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Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex., The oxidoreductase (NXR) multiprotein complex is a key enzyme in the nitrogen cycle. A detailed structural and biochemical characterization of NXR from the anammox bacterium Kuenenia stuttgartiensis shows that this complex is a filament-forming protein that catalysers both nitrite oxidation and nitrate reduction, and elucidates the mechanisms governing complex assembly and function.

Anaerobic ammonium-oxidizing (anammox) bacteria are important players in both the environment and industry where they contribute substantially to nitrogen removal from natural and man-made (eco)systems. Apart from their ecological value, anammox bacteria are extremely interesting from both a physiological and cell biological perspective. Cells of anammox bacteria contain a major membrane-bound compartment, the anammoxosome, which is dedicated to the anammox reaction and energy conversion. The anammox reaction converts the substrates ammonium and nitrite to the product dinitrogen gas via two reactive intermediates: nitric oxide and hydrazine. First, nitrite is converted to nitric oxide by a nitrite reductase, nitric oxide and ammonium are then combined by hydrazine synthase to form hydrazine, and, finally, hydrazine is oxidized to dinitrogen gas by hydrazine dehydrogenase. The hydrazine synthase and hydrazine dehydrogenase enzymes are biochemical novelties. Electrons released from hydrazine oxidation are proposed to be shuttled to an electron transport chain in the anammoxosome membrane resulting in the establishment of a proton motive force and subsequent ATP synthesis. The anammoxosome compartment has a highly curved membrane and contains tubule-like structures and electron-dense, iron-containing particles. Finally, anammox bacteria contain unique ladderane membrane lipids that are postulated to render anammox membranes less permeable than conventional membranes. In this chapter, the cell biology, physiology and biochemistry of the anammox reaction and anammoxosome compartment are discussed.

Anaerobic ammonium-oxidizing (anammox) bacteria, members of the “Candidatus Brocadiaceae” family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-enclosed compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic-, and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbor multiple copies of ammonium-, nitrite-, and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite-, and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells showed that four of the seven ammonium transporter homologs and two of the nine nitrite transporter homologs were significantly upregulated during ammonium-limited growth, while another ammonium transporter- and four nitrite transporter homologs were upregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2−) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. 15N-labeling experiments revealed that NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH4+ oxidation with electrode as electron acceptor. Complete NH4+ oxidation to N2 without accumulation of NO2− and NO3− was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen., Bacteria capable of anaerobic ammonium oxidation (anammox) produce half of the nitrogen gas in the atmosphere, but much of their physiology is still unknown. Here the authors show that anammox bacteria are capable of a novel mechanism of ammonium oxidation using extracellular electron transfer.

Planctomycetes are a bacterial phylum known for their complex intracellular compartmentalization. While most Planctomycetes have two compartments, the anaerobic ammonium oxidizing (anammox) bacteria contain three membrane-enclosed compartments. In contrast to a long-standing consensus, recent insights suggested the outermost Planctomycete membrane to be similar to a Gram-negative outer membrane (OM). One characteristic component that differentiates OMs from cytoplasmic membranes (CMs) is the presence of outer membrane proteins (OMPs) featuring a β-barrel structure that facilitates passage of molecules through the OM. Although proteomic and genomic evidence suggested the presence of OMPs in several Planctomycetes, no experimental verification existed of the pore-forming function and localization of these proteins in the outermost membrane of these exceptional microorganisms. Here, we show via lipid bilayer assays that at least two typical OMP-like channel-forming proteins are present in membrane preparations of the anammox bacterium Kuenenia stuttgartiensis. One of these channel-forming proteins, the highly abundant putative OMP Kustd1878, was purified to homogeneity. Analysis of the channel characteristics via lipid bilayer assays showed that Kustd1878 forms a moderately cation-selective channel with a high current noise and an average single-channel conductance of about 170-190pS in 1M KCl. Antibodies were raised against the purified protein and immunogold localization indicated Kustd1878 to be present in the outermost membrane. Therefore, this work clearly demonstrates the presence of OMPs in anammox Planctomycetes and thus firmly adds to the emerging view that Planctomycetes have a Gram-negative cell envelope.

Contains fulltext : 198859.pdf (Publisher’s version ) (Closed access) Anaerobic ammonium-oxidizing (anammox) bacteria are major players in the biological nitrogen cycle and can be applied in wastewater treatment for the removal of nitrogen compounds. Anammox bacteria anaerobically convert the substrates ammonium and nitrite into dinitrogen gas in a specialized intracellular compartment called the anammoxosome. The anammox cell biology, physiology and biochemistry is of exceptional interest but also difficult to study because of the lack of a pure culture, standard cultivation techniques and genetic tools. Here we review the most important recent developments regarding the cell structure - anammoxosome and cell envelope - and anammox energy metabolism - nitrite reductase, hydrazine synthase and energy conversion - including the trending topics electro-anammox, extracellular polymeric substances and ladderane lipids.

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.

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost-effective and environment-friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere.

Anaerobic ammonium oxidation (anammox) bacteria are a distinct group of Planctomycetes that are characterized by their unique ability to perform anammox with nitrite to dinitrogen gas in a specialized organelle. The cell of anammox bacteria comprises three membrane-bound compartments and is surrounded by a two-dimensional crystalline S-layer representing the direct interaction zone of anammox bacteria with the environment. Previous results from studies with the model anammox organism Kuenenia stuttgartiensis suggested that the protein monomers building the S-layer lattice are glycosylated. In the present study, we focussed on the characterization of the S-layer protein glycosylation in order to increase our knowledge on the cell surface characteristics of anammox bacteria. Mass spectrometry (MS) analysis showed an O-glycan attached to 13 sites distributed over the entire 1591-amino acid S-layer protein. This glycan is composed of six monosaccharide residues, of which five are N-acetylhexosamine (HexNAc) residues. Four of these HexNAc residues have been identified as GalNAc. The sixth monosaccharide in the glycan is a putative dimethylated deoxyhexose. Two of the HexNAc residues were also found to contain a methyl group, thereby leading to an extensive degree of methylation of the glycan. This study presents the first characterization of a glycoprotein in a planctomycete and shows that the S-layer protein Kustd1514 of K. stuttgartiensis is heavily glycosylated with an O-linked oligosaccharide which is additionally modified by methylation. S-layer glycosylation clearly contributes to the diversification of the K. stuttgartiensis cell surface and can be expected to influence the interaction of the bacterium with other cells or abiotic surfaces.

Anaerobic ammonium-oxidizing (anammox) bacteria are divided into three compartments by bilayer membranes (from out- to inside): paryphoplasm, riboplasm and anammoxosome. It is proposed that the anammox reaction is performed by proteins located in the anammoxosome and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by membrane-bound ATPases. To test this hypothesis, we investigated the location of membrane-bound ATPases in the anammox bacterium 'Candidatus Kuenenia stuttgartiensis'. Four ATPase gene clusters were identified in the K. stuttgartiensis genome: one typical F-ATPase, two atypical F-ATPases and a prokaryotic V-ATPase. K. stuttgartiensis transcriptomic and proteomic analysis and immunoblotting using antisera directed at catalytic subunits of the ATPase gene clusters indicated that only the typical F-ATPase gene cluster most likely encoded a functional ATPase under these cultivation conditions. Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with the anammox physiology model, and confirms the status of the outermost cell membrane as cytoplasmic membrane. The occurrence of ATPase in the anammoxosome membrane suggests that anammox bacteria have evolved a prokaryotic organelle; a membrane-bounded compartment with a specific cellular function: energy metabolism.

Anaerobic ammonium oxidation (anammox) is an ecologically and industrially important process and is performed by a clade of deeply branching Planctomycetes . Anammox bacteria possess an intracytoplasmic membrane-bounded organelle, the anammoxosome. In the present study, the ultrastructures of four different genera of anammox bacteria were compared with transmission electron microscopy and electron tomography. The four anammox genera shared a common cell plan and contained glycogen granules. Differences between the four genera included cell size (from 800 to 1,100 nm in diameter), presence or absence of cytoplasmic particles, and presence or absence of pilus-like appendages. Furthermore, cytochrome c proteins were detected exclusively inside the anammoxosome. This detection provides further support for the hypothesis that this organelle is the locus of anammox catabolism.

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.

Planctomycetes are intriguing microorganisms that apparently lack peptidoglycan, a structure that controls the shape and integrity of almost all bacterial cells. Therefore, the planctomycetal cell envelope is considered exceptional and their cell plan uniquely compartmentalized. Anaerobic ammonium-oxidizing (anammox) Planctomycetes play a key role in the global nitrogen cycle by releasing fixed nitrogen back to the atmosphere as N2. Here using a complementary array of state-of-the-art techniques including continuous culturing, cryo-transmission electron microscopy, peptidoglycan-specific probes and muropeptide analysis, we show that the anammox bacterium Kuenenia stuttgartiensis contains peptidoglycan. On the basis of the thickness, composition and location of peptidoglycan in K. stuttgartiensis, we propose to redefine Planctomycetes as Gram-negative bacteria. Our results demonstrate that Planctomycetes are not an exception to the universal presence of peptidoglycan in bacteria., Planctomycetes are unusual bacteria with complex intracellular compartments and an apparent lack of peptidoglycan in their cell walls. Here, van Teeseling et al. show that the cell wall of an anammox planctomycete does contain peptidoglycan, and propose to redefine planctomycetes as Gram-negative bacteria.

The demand for new and sustainable systems for nitrogen removal has increased dramatically in the last decade. It is clear that the conventional systems cannot deal with the increasing nitrogen loads in a cost effective way. As an alternative, the implementation of the anammox (anaerobic ammonium oxidation) process in the treatment of wastewater with high ammonium concentrations has been started. The compact anammox reactors can sustain high nitrogen loads without any problems. The highest observed anammox capacity is 8.9 kg N removed m-3 reactor day-1. The first 75 m3 anammox reactor is operating in Rotterdam, the Netherlands, combined with the partial nitrification process Single reaction system for High Ammonium Removal Over Nitrite (SHARON). Partial nitrification and anammox can also be combined in one reactor systems like Completely Autotrophic Nitrogen removal Over Nitrite (CANON) or Oxygen Limited Ammonium removal via Nitrification Denitrification (OLAND) where aerobic ammonium-oxidizing bacteria (AOB) and anammox bacteria cooperate under oxygen-limitation. These systems remove about 1.5 kg N m-3 reactor day-1. In addition to ammonium, urea can also be converted in the CANON system after a two-week adaptation period. The ecophysiological properties of the anammox bacteria make them very well suited to convert ammonium and nitrite. The Ks values for ammonium and nitrite are below 5 μM. However, nitrite above 10 mM is detrimental for the anammox process, and oxygen reversibly inhibits the process at concentrations as low as 1 μM. Acetate and propionate can be used by the anammox bacteria to convert nitrite and nitrate, whereas methanol and ethanol severely inhibit the anammox reaction. The enzyme hydroxylamine/hydrazine oxidoreductase (HAO), one of the key enzymes, is located in the anammoxosome, which is a membrane bound organelle. The membranes of the anammox bacteria contain unique ladderane lipids and hopanoids. The bacteria responsible for the anammox reaction are related to the Planctomycetes. The first anammox bacteria were isolated via Percoll centrifugation and characterized as Candidatus “Brocadia anammoxidans”. Survey of different wastewater treatment plants using anammox specific 16S rRNA gene primers and anammox specific oligonucleotide probes has revealed the presence of at least three other anammox bacteria, which have been tentatively named Candidatus “Kuenenia stuttgartiensis”, Candidatus “Scalindua wagneri” and Candidatus “Scalindua brodae”. A close relative of the latter, Candidatus “Scalindua sorokinii” was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the Black Sea, making the anammox bacteria an important player in the oceanic nitrogen cycle.

Anammox bacteria convert ammonium and nitrite to dinitrogen gas under anaerobic conditions to obtain their energy for growth. The anammox reaction was deemed impossible until its discovery in the early 1990s. Now, anammox bacteria are recognized as major players in the global nitrogen cycle and estimated to be responsible for up to 50% of the nitrogen in the air that we breathe. In addition, anammox bacteria are extremely valuable for wastewater treatment where they are applied for the removal of ammonium. Besides their importance in industry and the environment, anammox bacteria defy some basic biological concepts. Whereas most other bacteria have only one cell compartment, the cytoplasm, anammox bacteria have three independent cell compartments bounded by bilayer membranes, from out- to inside; the paryphoplasm, riboplasm and anammoxosome. The anammoxosome is the largest compartment of the anammox cell and is proposed to be dedicated to energy conservation. As such it would be analogous to the mitochondria of eukaryotes. This review will discuss the anammox cell plan in detail, with the main focus on the anammoxosome. The identity of the anammoxosome as a prokaryotic organelle and the importance of this organelle for anammox bacteria are discussed as well as challenges these bacteria face by having three independent cell compartments.

In marine oxygen minimum zones (OMZs), ammonia-oxidizing archaea (AOA) rather than marine ammonia-oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium-oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory-scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C18 anammox [3]-ladderane fatty acid was highly abundant in the oxygen-limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.

SUMMARY Anaerobic ammonium-oxidizing (anammox) bacteria defy many microbiological concepts and share numerous properties with both eukaryotes and archaea. Among their most intriguing characteristics are their compartmentalized cell plan and archaeon-like cell wall. Here we review our current knowledge about anammox cell biology. The anammox cell is divided into three separate compartments by bilayer membranes. The anammox cell consists of (from outside to inside) the cell wall, paryphoplasm, riboplasm, and anammoxosome. Not much is known about the composition or function of both the anammox cell wall and the paryphoplasm compartment. The cell wall is proposed to be proteinaceous and to lack both peptidoglycan and an outer membrane typical of Gram-negative bacteria. The function of the paryphoplasm is unknown, but it contains the cell division ring. The riboplasm resembles the standard cytoplasmic compartment of other bacteria; it contains ribosomes and the nucleoid. The anammoxosome occupies most of the cell volume and is a so-called “prokaryotic organelle” analogous to the eukaryotic mitochondrion. This is the site where the anammox reaction takes place, coupled over the curved anammoxosome membrane, possibly giving rise to a proton motive force and subsequent ATP synthesis. With these unique properties, anammox bacteria are food for thought concerning the early evolution of the domains Bacteria , Archaea , and Eukarya .

Anaerobic ammonium-oxidizing (anammox) bacteria are the last major addition to the nitrogen-cycle (N-cycle). Because of the presumed inert nature of ammonium under anoxic conditions, the organisms were deemed to be nonexistent until about 15 years ago. They, however, appear to be present in virtually any anoxic place where fixed nitrogen (ammonium, nitrate, nitrite) is found. In various mar`ine ecosystems, anammox bacteria are a major or even the only sink for fixed nitrogen. According to current estimates, about 50% of all nitrogen gas released into the atmosphere is made by these bacteria. Besides this, the microorganisms may be very well suited to be applied as an efficient, cost-effective, and environmental-friendly alternative to conventional wastewater treatment for the removal of nitrogen. So far, nine different anammox species divided over five genera have been enriched, but none of these are in pure culture. This number is only a modest reflection of a continuum of species that is suggested by 16S rRNA analyses of environmental samples. In their environments, anammox bacteria thrive not just by competition, but rather by delicate metabolic interactions with other N-cycle organisms. Anammox bacteria owe their position in the N-cycle to their unique property to oxidize ammonium in the absence of oxygen. Recent research established that they do so by activating the compound into hydrazine (N(2)H(4)), using the oxidizing power of nitric oxide (NO). NO is produced by the reduction of nitrite, the terminal electron acceptor of the process. The forging of the N-N bond in hydrazine is catalyzed by hydrazine synthase, a fairly slow enzyme and its low activity possibly explaining the slow growth rates and long doubling times of the organisms. The oxidation of hydrazine results in the formation of the end product (N(2)), and electrons that are invested both in electron-transport phosphorylation and in the regeneration of the catabolic intermediates (N(2)H(4), NO). Next to this, the electrons provide the reducing power for CO(2) fixation. The electron-transport phosphorylation machinery represents another unique characteristic, as it is most likely localized on a special cell organelle, the anammoxosome, which is surrounded by a glycerolipid bilayer of ladder-like ("ladderane") cyclobutane and cyclohexane ring structures. The use of ammonium and nitrite as sole substrates might suggest a simple metabolic system, but the contrary seems to be the case. Genome analysis and ongoing biochemical research reveal an only partly understood redundancy in respiratory systems, featuring an unprecedented collection of cytochrome c proteins. The presence of the respiratory systems lends anammox bacteria a metabolic versatility that we are just beginning to appreciate. A specialized use of substrates may provide different anammox species their ecological niche.

The most striking example of a complex prokaryotic intracytoplasmic organization can be found in the members of the phylum Planctomycetes. Among them are the anammox (anaerobic ammonium-oxidizing) bacteria, which possess a unique cell compartment with an unprecedented function in bacteria: the anammoxosome is a prokaryotic cell organelle evolved for energy metabolism. It is an independent entity, which is enclosed by a contiguous membrane. Several lines of evidence indicate its importance in the anammox reaction and the unusual subcellular organization may well be essential for the lifestyle of anammox bacteria. The present review summarizes our knowledge about the ultrastructure of anammox cells and the connection between the anammoxosome and the energy metabolism of the cell. In the future, much more research will be necessary to validate the current models and to answer questions on the functional cell biology of anammox bacteria.

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.

Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere's dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells. In most Bacteria, GTP hydrolysis drives the tubulin-analogue FtsZ to assemble into a ring-like structure at the cell division site where it functions as a scaffold for the molecular machinery that performs cell division. However, the genome of the anammox bacterium 'Candidatus Kuenenia stuttgartiensis' does not encode ftsZ. Genomic analysis of open reading frames with potential GTPase activity indicated a possible novel cell division ring gene: kustd1438, which was unrelated to ftsZ. Immunogold localization specifically localized kustd1438 to the cell division ring. Genomic analyses of other members of the phyla Planctomycetes and Chlamydiae revealed no putative functional homologues of kustd1438, suggesting that it is specific to anammox bacteria. Electron tomography also revealed that the bacterial organelle was elongated along with the rest of the cell and divided equally among daughter cells during the cell division process.

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.

The anaerobic oxidation of ammonium to dinitrogen gas (anammox) is a recently discovered pathway of the biological nitrogen cycle [1] and is currently estimated to be a major source of gaseous nitrogen on Earth [2]. Anammox is performed by a deep-branching clade of planctomycete bacteria, which have been applied in wastewater treatment for the removal of ammonium. Anammox bacteria divide only once per two weeks at maximum speed and possess a prokaryotic organelle, the “anammoxosome”, an intracytoplasmic compartment surrounded by a bilayer membrane. This major compartment is dedicated to anammox catabolism and is the locus of respiration by these bacteria, possibly analogous to the mitochondria in Eukaryotes. The anammoxosome membrane consists mainly of so-called ladderane lipids [3], which make this membrane more impermeable to protons and the valuable intermediates of anammox catabolism.

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C18 and C20 ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C20 alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of “Candidatus Kuenenia stuttgartiensis”, suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C27 hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".

Contains fulltext : 35005.pdf (Publisher’s version ) (Closed access) Anammox bacteria have unique intracellular membranes that divide their cytoplasm into three separate compartments. The largest and innermost cytoplasmic compartment, the anammoxosome, is hypothesized to be the locus of all catabolic reactions in the anammox metabolism. Electron tomography showed that the anammoxosome and its membrane were highly folded. This finding was confirmed by a transmission electron microscopy study using different sample preparation methods. Further, in this study electron-dense particles were observed and electron tomography showed that they were confined to the anammoxosome compartment. Energy dispersive X-ray analysis revealed that these particles contained iron. The functional significance of a highly folded anammoxosome membrane and intracellular iron storage particles are discussed in relation to their possible function in energy generation.

Ten years ago a fortuitous discovery led to the identification of oceanic bacteria capable of anaerobic ammonium oxidation (anammox). It was soon recognized that the anammox reaction has great ecological significance, as it is responsible for removing up to 50% of fixed nitrogen from the oceans. The genome of the anammox bacterium Kuenenia stuttgartiensis has now been sequenced in a remarkable feat of what is called environmental genomics. Anammox bacteria grow very slowly and are not available in pure culture. For genome analysis an inoculum of wastewater sludge was grown in a bioreactor for one year, clocking up 10–15 generations. The DNA of the whole microbial community was sequenced and the genome of this one anammox bacterium was deduced from the results. With the genome sequence known, it will be possible to gain insight into the metabolism and evolution of these important bacteria. The genome of Kuenenia stuttgartiensis has been sequenced to learn more about anaerobic ammonium oxidation. Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment1,2. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism3,4, the biosynthesis of ladderane lipids5,6 and the role of cytoplasm differentiation7 are unique in biology. Here we use environmental genomics8,9—the reconstruction of genomic data directly from the environment—to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis10 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.

The existence of anaerobic ammonium oxidation (anammox) was hypothesized based on nutrient profiles and thermodynamic calculations (5, 31, 44). It was first discovered about 1 decade ago (25) in a pilot plant treating wastewater from a yeast-producing company in Delft, The Netherlands. The anammox reaction is the oxidation of ammonium under anoxic conditions with nitrite as the electron acceptor and dinitrogen gas as the product. Hydroxylamine and hydrazine were identified as important intermediates (51). Due to their very low growth rates (doubling time in enrichments is at best 11 days) the cultivation of the anammox bacteria proved to be tedious and required very efficient biomass retention (41, 43). A physical purification of anammox organisms from enrichment cultures was achieved with percoll density centrifugation (42). The purified cells performed the anammox reaction after activation by hydrazine. Based on phylogenetic analysis, the discovered anammox organism branched deep in the Planctomycetes phylum (Fig. 1A and B, [42]) and was named “Candidatus Brocadia anammoxidans” (19). FIG. 1. (A) 16S rRNA gene-based phylogenetic tree reflecting the relationship of “Ca. Scalindua,” “Ca. Brocadia,” and “Ca. Kuenenia” to other Planctomycetes and other reference organisms. Tree reconstruction was ... After the first discovery, nitrogen losses, which could only be explained by the anammox reaction, were reported in other wastewater treatment facilities including landfill leachate treatment plants in Germany, Switzerland, and England (11, 14, 15, 36), as well as in semitechnical wastewater treatment plants in Germany (34), Belgium (30), Japan (12), Australia (48), and the United States (10, 45). Molecular techniques showed the presence of organisms affiliated with the anammox branch within the Planctomycetes in all these wastewater treatment plants. Nutrient profiles and 15N tracer studies in suboxic marine and estuarine environments indicated that anammox is also a key player in the marine nitrogen cycle (8, 46, 49). In addition, 16S rRNA gene analysis, fluorescence in situ hybridization (FISH), the distribution of specific anammox membrane lipids, nutrient profiles, and tracer experiments with [15N]ammonia showed the link between the anammox reaction and the occurrence of the anammox bacterium “Candidatus Scalindua sorokinii” in the suboxic zone of the Black Sea (20). The anammox reaction has also been tested for implementation for full-scale removal of ammonia in wastewater treatment (13, 52, 53). The detection and identification of active anammox organisms in environmental samples combined with information on environmental conditions can facilitate the search for possible biomass sources to be used as an inoculum for laboratory, semitechnical, or full-scale anammox reactors. Additionally, such information could provide insights into the niche differentiation of anammox organisms. This review summarizes the recent advances made in the 16S rRNA gene-based techniques for the detection of anammox bacteria. A convenient PCR detection method for anammox organisms is presented in which anammox-specific FISH probes were used as primers. Furthermore, methods which link activity and the detection of anammox bacteria, such as the combination of FISH and microautoradiography (FISH-MAR) (22) as well as FISH targeting the intergenic spacer region (ISR) between the 16S and 23S rRNA are discussed and compared to conventional methods to detect anammox activity. Each of these approaches by itself only addresses limited aspects, such as abundance, activity, or physiology. Thus, a combination of rRNA-based and non-rRNA-based methods is necessary to allow a comprehensive study of anammox bacteria in their ecosystems.

Contains fulltext : 60180.pdf (Publisher’s version ) (Closed access) Anammox bacteria belong to the phylum Planctomycetes and perform anaerobic ammonium oxidation (anammox); they oxidize ammonium with nitrite as the electron acceptor to yield dinitrogen gas. The anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane. The anammox bacteria, first found in a wastewater treatment plant in The Netherlands, have the potential to remove ammonium from wastewater without the addition of organic carbon. Very recently anammox bacteria were also discovered in the Black Sea where they are responsible for 30-50% of the nitrogen consumption. This review will introduce different forms of intracytoplasmic membrane systems found in prokaryotes and discuss the compartmentalization in anammox bacteria and its possible functional relation to catabolism and energy transduction. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Lipid membranes are essential to the functioning of cells, enabling the existence of concentration gradients of ions and metabolites. Microbial membrane lipids can contain three-, five-, six- and even seven-membered aliphatic rings, but four-membered aliphatic cyclobutane rings have never been observed. Here we report the discovery of cyclobutane rings in the dominant membrane lipids of two anaerobic ammonium-oxidizing (anammox) bacteria. These lipids contain up to five linearly fused cyclobutane moieties with cis ring junctions. Such 'ladderane' molecules are unprecedented in nature but are known as promising building blocks in optoelectronics. The ladderane lipids occur in the membrane of the anammoxosome, the dedicated intracytoplasmic compartment where anammox catabolism takes place. They give rise to an exceptionally dense membrane, a tight barrier against diffusion. We propose that such a membrane is required to maintain concentration gradients during the exceptionally slow anammox metabolism and to protect the remainder of the cell from the toxic anammox intermediates. Our results further illustrate that microbial membrane lipid structures are far more diverse than previously recognized.