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Keywords:

  • anammox bacteria;
  • anammoxosome;
  • proteomics;
  • immunogold labelling;
  • MS;
  • nitrogen cycling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N2. Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N2 production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N2 and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

During the last decades, observations of new links between biogeochemically important constituents and novel routes during nitrogen mineralization have challenged our understanding of nitrogen cycling in the biosphere (Dalsgaard et al., 2005; Hulth et al., 2005). For example, independent observations imply that anaerobic ammonium oxidation to N2 (anammox) is a globally significant pathway for nitrogen removal in marine environments (Thamdrup & Dalsgaard, 2002; Dalsgaard et al., 2003; Kuypers et al., 2003; Hamersley et al., 2007). All in all, anammox seems to account for between 25 and 50% of the total marine N2 production (Devol, 2003; Arrigo, 2005; Kuypers et al., 2005). Locally, however, the relative importance may be significantly higher (Thamdrup & Dalsgaard, 2002; Engström et al., 2005). Anammox was first discovered in a wastewater treatment plant (Mulder et al., 1995) and has since then been confirmed in a multitude of environments (Dalsgaard et al., 2005). In wastewater treatment, the anammox process is increasingly implemented as a potential alternative to denitrification for the low-cost removal of inorganic nitrogen (Op den Camp et al., 2006).

Anammox bacteria are monophyletic members of the phylum Planctomycetes (Strous et al., 1999; Jetten et al., 2005). So far, the anammox branch is associated with at least four genera: Candidatus‘Brocadia’, Candidatus‘Kuenenia’, Candidatus‘Anammoxoglobus’ and Candidatus‘Scalindua’ (Jetten et al., 2005; Kartal et al., 2007). In marine environments, however, anammox bacteria are suggested to be confined to the genus Candidatus‘Scalindua’. Planctomycetes, including anammox bacteria, generally contain internal organelle-resembling membranous compartments (Lindsay et al., 2001; van Niftrik et al., 2004; Fuerst, 2005). The intracellular membrane compartment of the anammox bacteria, the anammoxosome, has been suggested as the site for the anammox reaction (Fuerst, 2005). Interestingly, the anammoxosome membrane contains unique lipids with sequential structures of four-membered aliphatic cyclobutane rings arranged like a ‘staircase’ at the end of the hydrocarbon chains (i.e. ‘ladderane’ lipids; Sinninghe-Damstéet al., 2002). The ladderane lipids form an exceptionally dense membrane that likely provides a tight barrier against diffusion (Strous et al., 1999). Hydrazine, a toxic and mutagenic compound, is formed by the enzyme hydrazine hydrolase as an intermediate product in the anammox reaction (Strous et al., 2006). Because the reaction is suggested to be confined to the anammoxosome (Fuerst, 2005), the ladderane lipid membrane would protect the genetic material and the remainder of the anammox cell from exposure by trapping the hydrazine inside the anammoxosome.

The main routes of anammox include the reduction of nitrite to nitric oxide (NO) by nitrite reductase, the conversion of NO and ammonium to hydrazine by hydrazine hydrolase, the oxidation of hydrazine to N2 by a hydrazine-oxidizing enzyme (HZO) and the subsequent synthesis of ATP by an ATP synthase using the proton-motive force generated during the anammox cycle (Strous et al., 2006). The mechanisms that control the reduction of nitrite in the anammox reaction are under debate, and to date, there is no common agreement on whether the reaction proceeds by NO or hydroxylamine as intermediates (Strous et al., 2006; van der Star et al., 2008). Recently, the metagenome of the anammox bacterium Candidatus‘Kuenenia stuttgartiensis’ was obtained from an enrichment culture (Strous et al., 2006). The genome was found to code for a nitrite/NO oxidoreductase that would favor NO as an intermediate product during anammox. The hydrazine hydrolase is a protein unique to anammox bacteria. Based on the genome sequence, eight proteins (kuste2854–kuste2861) encode for the hydrazine hydrolase. This novel gene cluster contains domains involved in electron transfer and catalysis, with several cytochromes and a β-propeller complex (Strous et al., 2006). Similar structures have previously been found in, for example, nitrous oxide reductases (Brown et al., 2000; Strous et al., 2006). The hydrazine produced from the hydrazine hydrolase is converted to N2 by a HZO under the generation of protons (Fuerst, 2005). A multiheme protein with hydrazine-oxidizing capability was recently purified from an anammox enrichment culture (bacterial strain KSU-1) with a high sequence homology to the Candidatus‘Brocadia anammoxidans’ bacterium (Shimamura et al., 2007). Two genes were found coding for the HZO, demonstrating low sequence homologies (<30%) to any of the known hydroxylamine oxidoreductase (hao) genes, but very high (88% and 89%, respectively) to two of the hao genes found in the genome of C. ‘Kuenenia stuttgartiensis’ (kustc0694 and kustd1340). During the anammox reaction, protons are consumed in the cytoplasm and generated inside the anammoxosome (van Niftrik et al., 2004; Fuerst, 2005). This proton gradient could be used to catalyze and sustain the synthesis of ATP by membrane-bound ATP synthases. Identification of an ATP synthase in the anammoxosome membrane would support indirect experimental evidence that the anammoxosome is an energy-generating organelle-like compartment producing ATP.

The completion of the C. ‘Kuenenia stuttgartiensis’ metagenome (Strous et al., 2006) has provided important insights into the overall characteristics of anammox bacteria. However, to understand the microbial functionality and predict community feedbacks, for example caused by progressive changes in physical, chemical and biological forcings, it is necessary to identify and characterize the proteins actually expressed by the bacteria. The objective of this study was to identify key proteins associated with the anammox reaction and, by immunogold labelling electron microscopy (EM), determine the subcellular location of the hydrazine hydrolase. Since the anammox reaction has been suggested to be confined to the anammoxosome, we focused our investigation on proteins associated with this compartment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Anammox bacteria and FISH

Mixed aggregates of bacterial cells from the pilot anammox waste-water reactor at Himmerfjärdsverket, Sweden (http://www.syvab.se), were fixed and hybridized as described previously (Manz et al., 1992). Probes used for identification of anammox species were ‘S-*-AMX-0368-a-A-18’ (Schmid et al., 2003), ‘S-S-Kst-0157-a-A-18’ (Schmid et al., 2001) and ‘S-*-AMX-0820-a-A-22’ (Schmid et al., 2000). The slides were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA) containing 4′,6-diamidino-2-phenylindole (DAPI) for enumeration of total bacteria.

Preparation of anammoxosomes

Anammoxosomes were prepared according to Sinninghe-Damstéet al. (2002). Clusters of bacterial cells were transferred to a buffer solution (50 mM Tris-HCl, 60 mM EDTA and 0.25 M sucrose, pH 7). Bacterial cells were subjected to mild sonication in an ultrasonic bath (c. 5 min; 25% of maximum intensity, Ultrasonic, 500 W, NDI) until disruption into single cells. Disruption of aggregates was followed by a general confirmation of the procedure using ordinary light microscopy. If aggregates were still obvious, the mild sonication procedure was repeated. Following the addition of lysozyme (5 mg mL−1), the solution of single cells was rapidly cooled to c. 4 °C and treated at this temperature for 20 min to optimize the enzymatic activity. The solution was sonicated using a tip sonicator (5-s intervals between 5-s pulses; 20 times, 25% of maximum intensity; Sonics Vibracell, Model 501, 500 W). Sonication was again followed by light microscopy, and the sonication procedure was repeated until a homogeneous solution was obtained. The solution was centrifuged (4 °C, 10 000 g, 10 min), resulting in a red–orange pellet of anammoxosomes. We further established and confirmed the analytical protocol by transmission electron microscopy.

Anammoxosome protein analysis

The anammoxosome pellet was resuspended (10 mM Trizma, 300 mM NaCl, pH 8) and heavily sonicated (75% of maximum intensity, 500 W) using the tip sonicator, whereby proteoliposomes were formed from the lipid membrane of the anammoxosome. The optimum size range of proteoliposomes (100–200 nm in diameter) for downstream analysis of proteins was confirmed by light scattering experiments.

The proteoliposome solution (350 μL) was injected into the LPI FlowCell (Nanoxis AB, Göteborg, Sweden; patent WO 2006068619, Karlsson et al., 2004), where the proteoliposomes were immobilized onto specifically designed surfaces of the FlowCell. The proteins of the immobilized proteoliposomes were digested by incubating the sample with trypsin (5 μg mL−1 in 10 mM Trizma, 20 mM NaCl, pH 8) for 2 h at 37 °C. The resulting peptides were eluted with 700 μL buffer (10 mM Trizma, 20 mM NaCl, pH 8) and subsequently analyzed separately by LC-MS/MS (Proteomics Core Facility, University of Gothenburg). Before analysis, the sample was centrifuged in vacuum to dryness and reconstituted in 20 μL 0.1% formic acid in water. For LC, an Agilent 1100 binary pump was used and the tryptic peptides were separated on a 200 × 0.05 mm i.d. fused silica column packed in-house with 3 μm ReproSil-Pur C18-AQ particles (Dr Maisch GmbH, Ammerbuch, Germany). A 40-min gradient of 10–50% acetonitrile in 0.2% formic acid was used for separation of the peptides. Mass analyses were performed in a 7-T LTQ-FT mass spectrometer (Hybrid Linear Trap Quadrupole-Fourier Transform; Thermo Electron). All tandem mass spectra were searched by mascot (Matrix Science) against the bacterial database. In order to predict possible sequence homologies of the identified peptides between different types of bacteria, a search was performed against the protein database at the Swiss Institute of Bioinformatics using the blast network service (http://www.expasy.org, Altschul et al., 1997).

Antibody production for immunogold labelling

Two peptides from the proteins kuste2860 (single letter amino acid sequence KEFDTPTLRD) and kuste2861 (RSPYPLPDDRM), respectively, were chosen from the MS results as candidates for the immunogold labelling of the hydrazine hydrolase. The peptides were chosen due to their high abundance and multiple queries in the MS-database analysis. Synthetic duplicate peptides and antibodies against these peptides were produced by Innovagen AB (http://www.innovagen.se, Lund, Sweden). The production of antibodies was induced by immunization of specific pathogen-free rabbits. The antibodies were referred to as anti-kuste2860 and anti-kuste2861.

EM preparation

Bacterial aggregates in suspension were fixed for 2 h with a mixture of 4% paraformaldehyde, 0.5% glutaraldehyde and 0.02% Na azide in 0.05 M Na cacodylate buffer (pH 7.4). After washing in buffer, the samples were embedded in 5% gelatin. Dehydration and infiltration with Lowicryl K4M resin was performed as a progressive lowering of temperature routine in a Leica CS Auto cryosubstitution unit according to the recommendations by the manufacturer. The resin was polymerized under UV light at −20 °C. Semi-thin (1 μm, Richardson's stain) and ultrathin sections (setting c. 60 nm) were obtained using a Reichert Ultracut E ultramicrotome fitted with diamond knives.

Immunocytochemistry

Immunoreagents were diluted in phosphate-buffered saline with 0.1% cationized bovine serum albumin (Aurion), 1% normal goat serum, 5% dried milk powder, 20 mM glycine and 0.02% Na azide. After etching of sections with periodate and ammonium chloride, primary antisera were applied at 4 °C for 16.5 h (dilutions 1 : 100 or 1 : 1000). Rabbit immunoglobulin G (IgG) served as a negative control. The secondary antibody was Goat anti-rabbit IgG conjugated with 10 nM gold 1 : 50 for 70 min (Sigma-Aldrich). Sections were postfixed after immunoincubation with 1% glutaraldehyde, contrasted with uranylacetate and lead citrate and examined in a LEO 912AB transmission electron microscope equipped with a megaview III CCD camera for digital image capture.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The bacterial aggregate from the wastewater reactor contained c. 40% anammox bacteria, determined by FISH (Fig. 1). Following the enzymatic digestion in the LPI FlowCell (Fig. 2), MS/MS analysis of the acquired peptides gave rise to numerous hits in the bacterial database (Table 1 and Supporting Information). As only the genome of C. ‘Kuenenia stuttgartiensis’ has been sequenced (Strous et al., 2006), hits arising from anammox bacteria refer to this particular species. In our study, FISH studies demonstrated a high relative contribution of the anammox bacterium C. ‘Brocadia anammoxidans’ (c. 80%), while only <20% originated from C. ‘Kuenenia stuttgartiensis’. It is therefore likely that hits ascribed here to C. ‘Kuenenia stuttgartiensis’ are rather homologue amino acid sequences related to C. ‘Brocadia anammoxidans’. Using the blast network service (http://www.expasy.org), peptides originating from the hydrazine hydrolase were predominantly found in the genome of the C. ‘Kuenenia stuttgartiensis’ and, occasionally, in the KSU-1 anammox bacterium (Table 1; Shimamura et al., 2007). The search of peptides from the HZO displayed additional hits from uncultured Planctomycetes. As expected, several peptides in the protein database were found to be very similar to the peptides from the ATP synthase. The ATP synthase is one of the most conserved proteins of bacteria and sequence overlaps are therefore not surprising (Deckers-Hebestreit & Altendorf, 1996). Peptides from the β subunit, identified as C. ‘Kuenenia stuttgartiensis’, were also found in the KSU-1 bacterium and uncultured Planctomycetes. The α subunit demonstrated complete sequence overlap with several types of bacteria (Table 1).

image

Figure 1.  FISH image of a cluster of anammox bacteria. DNA staining by 4′,6-diamidino-2-phenylindole (DAPI) (blue) represents the total amount of bacteria. The probe ‘S-*-AMX-0368-a-A-18’ (Schmid et al., 2003; Thermo Electron Corporation, Ulm, Germany), labelled with Cy3 (red), was used for a general identification of anammox bacteria.

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image

Figure 2.  Schematic illustration of the LPI FlowCell (Nanoxis AB, Göteborg, Sweden) and general principles of the analytical protocol. In brief, a solution of vesicles containing target proteins is injected into the FlowCell and the vesicles are allowed to attach to the specifically designed membrane-attracting surfaces (1). The proteins associated with the membrane of the vesicles are digested into smaller peptide fragments using, for example, trypsin (2). The peptides are eluted from the FlowCell and analyzed by LC-MS/MS (3).

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Table 1.   Proteins and ORF notations of the hydrazine hydrolase complex, the HZO and the ATP synthase identified from the peptides found in the MS/MS database search
ProteinORFPeptidesblast hits
  1. Three different proteins of the hydrazine hydrolase were found: kuste2861, kuste2860 and kuste2859. The HZO was identified as kustd1340 and both the α (kuste3793) and the β (kuste3795) subunit of the globular domain of the ATP synthase were found. Identification followed extraction of anammoxosomes, preparation of proteoliposomes and enzymatic digestion of proteins in the LPIFlowCell. The peptides are given with amino acids in their single-letter abbreviation. The blast hits (Altschul et al., 1997) of the identified peptides provided the degree of sequence homology to other types of bacteria. Hits originating from Candidatus‘Kuenenia stuttgartiensis’ are abbreviated as K. stutt.

Hydrazine hydrolase
 Hypothetical (diheme) proteinkuste2861KITFGDRKIK. stutt.
 KFLTQDERYK. stutt. and KSU-1
 RMLVSYAERGK. stutt.
 RWINTFTAGKNK. stutt. and KSU-1
 RSPYPLPDDRMK. stutt. and KSU-1
 RSSSNSGTAFQQRGK. stutt.
 Hypothetical (diheme) proteinkuste2860RDPGFVFRSK. stutt. and KSU-1
 KEFDTPTLRDK. stutt. and KSU-1
 RALEFTGSPFRNK. stutt.
 RNADGSLTEAQKRGK. stutt. and KSU-1
 RALFSDAQTHDVGTGRVK. stutt.
 Hypothetical proteinkuste2859KVGDWPSGIKIK. stutt.
 RSIYTSDYGPRMK. stutt.
HZO
 Hydroxylamine oxidoreductasekustd1340KYRETFKVK. stutt., KSU-1, planctomycete
 KTGESGEFRMK. stutt., KSU-1, planctomycete
 RDEVRPSNPIKGK. stutt. and KSU-1
 RENLQAMDESVKDK. stutt., planctomycete
 KTGEWLDQLTGPYIVKNK. stutt., KSU-1, planctomycete
The ATP synthase
 ATP synthase subunit βkuste3795KVIDLLAPFARGK. stutt., KSU-1, planctomycete
 RQIAELGIYPAVDPLRSK. stutt.
 ATP synthase F1 subunit αkuste3793RELIIGDRAseveral species
 KAIDAMIPIGRGseveral species

Three proteins of the hydrazine hydrolase protein complex were identified, as were two proteins of the ATP synthase and the HZO. The hydrazine hydrolase proteins kuste2859, kuste2860 and kuste2861 were all highly abundant in the MS/MS analysis, of which two (kuste2859 and kuste2861) form β-propellers as part of the catalytic site of the hydrazine hydrolase enzyme (Strous et al., 2006). Converting ammonium and NO (or nitrite) into hydrazine, the hydrazine hydrolase is a central enzyme in the anammox reaction. The present investigation is the first proteomic study that has identified the putative hydrazine hydrolase. In order to experimentally verify that the anammox reaction takes place in the anammoxosome, efforts were made to determine the subcellular location of the hydrazine hydrolase through immunogold EM analysis. For the immunogold labelling, antibodies were produced against two of the hydrazine hydrolase peptides found in the MS analysis: kuste2860 (KEFDTPTLRD) and kuste2861 (RSPYPLPDDRM). In untreated bacterial colonies still enclosed in mucoid material, the anammox bacteria demonstrated a distorted polygonal form (Fig. 3a). The anammoxosomes of intact bacteria were invariably labelled with the anti-kuste2860 and anti-kuste2861 antibodies, whereas no labelling was observed in the cytosol outside the membranous compartment (Fig. 3a). This confirmed a clear association of the hydrazine hydrolase with the anammoxosomes. Interestingly, immunogold particles were not randomly distributed within the anammoxosomes, but were frequently found in ordered configurations, indicating an inner structural organization (Fig. 3a). Controls were devoid of gold labelling (Fig. 3b). Isolated anammoxosomes were rounded, less dense and homogeneous, and thus appeared swollen compared with the same structures of intact bacteria (Fig. 3c). The intensity of the immunogold signal varied, indicating leakage of the antigen. At the ultrastructural level, the preparation of anammoxosomes resulted in an altered integrity of this organelle-like compartment. Supportive evidence was thereby provided that the anammox reaction takes place in the anammoxosome and that hydrazine is released into the interior of the anammoxosome.

image

Figure 3.  (a) EM of an anammox bacterial cell after immuno-localization of the hydrazine hydrolase using anti-kuste2861 as the primary antibody. As the secondary antibody, gold-labelled goat anti-rabbit antibodies were used. The cytoplasmic membrane (CM, black arrows) and the convoluted inner anammoxosome membrane (AM, black arrowheads) are indicated. Note the polygonal shape of the cell and the dense homogeneous appearance of the anammoxosome content. Immunogold particles were abundant over the anammoxosome and were occasionally distributed in arranged sequences (white arrowheads). The cytosol surrounding the anammoxosome (bold white arrow) was devoid of gold labelling. (b) Immunogold EM image illustrating negative control staining using rabbit IgG. No staining of the anammoxosomes was observed. (c) Immunogold EM image of free anammoxosomes. The three structures demonstrated different intensities of binding of the anti-kuste2861 antibody (immunogold particles indicated by white arrowheads). The anammoxosome content appeared to be less homogeneous than in untreated cells. The intensity of the immunogold labelling of free anammoxosomes varied to a much greater degree compared with anammoxosomes of intact bacteria. This indicates a leakage of the antigen. Scale bars=0.5 μm.

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The hydrazine produced by the hydrazine hydrolase is sequentially oxidized to N2 by the HZO (Shimamura et al., 2007). In the present study, the protein kustd1340, denoted similar to hydroxylamine oxidoreductase, was highly abundant in all MS/MS runs. An enzyme with hydrazine-oxidizing capability was purified and characterized from a closely related anammox strain (KSU-1) (Shimamura et al., 2007). Because this protein had a very high sequence homology to kustd1340, but very low homology to other hao genes, kustd1340 was the gene expressed as the HZO in the mixed anammox community analyzed here. Immunogold labelling EM of a hydroxylamine oxidoreductase (hao), a close relative of the HZO, placed the hao inside the anammoxosome (Lindsay et al., 2001).

The oxidation of hydrazine by the HZO concomitantly releases protons inside the anammoxosome (Fuerst, 2005), thus rendering the anammoxosome acidic compared with the surrounding cytosol. The MS/MS analysis of this study identified at least two proteins assigned to a membrane-bound F-type ATP synthase (kuste3793 and kuste3795). ATP synthase produces ATP utilizing a proton gradient across the membrane in which it is situated. Kuste3793 was denoted strongly similar to the ATP synthase F1 α subunit, and kuste3795 was referred to as strongly similar to the ATP synthase subunit β (Table 1). Kuste3793 and kuste3795 form the globular ATP-synthesizing domains that connect to other parts of the ATP synthase complex. Identification of this membrane-bound ATP synthase in the anammoxosome membrane further supports the proposed reaction scheme where the proton gradient generated from the oxidation of hydrazine to N2 is utilized to synthesize ATP.

The deciphering of the C. ‘Kuenenia stuttgartiensis’ genome suggests that c. 200 genes are involved in anammox catabolism and respiration (Strous et al., 2006). This is by far more than normally found in other types of bacteria. The identification of a variety of respiration complexes strengthens the hypothesis that anammox bacteria are not specialists as suggested previously, but rather generalists utilizing a wide range of possible reaction pathways to thrive under a variety of stressors in aquatic systems and during wastewater treatment. Anammox bacteria are commonly exposed to a multitude of environmental conditions, for example, temperature, oxygen, pH and organic matter availability. Although the genome provides strong indications of functionality, this information alone is not sufficient to predict how the anammox bacteria respond to applied stressors. In order to determine environmental controls and better understand up-and-down regulations of key proteins, proteomic tools and protein expression profiling are crucial. The results presented here, including the identification and subcellular location of the putative hydrazine hydrolase, provide a major step towards this goal.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Financial support was provided by The Swedish Research Council (VR), The Foundation for Strategic Environmental Research (MISTRA) and The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS). S. Stridh and M. Tuvesson (SYVAB) provided access to the anammox pilot wastewater reactor. The EM equipment has been defrayed by grants from the Lundberg Research Foundation. The technical support by Yvonne Josefsson is acknowledged. The expertise at the Proteomics Core Facility, University of Gothenburg, is acknowledged.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Appendix S1. Detailed description of Materials and methods.

Appendix S2. List of proteins, possible function and ORF notation identified from the peptides found in the MS/MS database search. Identification followed extraction of anammoxosomes, preparation of proteoliposomes and enzymatic digestion of proteins in the LPITMFlowCell. The peptides are given with amino acids in their single-letter abbreviation.

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FML_1677_sm_appendixS1.doc35KSupporting info item
FML_1677_sm_appendixS2.doc23KSupporting info item

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