insight into the mechanism of biological hydrazine synthesis, which is of major significance for our understanding of the conversion of nitrogenous compounds in nature.
Asunto(s)
Bacterias/enzimología , Hidrazinas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hidroxilamina/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Modelos Moleculares , Óxido Nítrico/metabolismo , Multimerización de ProteínaRESUMEN
Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest scientific discoveries in the biogeochemical nitrogen cycle. These microorganisms are able to oxidize ammonium (NH4+) with nitrite (NO2-) as the oxidant instead of oxygen and form dinitrogen (N2) as the end product. Recent research has shed a light on the biochemistry underlying anammox metabolism with two key intermediates, nitric oxide (NO) and hydrazine (N2H4). Substrates and intermediates are converted exploiting the catalytic and electron-transfer potentials of c-type heme proteins known from numerous biochemical reactions and that have acquired new functionality in anammox biochemistry. On a global scale, anammox bacteria significantly contribute to the removal of fixed nitrogen from the environment and the process finds rapidly increasing interest in wastewater treatment.
Asunto(s)
Compuestos de Amonio/química , Proteínas Bacterianas/química , Hemoproteínas/química , Nitrógeno/química , Planctomycetales/metabolismo , Compuestos de Amonio/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Transporte de Electrón , Expresión Génica , Hemo/análogos & derivados , Hemo/química , Hemo/metabolismo , Hemoproteínas/genética , Hemoproteínas/metabolismo , Hidrazinas/química , Hidrazinas/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Nitritos/química , Nitritos/metabolismo , Nitrógeno/metabolismo , Oxidación-Reducción , Planctomycetales/genética , Planctomycetales/ultraestructura , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Especificidad por Sustrato , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Anaerobic oxidation of methane (AOM) is crucial for controlling the emission of this potent greenhouse gas to the atmosphere. Nitrite-, nitrate-, and sulfate-dependent methane oxidation is well-documented, but AOM coupled to the reduction of oxidized metals has so far been demonstrated only in environmental samples. Here, using a freshwater enrichment culture, we show that archaea of the order Methanosarcinales, related to "Candidatus Methanoperedens nitroreducens," couple the reduction of environmentally relevant forms of Fe3+ and Mn4+ to the oxidation of methane. We obtained an enrichment culture of these archaea under anaerobic, nitrate-reducing conditions with a continuous supply of methane. Via batch incubations using [13C]methane, we demonstrated that soluble ferric iron (Fe3+, as Fe-citrate) and nanoparticulate forms of Fe3+ and Mn4+ supported methane-oxidizing activity. CO2 and ferrous iron (Fe2+) were produced in stoichiometric amounts. Our study connects the previous finding of iron-dependent AOM to microorganisms detected in numerous habitats worldwide. Consequently, it enables a better understanding of the interaction between the biogeochemical cycles of iron and methane.
RESUMEN
Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms.
Asunto(s)
Compuestos de Amonio/química , Proteínas Bacterianas/química , Hidrazinas/química , Nitrógeno/química , Oxidorreductasas/química , Planctomycetales/enzimología , Compuestos de Amonio/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Hidrazinas/metabolismo , Nitrógeno/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Planctomycetales/genéticaRESUMEN
Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species.
Asunto(s)
Compuestos de Amonio/metabolismo , Anaerobiosis/fisiología , Bacterias/metabolismo , Transporte de Electrón/fisiología , Membranas/metabolismo , Fenómenos Bioquímicos/fisiología , Respiración de la Célula/fisiología , Electrones , Nitrógeno/metabolismo , Oxidación-Reducción , Fosforilación/fisiología , Proteómica/métodos , Compuestos de Amonio Cuaternario/metabolismoRESUMEN
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.
Asunto(s)
Anaerobiosis , Compuestos de Amonio Cuaternario/metabolismo , Amoníaco/metabolismo , Atmósfera/química , Bacterias Anaerobias/metabolismo , Biocatálisis , Hidrazinas/metabolismo , Nitrato-Reductasa/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Ciclo del Nitrógeno , Fijación del Nitrógeno , Oxidación-Reducción , Compuestos de Amonio Cuaternario/químicaRESUMEN
UNLABELLED: Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite as the terminal electron acceptor to form dinitrogen gas in the absence of oxygen. Anammox bacteria have a compartmentalized cell plan with a central membrane-bound "prokaryotic organelle" called the anammoxosome. The anammoxosome occupies most of the cell volume, has a curved membrane, and contains conspicuous tubule-like structures of unknown identity and function. It was suggested previously that the catalytic reactions of the anammox pathway occur in the anammoxosome, and that proton motive force was established across its membrane. Here, we used antibodies raised against five key enzymes of the anammox catabolism to determine their cellular location. The antibodies were raised against purified native hydroxylamine oxidoreductase-like protein kustc0458 with its redox partner kustc0457, hydrazine dehydrogenase (HDH; kustc0694), hydroxylamine oxidase (HOX; kustc1061), nitrite oxidoreductase (NXR; kustd1700/03/04), and hydrazine synthase (HZS; kuste2859-61) of the anammox bacterium Kuenenia stuttgartiensis. We determined that all five protein complexes were exclusively located inside the anammoxosome matrix. Four of the protein complexes did not appear to form higher-order protein organizations. However, the present data indicated for the first time that NXR is part of the tubule-like structures, which may stretch the whole length of the anammoxosome. These findings support the anammoxosome as the locus of catabolic reactions of the anammox pathway. IMPORTANCE: Anammox bacteria are environmentally relevant microorganisms that contribute significantly to the release of fixed nitrogen in nature. Furthermore, the anammox process is applied for nitrogen removal from wastewater as an environment-friendly and cost-effective technology. These microorganisms feature a unique cellular organelle, the anammoxosome, which was proposed to contain the energy metabolism of the cell and tubule-like structures with hitherto unknown function. Here, we purified five native enzymes catalyzing key reactions in the anammox metabolism and raised antibodies against these in order to localize them within the cell. We showed that all enzymes were located within the anammoxosome, and nitrite oxidoreductase was located exclusively at the tubule-like structures, providing the first insights into the function of these subcellular structures.
Asunto(s)
Bacterias/enzimología , Inmunohistoquímica , Orgánulos/fisiología , Compuestos de Amonio/metabolismo , Anaerobiosis , Bacterias/citología , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Oxidación-ReducciónRESUMEN
Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.
Asunto(s)
Proteínas Bacterianas/química , Óxido Nítrico/química , Oxidorreductasas/química , Planctomycetales/enzimología , Amoníaco/química , Amoníaco/metabolismo , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Hidrazinas/química , Hidrazinas/metabolismo , Óxido Nítrico/biosíntesis , Oxidación-Reducción , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Methanol dehydrogenase (MDH) catalyzes the first step in methanol use by methylotrophic bacteria and the second step in methane conversion by methanotrophs. Gram-negative bacteria possess an MDH with pyrroloquinoline quinone (PQQ) as its catalytic center. This MDH belongs to the broad class of eight-bladed ß propeller quinoproteins, which comprise a range of other alcohol and aldehyde dehydrogenases. A well-investigated MDH is the heterotetrameric MxaFI-MDH, which is composed of two large catalytic subunits (MxaF) and two small subunits (MxaI). MxaFI-MDHs bind calcium as a cofactor that assists PQQ in catalysis. Genomic analyses indicated the existence of another MDH distantly related to the MxaFI-MDHs. Recently, several of these so-called XoxF-MDHs have been isolated. XoxF-MDHs described thus far are homodimeric proteins lacking the small subunit and possess a rare-earth element (REE) instead of calcium. The presence of such REE may confer XoxF-MDHs a superior catalytic efficiency. Moreover, XoxF-MDHs are able to oxidize methanol to formate, rather than to formaldehyde as MxaFI-MDHs do. While structures of MxaFI- and XoxF-MDH are conserved, also regarding the binding of PQQ, the accommodation of a REE requires the presence of a specific aspartate residue near the catalytic site. XoxF-MDHs containing such REE-binding motif are abundantly present in genomes of methylotrophic and methanotrophic microorganisms and also in organisms that hitherto are not known for such lifestyle. Moreover, sequence analyses suggest that XoxF-MDHs represent only a small part of putative REE-containing quinoproteins, together covering an unexploited potential of metabolic functions.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Coenzimas/metabolismo , Bacterias Gramnegativas/enzimología , Cofactor PQQ/metabolismo , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Calcio/metabolismo , Bacterias Gramnegativas/genética , Metano/metabolismo , Metanol/metabolismo , Modelos Moleculares , Filogenia , Conformación Proteica , Subunidades de Proteína/metabolismoRESUMEN
BACKGROUND: Anaerobic ammonium oxidizing (anammox) bacteria may contribute up to 50% to the global nitrogen production, and are, thus, key players of the global nitrogen cycle. The molecular mechanism of anammox was recently elucidated and is suggested to proceed through a branched respiratory chain. This chain involves an exceptionally high number of c-type cytochrome proteins which are localized within the anammoxosome, a unique subcellular organelle. During transport into the organelle the c-type cytochrome apoproteins need to be post-translationally processed so that heme groups become covalently attached to them, resulting in mature c-type cytochrome proteins. RESULTS: In this study, a comparative genome analysis was performed to identify the cytochrome c maturation system employed by anammox bacteria. Our results show that all available anammox genome assemblies contain a complete type II cytochrome c maturation system. CONCLUSIONS: Our working model suggests that this machinery is localized at the anammoxosome membrane which is assumed to be the locus of anammox catabolism. These findings will stimulate further studies in dissecting the molecular and cellular basis of cytochrome c biogenesis in anammox bacteria.
Asunto(s)
Compuestos de Amonio/metabolismo , Bacterias/genética , Bacterias/metabolismo , Citocromos c/metabolismo , Redes y Vías Metabólicas/genética , Procesamiento Proteico-Postraduccional , Biología Computacional , Genoma Bacteriano , Proteínas de la Membrana/metabolismo , Orgánulos/enzimología , Orgánulos/metabolismo , Oxidación-ReducciónRESUMEN
'Candidatus Methylomirabilis oxyfera' is a denitrifying methanotroph that performs nitrite-dependent anaerobic methane oxidation through a newly discovered intra-aerobic pathway. In this study, we investigated the response of a M. oxyfera enrichment culture to oxygen. Addition of either 2% or 8% oxygen resulted in an instant decrease of methane and nitrite conversion rates. Oxygen exposure also led to a deviation in the nitrite to methane oxidation stoichiometry. Oxygen-uptake and inhibition studies with cell-free extracts displayed a change from cytochrome c to quinol as electron donor after exposure to oxygen. The change in global gene expression was monitored by deep sequencing of cDNA using Illumina technology. After 24 h of oxygen exposure, transcription levels of 1109 (out of 2303) genes changed significantly when compared with the anoxic period. Most of the genes encoding enzymes of the methane oxidation pathway were constitutively expressed. Genes from the denitrification pathway, with exception of one of the putative nitric oxide reductases, norZ2, were severely downregulated. The majority of known genes involved in the vital cellular functions, such as nucleic acid and protein biosynthesis and cell division processes, were downregulated. The alkyl hydroperoxide reductase, ahpC, and genes involved in the synthesis/repair of the iron-sulfur clusters were among the few upregulated genes. Further, transcription of the pmoCAB genes of aerobic methanotrophs present in the non-M. oxyfera community were triggered by the presence of oxygen. Our results show that oxygen-exposed cells of M. oxyfera were under oxidative stress and that in spite of its oxygenic capacity, exposure to microoxic conditions has an overall detrimental effect.
Asunto(s)
Bacterias/metabolismo , Ciclo del Nitrógeno/fisiología , Oxígeno/metabolismo , Anaerobiosis/fisiología , Bacterias/genética , Metano/metabolismo , Nitritos/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics--the reconstruction of genomic data directly from the environment--to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Evolución Biológica , Genoma Bacteriano , Compuestos de Amonio Cuaternario/metabolismo , Anaerobiosis , Bacterias/clasificación , Reactores Biológicos , Evolución Molecular , Ácidos Grasos/biosíntesis , Genes Bacterianos/genética , Hidrazinas/metabolismo , Hidrolasas/metabolismo , Operón/genética , Oxidorreductasas/metabolismo , Filogenia , TermodinámicaRESUMEN
Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the "anammoxosome". Thus far unique "ladderane" lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham's razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N(2) gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.
Asunto(s)
Bacterias/metabolismo , Nitrógeno/metabolismo , Microbiología del Agua , Anaerobiosis , Oxidación-Reducción , Compuestos de Amonio Cuaternario/metabolismoRESUMEN
The anaerobic nitrite-reducing methanotroph 'Candidatus Methylomirabilis oxyfera' ('Ca. M. oxyfera') produces oxygen from nitrite by a novel pathway. The major part of the O(2) is used for methane activation and oxidation, which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes, such as respiration. We have found that the genome of 'Ca. M. oxyfera' harbours four sets of genes encoding terminal respiratory oxidases: two cytochrome c oxidases, a third putative bo-type ubiquinol oxidase, and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed, albeit at low levels. Oxygen-uptake and inhibition experiments, UV-visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by 'Ca. M. oxyfera', notably the membrane-bound bo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems, and are an additional indication of the flexibility of terminal oxidases, of which the distribution among anaerobic micro-organisms may be largely underestimated.
Asunto(s)
Bacterias Anaerobias/fisiología , Metano/metabolismo , Nitritos/metabolismo , Oxidorreductasas/metabolismo , Anaerobiosis , Bacterias Anaerobias/enzimología , Bacterias Anaerobias/genética , Bacterias Anaerobias/metabolismo , Óxido Nítrico/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Oxígeno/metabolismo , Consumo de Oxígeno , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Biological methane oxidation proceeds either through aerobic or anaerobic pathways. The newly discovered bacterium Candidatus 'Methylomirabilis oxyfera' challenges this dichotomy. This bacterium performs anaerobic methane oxidation coupled to denitrification, but does so in a peculiar way. Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in sulfate-reducing systems, it produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas. The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane mono-oxygenase. The present mini-review summarizes the current knowledge about this process and the micro-organism responsible for it.
Asunto(s)
Aerobiosis/fisiología , Anaerobiosis/fisiología , Bacterias Anaerobias/metabolismo , Metano/metabolismo , Methylococcaceae/metabolismo , Nitritos/metabolismo , Bacterias Anaerobias/clasificación , Bacterias Anaerobias/genética , Metabolismo Energético , Methylococcaceae/clasificación , Methylococcaceae/genética , Oxidación-Reducción , FilogeniaRESUMEN
It has been less than two decades since anammox (anaerobic ammonium oxidation) coupled to nitrite reduction has been discovered. Already, this process has been recognized as an important sink for fixed nitrogen in the natural environment and has been implemented as a cost-effective ammonium removal technology. Still, little is known about the molecular mechanism of this remarkable reaction. In this mini review, we present an insight into how ammonium and nitrite are combined to form dinitrogen gas.
Asunto(s)
Bacterias Anaerobias/metabolismo , Proteínas Bacterianas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Adenosina Trifosfato/biosíntesis , Bacterias Anaerobias/genética , Transporte de Electrón/fisiología , Genoma Bacteriano , Familia de Multigenes , Nitratos/metabolismo , Nitritos/metabolismo , Nitrógeno/química , Nitrógeno/metabolismo , Oxidación-ReducciónRESUMEN
Methanomicrococcus blatticola is an obligately anaerobic methanogen that derives the energy for growth exclusively from the reduction of methylated compounds to methane with molecular hydrogen as energy source. Competition for methanol (concentration below 10 microM) and H(2) (concentration below 500 Pa), as well as oxidative stress due to the presence of oxygen are likely to occur in the peripheral region of the cockroach hindgut, the species' normal habitat. We investigated the ecophysiological properties of M. blatticola to explain how it can successfully compete for its methanogenic substrates. The organism showed affinities for methanol (K(m)=5 microM; threshold<1 microM) and hydrogen (K(m)=200 Pa; threshold <0.7 Pa) that are superior to other methylotrophic methanogens (Methanosphaera stadtmanae, Methanosarcina barkeri) investigated here. Thermodynamic considerations indicated that 'methanol respiration', i.e. the use of methanol as the terminal electron acceptor, represents an attractive mode of energy generation, especially at low hydrogen concentrations. Methanomicrococcus blatticola exploits the opportunities by specific growth rates (>0.2 h(-1)) and specific growth yields (up to 7 g of dry cells per mole of methane formed) that are particularly high within the realm of mesophilic methanogens. Upon oxygen exposure, part of the metabolic activity may be diverted into oxygen removal, thus establishing appropriate anaerobic conditions for survival and growth.
Asunto(s)
Sistema Digestivo/microbiología , Metano/metabolismo , Methanosarcinaceae/metabolismo , Periplaneta/microbiología , Animales , Sistema Digestivo/metabolismo , Hidrógeno/química , Hidrógeno/metabolismo , Metano/química , Metanol/química , Metanol/metabolismo , Methanosarcinaceae/efectos de los fármacos , Methanosarcinaceae/crecimiento & desarrollo , Oxígeno/farmacología , Periplaneta/metabolismo , TermodinámicaRESUMEN
Ammonium and methane are inert molecules and dedicated enzymes are required to break up the N-H and C-H bonds. Until recently, only aerobic microorganisms were known to grow by the oxidation of ammonium or methane. Apart from respiration, oxygen was specifically utilized to activate the inert substrates. The presumed obligatory need for oxygen may have resisted the search for microorganisms that are capable of the anaerobic oxidation of ammonium and of methane. However extremely slowly growing, these "impossible" organisms exist and they found other means to tackle ammonium and methane. Anaerobic ammonium-oxidizing (anammox) bacteria use the oxidative power of nitric oxide (NO) by forging this molecule to ammonium, thereby making hydrazine (N2H4). Nitrite-dependent anaerobic methane oxidizers (N-DAMO) again take advantage of NO, but now apparently disproportionating the compound into dinitrogen and dioxygen gas. This intracellularly produced dioxygen enables N-DAMO bacteria to adopt an aerobic mechanism for methane oxidation.Although our understanding is only emerging how hydrazine synthase and the NO dismutase act, it seems clear that reactions fully rely on metal-based catalyses known from other enzymes. Metal-dependent conversions not only hold for these key enzymes, but for most other reactions in the central catabolic pathways, again supported by well-studied enzymes from model organisms, but adapted to own specific needs. Remarkably, those accessory catabolic enzymes are not unique for anammox bacteria and N-DAMO. Close homologs are found in protein databases where those homologs derive from (partly) known, but in most cases unknown species that together comprise an only poorly comprehended microbial world.
Asunto(s)
Compuestos de Amonio/metabolismo , Bacterias Anaerobias/enzimología , Proteínas Bacterianas/metabolismo , Metaloproteínas/metabolismo , Metano/metabolismo , Anaerobiosis/fisiología , Bacterias Anaerobias/genética , Proteínas Bacterianas/genética , Bases de Datos de Proteínas , Metaloproteínas/genética , Oxidación-ReducciónRESUMEN
Anaerobic ammonium-oxidizing (anammox) bacteria primarily grow by the oxidation of ammonium coupled to nitrite reduction, using CO2 as the sole carbon source. Although they were neglected for a long time, anammox bacteria are encountered in an enormous species (micro)diversity in virtually any anoxic environment that contains fixed nitrogen. It has even been estimated that about 50% of all nitrogen gas released into the atmosphere is made by these 'impossible' bacteria. Anammox catabolism most likely resides in a special cell organelle, the anammoxosome, which is surrounded by highly unusual ladder-like (ladderane) lipids. Ammonium oxidation and nitrite reduction proceed in a cyclic electron flow through two intermediates, hydrazine and nitric oxide, resulting in the generation of proton-motive force for ATP synthesis. Reduction reactions associated with CO2 fixation drain electrons from this cycle, and they are replenished by the oxidation of nitrite to nitrate. Besides ammonium or nitrite, anammox bacteria use a broad range of organic and inorganic compounds as electron donors. An analysis of the metabolic opportunities even suggests alternative chemolithotrophic lifestyles that are independent of these compounds. We note that current concepts are still largely hypothetical and put forward the most intriguing questions that need experimental answers.
Asunto(s)
Bacterias Anaerobias/crecimiento & desarrollo , Bacterias Anaerobias/metabolismo , Metabolismo Energético , Microbiología Ambiental , Compuestos de Amonio Cuaternario/metabolismo , Adenosina Trifosfato/biosíntesis , Dióxido de Carbono/metabolismo , Hidrazinas/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxidación-ReducciónRESUMEN
The genomes of two phenotypically denitrifying type strains of the genus Bacillus were sequenced and the pathways for dissimilatory nitrate reduction were reconstructed. Results suggest that denitrification proceeds in the periplasmic space and in an analogous fashion as in Gram-negative organisms, yet with the participation of proteins that tend to be membrane-bound or membrane-associated. A considerable degree of functional redundancy was observed with marked differences between B. azotoformans LMG 9581(T) and B. bataviensis LMG 21833(T). In addition to the already characterized menaquinol/cyt c-dependent nitric oxide reductase (Suharti et al., 2001, 2004) of which the encoding genes could be identified now, evidence for another novel nitric oxide reductase (NOR) was found. Also, our analyses confirm earlier findings on branched electron transfer with both menaquinol and cytochrome c as reductants. Quite unexpectedly, both bacilli have the disposal of two parallel pathways for nitrite reduction enabling a life style as a denitrifier and as an ammonifying bacterium.